United States Environmental Protection Agency
Office of Water
Washington, DC
EPA# 841-F-19-004
National Coastal Condition Assessment
2020
Laboratory Operations Manual
Version 1,2
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NOTICE
The goal of the National Coastal Condition Assessment (NCCA) is to provide a comprehensive
assessment of the Nation's freshwater, marine shoreline and estuarine waters. The complete
documentation of overall project management, design, methods, and standards is contained in
four companion documents:
National Coastal Condition Assessment: Quality Assurance Project Plan (EPA # 841-F-19-003)
National Coastal Condition Assessment: Site Evaluation Guidelines (EPA # 841-B-20-001)
National Coastal Condition Assessment: Field Operations Manual (EPA # 841-F-19-005)
National Coastal Condition Assessment: Laboratory Operations Manual (EPA # 841-F-19-004)
This document (Laboratory Operations Manual) contains information on laboratory methods
for analyses of the samples collected during the National Coastal Condition Assessment (NCCA).
It also provides quality assurance objectives, sample handling procedures, and data reporting
requirements. Methods described in this document are to be used specifically in work relating
to the NCCA 2020. All NCCA cooperator laboratories must follow the guidelines presented in
the document.
With the exception of the requirements in Chapters 3 and 4 for evaluating algal toxins, mention
of trade names or commercial products in this document does not constitute endorsement or
recommendation for use. Chapters 3 and 4 requires use of a specific kit and supplemental
materials manufactured by a single firm.
More details on specific methods for site evaluation, and sampling and sample processing can
be found in the appropriate companion document (Site Evaluation Guidelines and Field
Operations Manual, respectively).
The suggested citation for this document is:
USEPA. National Coastal Condition Assessment 2020: Laboratory Operations Manual. U.S.
Environmental Protection Agency, Office of Water, Washington, DC. 2020. EPA# 841F19004
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VERSION HISTORY
Version
1.0
1.1
1.2
Date
April 10, 2020
April 22, 2020
March 9, 2021
Changes Made
n/a
Changed header of "Fish Tissue Fillet (Great Lakes)" to
"Great Lakes Human Health Fish Tissue Samples'"
Version Date corrected to read "April" 2020 in a few
page headers
Section 3.5.4 number 29.c.iv- added a definition for
holding time excursion.
Section 4.6.4 number 25.d.ii- added a definition for
holding time excursion. Removed quality flag wording
to combine with data flags.
Changed tables and wording within Chapter 6 to call for
300g of mass for fish tissue analyses. Standardized this
across the chapter and in multiple tables.
Section 9.5 number 7- language added for clarity in
laboratory responsibilities.
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TABLE OF CONTENTS
NOTICE II
VERSION HISTORY Ill
TABLE OF CONTENTS IV
FIGURES VII
TABLES VII
LIST OF ACRONYMS VIII
1.0 INTRODUCTION 11
2.0 GENERAL LABORATORY GUIDELINES 13
2.1 Responsibility and Personnel Qualifications 13
2.2 Roles and Contact Information 13
2.3 Sample Tracking 14
2.4 Reporting 14
3.0 ALGAL TOXIN: CYLINDROSPERMOPSIN IMMUNOASSAY PROCEDURE 17
3.1 Definitions and Required Personnel Qualifications 17
3.1.1 Definitions 17
3.2 Precautions 19
3.3 Equipment/Materials 19
3.4 Sample Receipt 20
3.5 Procedure 21
3.5.1 Sample Preparation 21
3.5.2 Additional Sample Preparation for Samples with Salinity greater than 8 ppt 22
3.5.3 Kit Preparation 22
3.5.4 Insertion of Contents into Wells 23
3.5.5 Dilutions (if needed) 29
3.6 Pertinent QA/QC Procedures 30
3.6.1 QC Samples 30
3.6.2 Summary of QA/QC Requirements 30
3.7 Sample and Record Retention 32
3.8 References 33
4.0 ALGAL TOXIN: MICROCYSTIN IMMUNOASSAY PROCEDURE 34
4.1 Summary of the Procedure 34
4.2 Health and Safety Warnings 34
4.3 Definitions and Required Resources (Laboratories and Equipment) 35
4.3.1 Definitions 35
4.4 General Requirements for Laboratories 37
4.4.1 Expertise 37
4.4.2 Quality assurance and quality control requirements 37
4.4.3 Equipment/Materials 38
4.5 Sample Receipt 39
4.6 Procedure 40
4.6.1 Sample Preparation: Freeze-Thaw Steps 40
4.6.2 Additional Sample Preparation for Samples with Salinity>3.5 parts per thousand 41
4.6.3 Kit Preparation 42
4.6.4 Insertion of Contents into Wells 43
4.6.5 Dilutions (if needed) 50
4.7 Quality Measures 51
4.7.1 Assistance Visits 51
4.7.2 QC Samples 51
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4.7.3 Summary ofQA/QC Requirements 51
4.8 Sample and Record Retention 53
4.9 References 54
5.0 BENTHIC MACROINVERTEBRATES 55
5.1 Summary of Method 55
5.2 Health and Safety Warnings 55
5.3 Definitions and Required Resources (Laboratory, Personnel, and Equipment) 56
5.3.1 Definitions 56
5.3.2 Laboratory 59
5.3.3 Personnel 59
5.3.4 Equipment/Materials 60
5.4 Sample Receipt 61
5.5 Sample Preparation and Picking Organisms 62
5.6 Taxonomic Identification 64
5.7 Data Entry 71
5.8 Sample and Record Retention 71
5.9 External Taxonomic Quality Control 71
5.10 Quality Assurance/Quality Control (QA/QC) 76
5.11 References 77
6.0 WHOLE BODY FISH PROCESSING AND CONTAMINANT ANALYSIS 80
6.1 Summary of the Procedure 80
6.2 Health and Safety Warnings 80
6.3 Definitions and Required Resources (Personnel, Laboratories, and Equipment) 81
6.3.1 Definitions 81
6.3.2 General Requirements for Laboratories 82
6.3.3 Equipment/Materials 83
6.4 Sample Receipt 83
6.5 Whole Fish Preparation and Homogenization Procedures 85
6.5.1 Sample Classification: Routine or Non-Routine 85
6.5.2 Fish Examination and Preparation 85
6.5.3 Equipment Cleaning and Rinsate Collection 87
6.5.4 Compositing and Homogenization Procedure 88
6.6 Contaminant Analysis: Requirements 90
6.7 Data Entry 94
6.8 Quality Measures 95
6.8.1 Assistance Visits 95
6.8.2 Summary ofQA/QC Requirements 96
6.9 Sample and Record Retention 99
6.10 References 100
7.0 SEDIMENT CONTAMINANT, GRAIN SIZE, AND TOC ANALYSES 101
7.1 Summary of the Procedure 101
7.2 Health and Safety Warnings 101
7.3 Definitions and Required Resources (Personnel, Laboratories, and Equipment) 101
7.3.1 Definitions 101
7.3.2 General Requirements for Laboratories 103
7.3.3 Equipment/Materials 103
7.3.4 Sample Receipt 104
1A Laboratory Analysis: Requirements 105
7.5 Data Entry 109
7.6 Quality Measures Ill
7.7 Assistance Visits Ill
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7.7.1 Summary of QA/QC Requirements Ill
7.8 Sample and Record Retention 114
7.9 References 115
8.0 WATER CHEMISTRY AND CHLOROPHYLL A 116
8.1 Health and Safety Warnings 116
8.2 Definitions and Required Resources (Personnel, Laboratories, and Equipment) 116
8.2.1 Definitions 116
8.2.2 General Requirements for Laboratories 118
8.2.3 Equipment/Materials 118
8.3 Sample Receipt 119
8.4 Preparation of Water Chemistry Aliquots 120
8.5 Water Chemistry and Chlorophyll a Analysis: Requirements 122
8.6 Data Entry 126
8.7 Quality Measures 127
8.8 Sample and Record Retention 133
8.9 References 133
9.0 SEDIMENT TOXICITY TESTING 135
9.1 Summary of the Procedure 135
9.2 Health and Safety Warnings 135
9.3 Definitions and Required Resources (Personnel, Laboratories, and Equipment) 135
9.3.1 Definitions 135
9.3.2 General Requirements for Laboratories 136
9.3.3 Equipment/Materials 137
9.4 Sample Receipt 137
9.5 ToxicityTesting: Requirements 139
9.6 Data Entry 142
9.7 Quality Measures 144
9.7.1 Assistance Visits 144
9.7.2 Summary of QA/QC Requirements 144
9.8 Sample and Record Retention 146
9.9 References 147
10.0 GREAT LAKES HUMAN HEALTH FISH TISSUE SAMPLES 148
11.0 MERCURY IN FISH TISSUE PLUGS 149
11.1 Summary of the Procedure 149
11.2 General Requirements for Laboratories 149
11.2.1 Equipment/Materials 149
11.3 Sample Receipt 149
11.4 Quality Measures 151
11.4.1 Assistance Visits 151
12.0 FECAL INDICATOR: ENTEROCOCCI 154
APPENDIX A: MICROCYSTINS-ADDA SAES ELISA TEST KIT PROTOCOL 155
APPENDIX B: TARGET FISH SPECIES FOR WHOLE FISH ANALYSES 156
APPENDIX C: EXAMPLE SOPS FOR MERCURY IN FISH TISSUE PLUG ANALYSES 160
APPENDIX D: RESEARCH INDICATOR - MICROPLASTICS IN SEDIMENT 161
APPENDIX E: RESEARCH INDICATOR- TOTAL ALKALINITY 162
APPENDIX F: RESEARCH INDICATOR- A N15 ISOTOPE IN BENTHIC ORGANIC MATTER 163
APPENDIX G: LABORATORY REMOTE EVALUATION FORMS 164
NCCA2020 DOCUMENT REQUEST FORM - CHEMISTRY LABORATORIES 164
NCCA 2020 DOCUMENT REQUEST FORM - BIOLOGY LABORATORIES 167
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FIGURES
Figure 3.1Cylindrospermopsin: sample template 24
Figure 4.1 Microcystis sample template 44
Figure 8.1 Water Chemistry and Dissolved Nutrient Samples: Receipt and Holding Times 121
TABLES
Table 1.1 NCCA: Indicators 12
Table 2.1 NCCA: Contact Information 13
Table 3.1 Cylindrospermopsin login: required data elements 21
Table 3.2: Cylindrospermopsin: required data elements- data submission 26
Table 3.3: Cylindrospermopsin: quality control- sample analysis 31
Table 4.1 Microcystes Login: Required Data Elements 40
Table 4.2 Microcystes: Required Data Elements 47
Table 4.3 Microcystes: Sample analysis quality control activities and objectives 52
Table 5.1 Benthic Macroinvertebrates Login: Required Data Elements 62
Table 5.2 Benthic Macroinvertebrates Taxonomic Identification: Required Data Elements 68
Table 5.3 Benthic Macroinvertebrates: Measurement Data Quality Objectives 76
Table 5.4 Benthic Macroinvertebrates: Laboratory Quality Control 76
Table 6.1 Whole Body Fish Login: Required Data Elements 84
Table 6.2 Whole Body Fish: Data Elements for Each Fish Specimen 86
Table 6.3 Whole Body Fish: Data Elements from Examination of Each Sample 87
Table 6.4 Whole Body Fish: Initial Aliquot Requirements 90
Table 6.5 Whole Body Fish: Analytical Methods 91
Table 6.6 Whole Body Fish: Lipids and Required Contaminants 91
Table 6.7 Whole Body Fish: Data Elements for Each Sample 94
Table 6.8 Whole Body Fish: Quality Control Activities 96
Table 7.1: Sediment Chemistry, Grain Size, andTOC Login: Required Data Elements 104
Table 7.2 Sediment Chemistry, Grain Size, andTOC: Analytical Methods 105
Table 7.3 Sediment Chemistry, Grain Size, andTOC: Required Parameters 106
Table 7.4 Sediment Chemistry, Grain Size, andTOC: Data Elements for Each Sample 109
Table 7.5 Sediment Chemistry, Grain Size, andTOC: Quality control activities for samples Ill
Table 8.1 Water Chemistry Login: Required Data Elements 120
Table 8.2 Water chemistry: acid preservatives added for various indicators 121
Table 8.3 Water Chemistry and Chlorophyll-^: Laboratory Method Performance Requirements 123
Table 8.4 Water Chemistry and Chlorophyll-^: Analytical Methods Used by Central Laboratory, EPA ORD-Corvallis)
124
Table 8.5 Water Chemistry and Chlorophyll-^: Data Elements for Each Sample 126
Table 8.6 Water Chemistry and Chlorophyll-^: Quality control activities for water quality samples 127
Table 9.1 SedimentToxicity Login: Required Data Elements 138
Table 9.2 Test Conditions for Conducting 10-d Sediment Toxicity Tests for estuarine sediments 140
Table 9.3 Test Conditions for Conducting 10-d Sediment Toxicity Tests for freshwater sediments 141
Table 9.4 SedimentToxicity Replicates: Laboratory method performance data deliverable requirements 142
Table 9.5 Laboratory method performance data deliverable requirements for sedimenttoxicity batch summaries 143
Table 9.6 Quality control activities for sedimenttoxicity samples 144
Table 11.1 Fish Tissue Plugs Login: Required Data Elements 150
Table 11.2 Measurement data quality objectives 151
Table 11.3 Fish Tissue Plugs Quality Control 151
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LIST OF ACRONYMS
A
ASTM
Avg
CAS
Chi-a
CI
CRM
CV
D
DDT
Dl
DO
DOC
DW
ELISA
EPA
ETOH
FOM
g
HDPE
HN03
HRP
H2S04
ISBN
ISO
ITIS
KC
kg
L
LCR
LCS
LIMS
LOM
LRL
mg
mg/kg
mL
MDL
MS
NABS
NARS
NC
ND
NELAC
Absorbance
American Society for Testing and Materials
Average
Chemical Abstracts Service Assigns Unique Identifiers to Chemicals
Chlorophyll-A
Chloride
Certified Reference Material
Curriculum Vitae
Duplicate Sample
Dichloro-Diphenyl-Trichloroethane
De-Ionized Water
Dissolved Oxygen
Dissolved Organic Carbon
Distilled Water
Enzyme-Linked Immunosorbent Assay
Environmental Protection Agency
Ethyl Alcohol
Field Operations Manual
Grams
High Density Polyethylene
Nitric Acid
Antibody-Horseradish Peroxidase
Sulphuric Acid
International Standard Book Number
International Organization for Standardization
Integrated Taxonomic Information System
Kit Control
Kilograms
Liters
Labeled Compound Recovery
Laboratory Control Sample
Laboratory Information Management System
Laboratory Operations Manual
Laboratory Reporting Limit
Milligrams
Milligrams per Kilogram
Milliliters
Method Detection Limit
Matrix Spike
North American Benthological Society
National Aquatic Resource Surveys
Negative Control
Non-Detect
National Environmental Laboratory Accreditation Conference
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ng Nanograms
NH4 Ammonium
NIST National Institute of Standards
N02 Nitrite
N03 Nitrate
ORD EPA's Office of Research and Development
OW EPA's Office of Water
PAH Polycyclic Aromatic Hydrocarbons
PCB Polychlorinated Biphenyl
PDE Percent Difference in Enumeration
ppb Parts per Billion
ppm Parts per Million
ppt Parts per Trillion
P Primary Sample
PSE Percent Sorting Efficiency
PT Performance Testing
PTD Percent Taxonomic Disagreement
QA Quality Assurance
QAPP Quality Assurance Project Plan
QA/QC Quality Assurance/Quality Control
QC Quality Control
QCCS Quality Control Check Sample
QCF Quality Control Failure
QMP Quality Management Plan
RL Reporting Limit
RO Reverse-Osmosis
RPD Relative Percent Difference
RQM Relative Quantitation Method
RSD Relative Standard Deviation
RTH Richest Targeted Habitat
S Standard Deviation
Sb Antimony
SEG Site Evaluation Guidelines
SFS Society of Freshwater Science
Si02 Silica
S04 Sulphate
SOPs Standard Operating Procedures
SPC Sample Processing Control
S-R Sedgewick-Rafter Count
SRM Standard Reference Material
SS Salmon Sperm
TMB Tetramethylbenzidine
TN Total Nitrogen
TOC Total Organic Carbon
TOCOR Task Order Contracting Officer's Representative
TP Total Phosphorus
TRANS Transect
TSN Taxonomic Serial Number
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TSS
Total Suspended Solids
TVS
Total Volatile Solids
Hg
Micrograms
M-g/g
Micrograms per Gram
Hg/L
Micrograms per Liter
U
Unknown or REGULAR
USGS
United States Geological Survey
WSA
Wadeable Streams Assessment
WQX
Water Quality Exchange
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1.0 INTRODUCTION
This manual describes methods for laboratory analyses of the samples to be collected during
the National Coastal Condition Assessment (NCCA). The manual includes quality assurance
objectives, sample handling specifications, and data reporting requirements.
The NCCA is one of a series of water assessments conducted by states, tribes, the U.S.
Environmental Protection Agency (EPA), and other partners. In addition to coastal waters, the
National Aquatic Resource Surveys (NARS) also focuses on rivers and streams, lakes, and
wetlands in a revolving sequence. The purpose of these assessments is to generate statistically
valid reports on the condition of our Nation's water resources and identify key stressors to
these systems.
The goal of NCCA is to address three key questions about the quality of the Nation's coastal
waters:
• What percent of the Nation's coastal waters are in good, fair, and poor condition for key
indicators of water quality, ecological health, and recreation?
• Is the condition of our Nation's coastal waters changing over time?
• What is the relative importance of key stressors such as nutrients and contaminated
sediments?
The NCCA is a probability-based survey of our Nation's coastal and estuarine waters, and
designed to:
• Assess the condition of the Nation's coastal and estuarine waters at national and
regional scales, including the Great Lakes;
• Evaluate changes in condition from previous National Coastal Assessments (NCA)
starting in 2000; and
• Identify the relative importance of selected stressors to coastal and estuarine water
quality;
• Help build state and tribal capacity for monitoring and assessment and promote
collaboration across jurisdictional boundaries.
EPA selected the sampling locations using a probability-based survey design. Sample surveys
have been used in a variety of fields (e.g., monthly labor estimates, forest inventory analysis) to
determine the status of populations or resources of interest using a representative sample of a
relatively few members or sites. Using this survey design allows data from the subset of
sampled sites to be applied to the larger target population, and assessments with known
confidence bounds to be made.
The NCCA field sampling season will be during the index period of June 1st through September
30th. Field crews will collect a variety of measurements and samples from the statistically
selected sampling locations identified by geographical coordinates. The samples are shipped to
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laboratories to evaluate the indicators identified in Table 1.1. The indicators are similar to those
evaluated in previous NCCA.
Table 1.1 NCCA: Indicators
Measure/Indicator
Assessment outcome
Water
Dissolved oxygen
Hypoxia/anoxia
Quality
PH
Temperature
Depth
Conductivity (freshwater)
Salinity (marine)
Water column characterization
Secchi/light measurements
Societal value and ecosystem
PAR
production
Nutrients:
Nutrient enrichment
Dissolved inorganic NO2NO3,
N03, NH3, P04;
Total N and P
Chlorophyll 0
Sediment
Grain size (Silt/Clay content)
Influencing factor for extent and
Quality
severity for contamination
Total Organic Carbon (TOC)
Influencing factor for extent and
severity for contamination
Sediment chemistry
Risk of biological response to
• 16 metals
sediment
• 25 PAHs
contamination
• 20 PCBs
• 14 pesticides
• 6 DDT metabolites
Sediment toxicity (10-day static
Biological response to sediment
bioassay
exposure
with Leptocheirus or Hyalella)
Ecological
Whole body fish contaminants
Environmentally available
Fish Tissue
• 13 metals (no Sb or Mn)
contaminant
Contamination
• 20 PCBs
• 14 pesticides
• 6 DDT metabolites
exposure
Biological
Benthic community structure
Biological response to site conditions
Quality
Human
Algal Toxins
Societal value
Health
Microcystin
Cylindrospermopsin
Enterococci
Mercury in Fish Plugs (Fillet
Tissue)
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2.0 GENERAL LABORATORY GUIDELINES
This chapter describes the general laboratory guidelines with an overview to the quality
assurance / quality control (QA/QC) requirements. Each of the following chapters describes
analytical procedures, and the relevant QA/QC requirements, for a different indicator. In
addition, the Quality Assurance Project Plan (QAPP) provides comprehensive descriptions of the
QA/QC requirements for NCCA 2020.
2.1 Responsibility and Personnel Qualifications
Each laboratory shall train its laboratory personnel in advance in the use of equipment and
procedures used for the standard operating procedure (SOP) for which they are responsible. All
personnel are responsible for complying with all QA/QC requirements that pertain to the
samples to be analyzed. Each laboratory follows its institutional or organizational requirements
for instrument maintenance. Additionally, each laboratory should have documentation of
experience of working with samples collected in estuarine/marine and/or the Great Lakes
environments. Appendix G identifies the specific documentation that each laboratory must
submit to demonstrate its qualifications for performing the analyses.
2.2 Roles and Contact Information
The EPA Headquarters Project Management Team consists of the Project Leader, Alternate
Project Leaders, Project QA Lead, and Laboratory Review Coordinator (Table 2.1). The Team is
responsible for overseeing all aspects of the project and ensuring that the laboratories properly
adhere to the technical and quality assurance requirements. The team is the final authority on
all decisions regarding laboratory analysis.
Table 2.1 NCCA: Contact Information
TITLE*
NAME
CONTACT INFORMATION
EPA HQ NCCA Project Lead
Hugh Sullivan, OW
sullivan.hugh@epa.gov
202-564-1763
EPA HQ NCCA Project QA
Coordinator
Danielle Grunzke, OW
grunzke.danielle@epa.gov
202-566-2876
EPA HQ NCCA Laboratory
Review Coordinator
Kendra Forde, OW
forde.kendra@epa.gov
202-564-0417
EPA HQ NARS Team Leader
Sarah Lehmann, OW
lehmann.sarah@epa.gov
202-566-1379
Information Management
Center Coordinator
Michelle Gover, ORD
gover.michelle@epa.gov
541-754-4793
*For any technical direction, laboratories under contract to EPA must contact the Task Order's
Contracting Officer's Representative (TOCOR) instead of the contacts provided in this table. For any
technical information or sample tracking, the laboratories are permitted to contact these persons.
The NARS Information Management (IM) Coordinator tracks the location of each NCCA sample
that involves post-processing. The coordinator will be the lab's main point of contact regarding
sample tracking and data submission.
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The following personnel will be required for all indicators outlined in the chapters below.
Additional personnel will be required for individual indicators and will be listed within those
chapters.
Laboratory Technician is one who is familiar and qualified to conduct the procedure outlined in
the indicator chapters within this NCCA Laboratory Operations Manual and the NCCA Quality
Assurance Project Plan.
External QC Coordinator is an EPA staff person who is responsible for selecting and managing
the "QC contractor."
QC Contractor is a person who must be dedicated to QA/QC functions and must not be a
primary laboratory or a field sampling contractor for NCCA. This is done to eliminate inherent
bias. 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 results from the laboratories; and preparing brief
summary reports.
2.3 Sample Tracking
Samples are collected by many different field crews during the index period (June 1st through
September 30th). The actual number of sites sampled on a given day will vary widely during this
time. Field crews will submit data via the NCCA app or in rare instances, on electronic forms.
When sample information is submitted on the app, it will immediately update 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 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. Each laboratory will plan with the NARS IM Coordinator,
listed above, to ensure access is granted.
When the samples arrive from the field crews, the shipments will include tracking forms (refer
to the NCCA Field Operations Manual (FOM)). These forms will list the samples included in the
shipment. Laboratory personnel must 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 labs must provide data analysis information to the HQ Project Management Team and the
NARS IM Center by March 1st, 2021 or as stipulated in contractual agreements. These reports
must include the data elements specified for each analytical method in this manual. The
submitted filename must use the following naming convention:
• Indicator name (ex: microcystins)
• Date of files submission to NARS IM Center by year, month, and day (ex: 2020_11_01)
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• Laboratory name (ex: MyLab)
Combined, the file name would look as follows: Microcystins_2020_ll_01_MyLab.xlsx
Before the laboratory submits the batch data to EPA, the analyst who generated the data and
an experienced data reviewer independently check and review the data, as follows:
The analyst shall review the data to ensure that:
• Sample preparation information is correct and complete;
• Analysis information is correct and complete;
• The appropriate method and standard operating procedures were followed;
• Analytical results are correct and complete;
• Appropriate QA codes are reported when necessary;
• Quality control samples were within established control limits;
• Blanks (where appropriate) were within the appropriate QC limits; and
• Documentation is complete.
The data reviewer shall review the data package to verify that:
• Calibration data (where appropriate) are scientifically sound and appropriate;
• QC samples were within established control limits;
• Qualitative and quantitative results are correct; and
• Documentation is complete.
Accompanying its data submission for each batch, the laboratory shall provide a short narrative
that includes the following information:
• Project summary referencing the batch QC identification number, total number of
samples in the batch and their sample numbers, and the analytical methodology used
for analysis;
• Discussion of any protocol deviations that may have occurred during sample testing;
• Discussion of QC questions or issues that were encountered and the corrective
measures taken;
• Definitions of any laboratory QC codes used in the data;
• Summary and discussion of samples that are diluted by the presence of an interference,
non-target analyte, or target analyte; and
• QC samples exceeding established control limits or parameters required by laboratory
internal analytical SOPs and an explanation of why, if known.
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 NCCA 2020 Project Lead. Unless
otherwise authorized by the Project Lead, the laboratory shall retain:
• The sample materials, including vials, for a minimum of three (3) years from the date the
EPA publishes the 2020 NCCA report. During this time, the laboratory shall maintain the
materials at the temperature specified in its laboratory method. The laboratory shall
periodically check the sample materials for degradation. Unless the Project Lead
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arranges for transfer of sample materials to EPA, at the end of the retention period, the
laboratory shall follow its internal protocols for disposal.
• Original records, including laboratory notebooks and raw data files (including logbooks,
bench sheets, and instrument tracings), for a minimum of ten (10) years from the date
that EPA publishes the final report.
The Project Lead is responsible for maintaining the following:
• Deliverables from contractors and cooperators, including raw data, which are
permanent as per EPA Record Schedule 258.
• EPA's project records which under Schedule 501 are permanent.
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3.0 ALGAL TOXIN: CYLINDROSPERMOPSIN IMMUNOASSAY PROCEDURE
This chapter describes an enzyme-linked immunosorbent assay procedure that measures
concentrations of total cylindrospermopsin in water samples. The laboratory uses Abraxis
Cylindrospermopsin Test Kits ("kits") to conduct the analyses.
Frozen cylindrospermopsin samples will be shipped on dry ice from the field crews to the
contract batching laboratory. The contract batching laboratory will send the batched samples to
the analysis laboratory in coolers on dry ice. Samples will arrive in the analytical laboratory
frozen where they can be held in a freezer for several months (up to 90 days) after collection
date.
The procedure is an adaption of the instructions provided by Abraxis for determining total
cylindrospermopsin concentrations using its ELISA kits. For freshwater samples, the procedure
reporting range is 0.1 |ag/L to 2.0 |-ig/L, although, theoretically, the procedure can detect, but
cannot quantify, cylindrospermopsin concentrations as low as 0.05 |ag/L. For samples with
concentrations higher than 2.0 |ag/L of cylindrospermopsin, the procedure includes the
necessary dilution steps. Laboratories must dilute samples with salinities greater than or equal
to 8 parts per thousand (ppt) to less than 8 ppt prior to running the kit. Should a dilution be
made, calibration ranges must be adjusted to fit the data.
3.1 Definitions and Required Personnel Qualifications
This section provides definitions and required resources for using the procedure.
3.1.1 Definitions
The following terms are used throughout the procedure:
Absorbance (A) is a measure of the amount of light that is absorbed in a sample. A standard
statistical curve is used to convert the absorbance value to the concentration value of
cylindrospermopsin.
Brackish and Seawater Samples, are defined for the purposes of the Abraxis
cylindrospermopsin test procedure, are those with salinity greater than 8 parts per thousand
(ppt). (EPA is using different definitions for the water chemistry samples.) EPA recognizes that
brackish water is usually defined as greater than or equal to 0.5 ppt, and seawater as greater
than or equal to 35 ppt. However, Abraxis prescribes that this immunoassay procedure requires
the additional steps described in Section 3.5.2 for any sample with salinity greater than 8 ppt.
The salinity level for each sample is documented on the sample label.
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 0.1 |ag/L to a maximum value of 2.0
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l-ig/L. Please note, NARS IM cannot accept characters within numeric fields. Values outside the
range are handled as follows. If the value is:
• < 0.05 |-ig/L, then the laboratory reports the result as is (without characters) and flags
the sample as a non-detect (i.e. DATA_FLAG=ND).
• Between 0.05 |ag/L and the reporting limit of 0.1 |ag/L (i.e., >0.05 |ag/L and <0.1 |ag/L),
the laboratory should record the value, but assign a Quality Control (QC) code to the
value indicating that the result is between the detection limit and the reporting limit
(i.e., DATA_FLAG=J).
• >2.0 |-ig/L, the result 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.
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 S (standard deviation) as follows:
Equation 3.1 Standard deviation
1/2
5 =
rrl>< - X)
n
i=1
where n is the number of replicate samples, A, is the absorbance measured for the replicate.
Per Section 3.5.4, 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:
Equation 3.2 Percent (%) coefficient of variation
5
%CV =
x 100
A
Dark or Dimly Lit: Away from sunlight, but under incandescent lighting is acceptable.
Duplicate samples (D): are defined as the second aliquot of an individual sample within a well
plate. Each sample including the standards are run in pairs and both results for the primary and
duplicate sample are reported in the result column of the lab deliverable.
Method Detection Limit (MDL) is the minimum concentration at which the analyte can be
detected with confidence (0.05 |-ig/L). In other words, the outcome can be reported with
confidence that it is greater than zero (i.e., present in the sample). The method detection limit
is less than the reporting limit of 0.1 |ag/L, at which the measured value of the analyte can be
reported with confidence. Also see "Sample-Specific Detection Limit" below.
Primary samples (P): are defined as the first aliquot of a sample within a well plate. Each
sample is analyzed in pairs. The results of both this aliquot and the secondary, duplicate aliquot
are reported in the result column of the lab deliverable.
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Relative Standard Deviation (RSD) is the same as the coefficient of variation (%CV). Because
many of the plate reader software programs provide the CV in their outputs, the procedure
presents the quality control requirement in terms of %CV instead of RSD.
Reporting Limit (RL): For undiluted samples, the reporting limit is 0.1 |-ig/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 limit equal
to the method detection limit of 0.05 |ag/L. For diluted samples, the sample-specific detection
limit will be the product of the method detection limit of 0.05 |ag/L and the dilution factor.
Typical values for the dilution factor will be 10 or 100.
3.2 Precautions
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., laboratory coat, protective eyewear, 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.
3.3 Equipment/Materials
The procedures require the following equipment and information:
• Abraxis Cylindrospermospin ELISA (Microtiter) Test Kit, Product # 522011 (see items in
Section 3.5.3).
• Adhesive Sealing Film (Parafilm) for Micro Plates: Used to cover plates during
incubation.
• Data Template - See Figure 3.1
• Distilled or Deionized Water: For diluting samples when necessary.
• ELISA evaluation software.
• 2 glass scintillation vials (20 mL).
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• Multichannel Pipette & Tips: An 8-channel pipette is used for this method. Proficient use
of the multichannel pipette is necessary to achieve reliable results. Practice with water if
you have never used this before.
• 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 (such as Metertech, Model M965 AccuReader): Complete with Metertech
PC Mate software for operation of machine. This machine reads the microtiter plates.
• Project Quality Control Samples.
• Reagent Reservoirs (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 |am filter).
3.4 Sample Receipt
Because USEPA 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 to the NARS IM Team by completing and emailing the sample
tracking spreadsheet with the sample login and sample condition information. (See
Section 2.2 of the manual for contact information).
2. Inspect each sample THE SAME DAY THEY ARE RECEIVED:
o Verify that the sample IDs in the shipment match those recorded on the:
¦ Chain of custody forms when the batching laboratory sends the samples to the
cylindrospermopsin laboratory; or
¦ Sample tracking form if the field crew sends the shipment directly to the state.
o For each sample, record the date received and lab comment (including Condition
Code as described below) in the sample tracking spreadsheet with the appropriate
Site ID/ Sample ID for the NARS IM Team.
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 is not frozen
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o If any sample is damaged or missing, contact the USEPA HQ Laboratory Review
Coordinator to discuss whether the sample can be analyzed. (See contact
information in Section 2.2 of the LOM).
3. Store samples in the freezer until sample preparation begins.
4. Maintain the sample tracking forms with the samples.
Table 3.1 Cylindrospermopsin login: required data elements
FIELD
FORMAT
DESCRIPTION
LAB NAME
Text
Name or abbreviation for QC laboratory
DATE RECEIVED
MMDDYY
Date sample was received by laboratory
SITE ID
text
NCCA site ID as used on sample label
VISIT_NO
numeric
Sequential visits to site (1 or 2)
SAMPLE J D
numeric
Sample ID as used on field sheet (on sample label)
DATE_COL
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
NF
Sample is not frozen
Q
Other quality concerns, not identified
above
COND_COMMENT
text
Comments about the condition of the sample.
3.5 Procedure
The following sections describe the sample, kit preparation and analysis.
3.5.1 Sample Preparation
For each frozen sample (500 mL per sample), the laboratory technician runs it through a freeze-
thaw cycle three times to lyse the cells as follows:
5. All cycles: Keep the samples in dark or dimly lit areas (i.e., away from sunlight, but under
incandescent lighting is acceptable).
6. First freeze-thaw cycle:
o Start with a frozen 500 ml sample.
o 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.
o Shake well to homogenize the sample, then transfer 10 mL to an appropriately
labeled clean 20 mL glass vial.
7. Second freeze-thaw cycle:
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o Freeze the vial.
o Keep the large sample bottle (from the 500 mL initial sample) frozen for future use.
o Thaw the sample vial contents to room temperature.
8. Third freeze-thaw cycle:
o Freeze the vial.
o Thaw the vial contents to room temperature.
o Filter the vial contents through a new, syringe filter (0.45 |am) into a new, labeled 20
mL glass scintillation vial. Norm-ject syringes and Whatman Glass fiber syringe filters
(25mm, GF 0.45 |am filter) or similar alternatives are acceptable. One new syringe
and filter should be used per sample.
3.5.2 Additional Sample Preparation for Samples with Salinity greater than 8 ppt
Seawater samples must also be diluted to a concentration less than or equal to 8 ppt to avoid
matrix effects (the salinity will be marked on sample vials). For all other samples (i.e. with
salinity less than 8 ppt), the technician shall not perform procedures in Section 3.5.1 and goes
directly to kit preparation as described in Section 3.5.3. For samples with salinity greater than 8
ppt the technician should follow the instructions:
1. Check the salinity of the sample, if the salinity of the sample is greater than 8 ppt, the
sample should be diluted to less than 8 ppt and the dilution factor should be denoted in
the data.
2. Adjust the detection ranges for the Sample-Specific Detection Limit by multiplying the
lowest standard (0.05 ppb) and highest standard (2.0 ppb) by the dilution factor.
3.5.3 Kit Preparation
The technician prepares the kits using the following instructions:
Check the expiration date on the kit box and verify that it has not expired. If the kit
has expired, discard and select a kit that is still within its marked shelf life. (Optional:
Instead of discarding the kit clearly mark all expired components as expired and
consider keeping it for training activities.)
3. Verify that each kit contains all the required contents:
• Microtiter plate
• Standards (7) referenced in this procedure as follows with the associated
concentration:
o SO: 0 |ag/L
o SI: 0.05 |ag/L
o S2: 0.1 |-ig/L,
o S3: 0.25 |ag/L
o S4: 0.5 |ag/L
o S5:1.0 |ag/L
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o S6: 2.0 |ag/L
• Kit Control (KC): 0.75 |ag/L
• Sample Diluent (distilled or deionized water)
• Cylindrospermospin-HRP conjugate Solution (vortex before use)
• Antibody solution (rabbit anti-Cylindrospermopsin)
• Wash Solution 5X Concentrate
• Substrate (Color) Solution
• Stop Solution
4. 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.
5. Adjust the microtiter plate, samples, standards, and the reagents to room temperature.
6. 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 store in the
refrigerator (4-8 °C).
7. Prepare a negative control (NC) using distilled or deionized 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 distilled or 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 distilled or deionized water). Mix thoroughly. Set aside
the diluted solution to wash the microtiter wells later.
10. Handle the stop solution containing diluted H2SO4 (sulfuric acid) with care.
3.5.4 Insertion of Contents into Wells
This section describes the steps for placing the different solutions into the 96 wells. Because of
the potential for cross contamination using a shaker table, the following steps specify manual
shaking of the kits instead mechanized shaking.
11. 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.
12. Turn on the computer so that it can control and access the plate reader.
13. Print the template (Figure 3.1) to use as reference when loading the standards, controls,
and samples as described in the next step. Templates contain rows, labeled with a
marking pen, of strips of 8 wells that snap into the blank frame. If the laboratory wishes
to use a different template, provide a copy to the USEPA HQ Laboratory Review
Coordinator for approval prior to first use. (See Section 2.2 of the manual for contact
information.)
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14. Using the 100-|aL pipette, add 50 |aL, each, of the standards, controls, and samples to the
appropriate wells in the plate. Place all seven standards (0.00, 0.05, 0.10, 0.25, 0.50,1.0
and 2.0 |-ig/L), the kit control (0.75 |aL), and negative control, in pairs (duplicate), starting
in the well in the upper left-hand corner of the kit as shown in Figure 3.1. Verify that the
software displays the same template or make any necessary corrections. Laboratories
with access to an autopipetter may use said machinery after proper documentation of
set up, training and calibration has been provided and approved by EPA HQ Laboratory
Review Coordinator prior to first use. (See Section 2.2 of the manual for contact
information).
A
SO
S4
NC
P4
P8
P12
P16
P20
P24
P28
P32
P36
B
SO
S4
NC
D4
D8
D12
D16
D20
D24
D28
D32
D36
C
SI
S5
PI
P5
P9
P13
P17
P21
P25
P29
P33
P37
D
SI
S5
D1
D5
D9
D13
D17
D21
D25
D29
D33
D37
E
S2
S6
P2
P6
P10
P14
P18
P22
P26
P30
P34
P38
F
S2
S6
D2
D6
D10
D14
D18
D22
D26
D30
D34
D38
G
S3
KC
P3
P7
Pll
P15
P19
P23
P27
P31
P35
P39
H
S3
KC
D3
D7
Dll
D15
D19
D23
D27
D31
D35
D39
Figure 3.1 Cylindrospermopsin: sample template
Key: S0-S6 = Standards; KC = Control supplied with Kit (i.e., Kit Control); NC = Negative Control
(Laboratory Reagent Blank); P = Primary run for each unknown sample collected by field crew;
D= "DUPLICATE" run for each matching unknown Primary sample
15. Add 50 |_iL of the conjugate solution to each well using the multi-channel pipettor and a
reagent reservoir. Add 50 |aL of the cylindrospermopsin 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.
16. Place the sealing Parafilm over the wells.
17. 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.
18. Place the plate in an area away from light for 45 minutes.
19. After 45 minutes, carefully remove the Parafilm.
20. Empty the contents of the plate into the sink, pat inverted plate dry on a stack of paper
towels, and then wash the wells of the plate four times with 250 |aL of washing solution
using the multi-channel pipette. After adding the washing solution each time, empty the
solution into the sink and use the paper towels as before.
21. Add 100 |_iL of substrate/ color solution to all wells using the multi-channel pipettor.
22. Cover the wells with Parafilm.
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23. 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.
24. Place the strip holder in an area away from light for 30-45 minutes.
25. After 30-45 minutes, remove the Parafilm, add 100 |aL of stop solution to the wells using
the multi-channel pipette and reagent reservoir in the same sequence as the substrate
solution.
26. Use a 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.
27. Dispose of solution in plates in a laboratory sink. Rinse plates and sink with water to
dilute the weak acid present.
28. Perform QC evaluations of the data as follows:
a. If the following failures occur in the standards and controls, 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-S6 must have decreasing absorbance values. First, calculate the
average values for each standard. That is, if Ai is the absorbance average for Si,
then the absorbance averages must be: Ao> Ai > A2 > A3 > A4 >As>A6
iii. The average absorbance of the standard SO less than 0.8 (i.e., Ao< 0.8).
iv. Two or more negative control samples with detectable concentrations of
Cylindrospermopsin (i.e., values > 0.1 |-ig/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.
v. Results for control samples of outside the acceptable range of 0.75 +/- 0.15 ppb.
That is, results must be between 0.60 and 0.90.
b. If either, or both, of the following failures occur for the sample, 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 3.5.5.
ii. The %CV > 15% between the duplicate absorbance values for a sample.
29. Record the results, even if the data failed the quality control requirements in #18b, for
each well in the USEPA's data template (Table 3.2). The required entries are for the
following columns:
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a. SAM_CODE should be one of the following codes: S0-S6 for standards; KC or NC, for
controls; QC for quality control samples; P for primary run of unknown samples, D
for duplicate/secondary run of unknown samples within a well plate.
b. CONC contains the numeric concentration value. Two special cases:
i. Non-detected concentrations: If the sample is non-detected, provide the result
within CONC column, record the data as 'ND' in the DATA FLAG column and
provide the sample-specific detection limit (0.05 |ag/L for undiluted samples with
salinity less than 8 ppt) in the method detection limit column (MDL). See Section
3.5.5 for calculating the sample-specific detection limit for a diluted sample.
ii. If the result shows that it is "HIGH," 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.1
c. QUALITY FLAGS have codes for the following special cases:
i. ND if the sample was non-detected;
ii. J if the value is detected (<0.05 |-ig/L) but at a level below the reporting limit of
0.1 |ag/L (for undiluted samples);
iii. HI if the concentration value registers as HIGH (exceeds the calibration range).
iv. H if the sample did not meet the holding time and was not analyzed within 90
days of collection
v. QCFif there is a QC failure per step 18 above. The QCF code must be used for all
failures to facilitate data analysis.
vi. Q for any other quality issue (describe in LAB_COMMENT)
d. DILUTION FACTOR is only required if the sample was diluted.
e. AVG_CONC and CV_ABSORB are required for all duplicate runs (use all three values
if the controls are used in triplicate).
Table 3.2: Cylindrospermopsin: required data elements- data submission
FIELD
COLUMN
HEADING
FORMAT
DESCRIPTION
LABORATORY ID
LABJD
Text
Name or abbreviation for QC
laboratory
DATE RECEIVED
DATE_RECEIVED
MMDDYY
Date sample was received by lab
SITE ID
SITE J D
Text
NCCA site ID code as recorded on
sample label or tracking form (blank if
standard or control)
1 EPA compares the cylindrospermopsin data values to 15 |ig/L. which is the magnitude of the EPA criteria for
recreational waterbodies in Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcvstins and Cylindrospermopsin. 2019. EPA 822-R-19-001. Retrieved June 5, 2019.
https://www.epa.gov/sites/production/files/2019-05/documents/lili-rec-criteria-habs-document-2019.pdf
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VISIT NUMBER
SAMPLE ID
VISIT NO
Numeric
SAMPLE ID
Numeric
DATE COLLECTED DATE COL
MMDDYY
CONDITION CODE CONDITION CODE Text
CONDITION
COMMENT
COND COMMENT Text
BATCH
IDENTIFICATION
TECHNICIAN
BATCH ID
Numeric
TECHNICIAN
Text
DATE ANALYZED
DATE ANALYZED MMDDYY
KIT EXPIRE DATE
KIT ID
KIT EXPIRE DATE MMDDYY
KIT ID
Text
R2
R2
Numeric
SAMPLE CODE
SAM CODE
Text
Sequential visits to site (1 or 2) (blank if
standard or control)
6-digit Sample ID number as recorded
on sample jar or tracking form (blank if
standard or control)
Date sample was collected (blank if
standard or control)
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
Sample container
is cracked
Sample or
container is
leaking
ML
NF
Sample label is
missing
Sample is not
frozen
well
Code
KC
NC
SO, SI, S2, S3, S4,
S5, S6
QC
Any comment based on the condition
code flags
Batch identification code; assigned by
lab
Name or initials of technician
performing the procedure
Date when samples are inserted into
the wells
Expiration date on kit box
Kit identification code. If one does not
exist, assign a unique code to each kit.
R2 from curve fit to the average
absorbance values for the standards.
Value is between 0 and 1.
Type of solution being tested in the
Definition
Kit Control
Negative Control
Standard
Quality Control
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LOCATION
LOCATION
PRIMARY OR
DUPLICATE
PRIM_DUP
SALINITY
SALINITY
CONCENTRATION
CONC
UNITS
UNITS
MDL*
MDL
RL
RL
ABSORBANCE
ABSORBANCE
DILUTION
FACTOR
DILUTION_FACTO
R
CV ABSORBANCE
CV_ABSORB
AVERAGE
ABSORBANCE
AVG_ABSORB
AVERAGE
CONCENTRATION
AVG_CONC
QA FLAG (if
appropriate)
QA_FLAG
Text
Text
Numeric
Numeric
Text
Numeric
Numeric
Numeric
Numeric
Numeric
Numeric
Text
Location of well in the kit (e.g., B5
would be the fifth well from the left in
the second row B)
Regular samples are listed as "P" for
Primary/first run or "D" for second run
(see Figure 3.1)
If the sample vial has the salinity
marked on the vial, record the value in
units of parts per thousands.
Otherwise, leave blank.
Concentration or sample-specific
detection limit of contents of well in
Hg/L. Sample-specific detection limit
should be 0.1 ng/L if the sample hasn't
been diluted.
The units of the concentration of the
CONC column
Method detection limit of the machine
in same units as the CONC column
Reporting limit in same units as the
CONC column
Absorbance value
10, 100, etc for number of times the
sample was diluted. If not diluted,
leave blank or record 1
Calculated %CV of duplicate values of
absorbance for all runs. Enter %CV.
Value is between 0 and 100%.
Calculated average of absorbance
values for all samples and standards.
Average value of the original sample
and its duplicate (or replicates for KC
and NC).
Calculated average of concentration
values for a sample. Substitute 0.1 ng/L
for any result recorded as <0.1 ng/L
Data qualifier codes associated with
specific identifications of voucher
samples. These codes provide more
information than 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
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ND
HI
QCF
Q
Concentration
below detection.
Unless the sample
was diluted, the
concentration will
be 0.05 jJLg/L
Sample did not
meet the holding
time and was not
analyzed within 90
days.
Result indicated
that a high
concentration
(i.e., outside
calibration
range)> 2.0 ng/L
Concentration
above detection
but below
reporting limit.
Without dilution,
these values are
between 0.05 and
0.1 M-g/L
QC failure
Other quality
concerns, not
identified above
LABORATORY
COMMENT
LAB COMMENT
Text
Explanation for data flag(s) (if needed)
or other comments.
*ln the event for sample dilution is necessary to overcome the matrix effect, please notify EPA
Laboratory Coordinator
3.5.5 Dilutions (if needed)
Dilutions if needed are prepared as follows (using clean glass tubes):
1. 1:10 dilution
a. Add 900 |aL of distilled or deionized water to a clean 20 mL vial. (Note: Dilutions may
also be made using the kit's diluent rather than distilled or deionized water.)
b. Pipette 100 |aL 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.
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d. Multiply final concentration and Abraxis' method detection limit of 0.05 |ag/L by 10
to obtain the sample-specific detection limit of .5 |-ig/L.
2. 1:100 dilution
a. Add 3.96 mL of distilled or deionized water to a clean, appropriately labeled glass
vial. (Note: Dilutions may also be made using the kit's diluent rather than distilled or
deionized water.)
b. Vortex the sample to mix thoroughly, then pipette 40 |aL from the sample and add to
the water (or diluent) in the appropriate labeled vial. Vortex the sample again.
Multiply the final concentration and Abraxis' method detection limit of 0.05 |ag/L by
100 to obtain the sample-specific detection limit of 50 |-ig/L.
3. Other dilutions can be calculated in the same manner as #1 and #2 if needed.
3.6 Pertinent QA/QC Procedures
This section describes the quality assurance and quality control measures used to ensure that
the data will meet NCCA requirements.
3.6.1 QC Samples
The External QC Coordinator will instruct the QC contractor to provide one or two identical sets
of freshwater QC samples (labeled as performance test (PT) samples) to all participating
laboratories. Each set will contain five samples to test the expected range of concentrations in
the NCCA samples.
For the contract laboratory, the QC contractor will provide the first set to be run with the first
set of samples and a second set to be run at the midpoint of the assigned samples. If available,
a third set will be run with the final batch of samples. Because most state laboratories will have
relatively few samples that can be analyzed using a single kit, the QC contractor will send only
one set to each state laboratory.
Each laboratory will run the QC samples following the same procedures used for the other
samples. The External QC Coordinator will compare the results and assess patterns in the data
(e.g., one laboratory being consistently higher or lower than all others). 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, which may include
no action, flagging the data, or excluding some or all the laboratory's data.
3.6.2 Summary of QA/QC Requirements
Table 3.3 provides a summary of the quality control requirements described in Sections 3.5.3
and 3.5.4. For cylindrospermopsin, the precision for a sample is reported in terms of the
percent coefficient of variation (%CV) of its absorbance values. Relative Standard Deviation
(RSD) is the same as the %CV. Because many of the plate reader software programs provides
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the CV in their outputs, the procedure presents the quality control requirement in terms of %CV
instead of RSD. Accuracy is calculated by comparing the average concentration of the kit
control with the required range (0.75 +/- 0.15).
Table 3.3: Cylindrospermopsin: 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, A0, for SO
must be >0.80; and
o Standards S0-S6 must have decreasing
average absorbance values. That is, if
A, is the average of the absorbance
values for S,, then the absorbance
average values must be: A0> Ai> A2>
A3 > A4 >As>A6
If any requirement fails:
Results from the analytical run are
not reported.
All samples in the analytical run
are reanalyzed until calibration
provides acceptable results. At its
discretion, the laboratory may
consult with USEPA for guidance
on persistent difficulties with
calibration.
Kit Control
The average concentration value of the
duplicates (or triplicate) must be within the
range of 0.75 +/- 0.15 ng/L. That is, results
must be between 0.60 and 0.90.
If either requirement fails:
• Results from the analytical run
are not reported
• The laboratory evaluates its
processes, and if appropriate,
modifies its processes to
correct possible
contamination or other
problems.
• The laboratory 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 < 0.1
Hg/L (i.e., the reporting limit); and
0 One or more concentration results
must be nondetectable (i.e., <0.05
Hg/L)
Sample
Evaluations
All samples are run in duplicate. Each
duplicate pair must have %CV<15% between
its absorbance values.
If %CV of the absorbance for the
sample>15%, then:
• Record the results for both
duplicates using different start
dates and/or start times to
distinguish between the runs.
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All samples are run in duplicate. If both of the
values are less than the upper calibration
range (i.e., 2.0 ng/Lfor undiluted samples),
then the requirement is met.
• Report the data for both
duplicate results using Quality
Control Failure flag "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 (which will have
been flagged with "QCF"). 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%).
Results Within
Calibration
Range
If a result registers as "HIGH", then
record the result with a data flag
of "HI." If one or both duplicates
register as 'HIGH,' then the sample
must be diluted and re-run. No
samples are to be run more than
twice. If samples are re-run, do
not enter concentration
information of the first run.
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.
3.7 Sample and Record Retention
The laboratory shall retain:
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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.
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.
3.8 References
Eurofins (Abraxis), "Cylindrospermopsin ELISA (Microtiter Plate)," Product 522011, Undated.
Retrieved March 12, 2020 from: https://www.eurofins-technologies.com/cylindrospermopsin-
elisa-96-tests.html
Kamp, L. (Abraxis) "Re: Abraxis CYL"; Email to D. Grunzke (EPA). February 14, 2019.
Recommended Human Health Recreational Ambient Water Quality Criteria or Swimming
Advisories for Microcystins and Cylindrospermopsin. 2019. EPA 822-R-19-001. Retrieved June 5,
2019. https://www.epa.gov/sites/production/files/2019-05/documents/hh-rec-criteria-habs-
document-2019.pdf.
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4.0 ALGAL TOXIN: MICROCYSTIN IMMUNOASSAY PROCEDURE
This chapter describes an immunoassay procedure that measures concentrations of total
microcystins in water samples using Abraxis' Microcystins-ADDA ELISATest Kits ("kits"). Each kit
is an enzyme-linked immunosorbent assay (ELISA) for the determination of microcystins and
nodularins in water samples. In some cases, EPA may approve the use of Microcystins-ADDA
SAES ELISA Test Kit2 after discussion and coordination with the NCCA Laboratory Review
Coordinator. This process will not be reviewed in the chapter, however, states may refer to the
attached Appendix A: Microcystins-ADDA SAES ELISA Test Kit Protocol to read the protocol.
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.
4.1 Summary of the Procedure
The procedure is an adaptation of the instructions provided by Abraxis for determining total
microcystins concentrations using its ELISA-ADDA kits.3 For samples with salinity less than 3.5
parts per thousand (ppt), the procedure's reporting range is 0.15 |ag/L to 5.0 |ag/L, although,
theoretically, the procedure can detect, not quantify, microcystins concentrations as low as
0.10 |ag/L (i.e., the method detection limit is 0.10 |ag/L in samples with undiluted salinity of less
than 3.5 ppt). For samples with higher concentrations of microcystins, the procedure includes
the necessary dilution steps. The procedure also provides additional sample preparation steps
for samples with salinities greater than or equal to 3.5 ppt. The results then are adjusted by a
factor of 1.75 for a reporting range of 0.263 |ag/L to 8.75 |ag/L.
4.2 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:
2 Eurofins Technologies, "Microcystins-ADDA SAES ELISA (Microtiter Plate): Product No. 520011SAES" Retrieved on
March 12, 2020 from https://www.eurofins-technologies.com/microcvstins-nodularins-adda-saes-elisa-96-
tests.html
3 Eurofins Technologies, "Microcystins-ADDA ELISA (Microtiter Plate): Product No. 520011." Retrieved on
March 12, 2020 from https://www.eurofins-technologies.com/microcvstins-nodularins-adda-epa-etv-epa-method-
546-elisa-96-tests.html
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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).
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.
4.3 Definitions and Required Resources (Laboratories and Equipment)
This section provides definitions and required resources for using the procedure.
4.3.1 Definitions
The procedure uses the following terms:
Absorbance (A) is a measure of the amount of light absorbed by a sample. A standard statistical
curve is used to convert the absorbance value to the concentration value of microcystins.
Brackish and Seawater Samples, for the purposes of the Abraxis microcystins test procedure,
are defined as samples with salinity greater than or equal to 3.5 parts per thousand (ppt). Labs
must use additional steps described in Section 4.6.2 for any sample with salinity greater than or
equal to 3.5 ppt. The sample labels provide the salinity levels.
Calibration Range is the assay range for which analysis results can be reported with confidence.
For example, assays of undiluted samples with salinities less than 3.5 ppt range from the
reporting limit of 0.15 |ag/L to a maximum value of 5.0 |ag/L. Please note, NARS IM cannot
accommodate character values within numeric fields.
Given the salinity is less than 3.5 ppt, if the result value is:
• Less than 0.10 |ag/L, then the laboratory reports the result as is and flags the sample as
being a non-detect (DATA_FLAG=ND).
• Between 0.10 |ag/L and the reporting limit of 0.15 |ag/L (i.e., >0.10 |ag/L and <0.15 |ag/L),
the laboratory should record the value, but assign a QC code to the value (i.e.,
DATA_FLAG=J).
• 5.0 |ag/L or greater, the laboratory must flag the samples as HI, leave the CONC column
blank, dilute and reanalyze the sample (DATA_FLAG= HI).
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Given the salinity greater than or equal to 3.5 ppt, the technician must follow the seawater
cleanup procedures in Section 4.6.2 and then analyze the sample. The results are then adjusted
by a factor of 1.75 for a reporting range of 0.263 |ag/L to 8.75 |ag/L. If the adjusted result value
is:
• Less than 0.175 |-ig/L, then the laboratory reports the result as is and flag the sample as
a non-detect (DATA_FLAG=ND).
• Between 0.175 |ag/L and the reporting limit of 0.263 |ag/L (i.e., >0.175 |ag/L and <0.263
l-ig/L), the laboratory should record the value, but assign a QC code to the value (i.e.,
DATA_FLAG=J).
• Greater than 8.75 |ag/L, the laboratory must flag the data as "HI" in the Data Flag, leave
the CONC column blank, dilute and reanalyze the sample (DATA_FLAG= HI).
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 the standard deviation, S, as
follows:
1/2
5 = ~z~[ZiAi ~ 1)7
n
i=1
where n is the number of replicate samples, A, is the absorbance measured for the replicate.
Per Section 4.6.4, 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:
5
o/o CV = -= X 100
A
Dark or Dimly Lit: Away from sunlight, but under incandescent lighting is acceptable.
Duplicate samples (D): are defined as the second aliquot of an individual sample within a well
plate. Each sample including the standards are run in pairs and both results for the primary and
duplicate aliquot are reported in the result column of the lab deliverable.
Method Detection Limit: 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 method detection limit is less than the reporting
limit at which the measured value of the analyte can be reported with confidence. Also see
"Sample-Specific Detection Limit."
NARS: National Aquatic Resource Surveys. The National Coastal Condition Assessment (NCCA) is
part of the NARS program.
NARS Information Management System (NARS IM): The IM system established to support all
surveys, including NCCA, in the NARS program. The IM system is used to track the samples from
field collection to the laboratory.
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NCCA: National Coastal Condition Assessment. Freshwater and estuarine/marine samples will
be collected during the field stage of NCCA.
Primary samples (P): are defined as the first aliquot of a sample within a well plate. Each
sample is analyzed in pairs. The results of both this aliquot and the secondary, duplicate aliquot
are reported in the result column of the lab deliverable.
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: A reporting limit is the point at which the measured value of the analyte can
be reported with confidence. For undiluted samples with a salinity <3.5 ppt, the reporting limit
is 0.15 |-ig/L, for undiluted samples with a salinity >3.5 ppt, the reporting limit is 0.263 |ag/L. The
reporting limit for diluted samples is the product of the result multiplied by the dilution factor.
Sample-Specific Detection Limit: Most samples will have a sample-specific detection equal to
the method detection limit (salinity <3.5 ppt = < 0.10 |ag/L or salinity >3.5 ppt = < 0.175 |-ig/L).
For diluted samples, the sample-specific detection limit will be the product of the method
detection limit and the dilution factor. Typical values for the dilution factor will be 10 or 100.
Seawater Sample: See definition for brackish and seawater samples.
Standard Deviation (S) shows variation from the average.
4.4 General Requirements for Laboratories
4.4.1 Expertise
To demonstrate its expertise, the laboratory shall provide EPA with one or more of the
following:
• 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.
4.4.2 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).
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To demonstrate its ongoing commitment, the person in charge of quality issues for the
organization shall sign the NCCA QAPP Certification Page.
4.4.3 Equipment/Materials
The procedures require the following equipment and information:
• Abraxis ADDA Test Kit, Product #520011 (see items in Section 4.6.2)
• Adhesive Sealing Film (Parafilm) for Micro Plates (such as Rainin, non-sterile, Cat.
No. 96-SP-100): Used to cover plates during incubation.
• Data Template - Figure 4.1 Microcystin: sample template.
• Distilled or Deionized Water: For diluting samples when necessary.
• ELISA evaluation software
• 2 Glass scintillation vials (20 mL each)
• Glass vials with Teflon-lined caps of size:
o 20 mL
o 4 mL (for dilutions)
• Multichannel Pipette & Plastic 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 (glass): For dilutions, if needed.
• Timer: For measuring incubation times.
• Vortex Genie: For mixing dilutions.
• Whatman Glass fiber syringe filter (25mm, GF 0.45 |am filter)
Analysis of samples with salinity >3.5 ppt require additional equipment and supplies, as follows:
o Microcystins-ADDA Seawater Sample Clean-Up Kit (Product #529912) which includes
the following supplies:
¦ Disposable 5 %" glass Pasteur pipettes
¦ Disposable 9" glass Pasteur pipettes
¦ Glass wool
¦ Pasteur pipette bulb
¦ Microcystins-ADDA Seawater Sample Treatment Solution
¦ Microcystins-ADDA Seawater Sample Clean-up Resin
o 12x75 mm test tubes
o Scoopula
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o Micropipettes with disposable plastic tips
o Vortex mixer
4.5 Sample Receipt
Field crews hold the microcystins samples on ice 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 will keep the samples frozen upon receipt. Periodically, the
batching laboratory ships samples to the microcystins laboratory. Samples will arrive in the
analytical laboratory frozen where they can be held in a freezer for several months (up to 90
days) after collection date.
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). 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 (see Section 2.2 for contact information).
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 4.1 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 is not frozen
c. If any sample is damaged or missing, contact the EPA HQ Laboratory Review
Coordinator to discuss whether the sample can be analyzed. (See contact
information in Section 2.2).
3. Store samples in the freezer until sample preparation begins.
4. Maintain the chain of custody or sample tracking forms with the samples.
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Table 4.1 Microcystins Login: Required Data Elements
FIELD
FORMAT
DESCRIPTION
LAB NAME
text
Name or abbreviation for QC laboratory
DATE RECEIVED
MMDDYY
Date sample was received by lab
SITE J D
text
NCCA site ID as used on sample label
VISIT_NO
numeric
Sequential visits to site (1 or 2)
SAMPLE ID
numeric
Sample ID as used on field sheet (on sample label)
DATE_COL
MMDDYY
Date sample was collected
CONDITION_CODE
text
Condition codes describing the condition of the sample
upon arrival at the laboratory.
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
NF
Sample is not frozen
Q
Other quality issue to which the above
flags are not applicable.
COND_COMMENT
text
Comments about the condition of the sample Required
for "Q". Optional for others.
4.6 Procedure
The following sections describe the sample and kit preparation and analysis.
4.6.1 Sample Preparation: Freeze-Thaw Steps
For each frozen sample (500 mL per sample), the laboratory technician runs it through a freeze-
thaw cycle three times to lyse the cells as follows:
1. All cycles: Keep the samples in dark or dimly lit areas (i.e., away from sunlight, but under
incandescent lighting is acceptable).
2. First freeze-thaw cycle:
a. Start with a frozen 500 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.
c. Shake well to homogenize the sample, then transfer 10 mL to an appropriately
labeled clean 20 mL glass vial.
3. Second freeze-thaw cycle:
a. Freeze the vial.
b. Keep the large sample bottle (from the 500 mL initial sample) frozen for future
use.
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c. Thaw the sample vial contents to room temperature.
4. Third freeze-thaw cycle:
a. Freeze the vial.
b. Thaw the vial contents to room temperature.
c. Filter the vial contents through a new, syringe filter (0.45 |am) into a new, labeled
20 mL glass scintillation vial. Norm-ject syringes and Whatman Glass fiber syringe
filters (25mm, GF 0.45 |am filter) or another similar alternative are acceptable.
Use one new syringe and filter per sample.
4.6.2 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. 4 For all other samples (i.e. with salinity less than 3.5 ppt), the
technician skips this section (i.e., Section 4.6.2) and goes directly to kit preparation as described
in Section 4.6.3. For samples with salinity greater than 3.5 ppt the technician should follow the
instructions:
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 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.
e. Allow the deionized or distilled water to drain from the column5. 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
4 Reformatted from Eurofins Technologies, "Microcystins in Brackish Water or Seawater Sample Preparation"
Retrieved on March 12, 2020 from https://abraxis.eurofins-technologies.com/media/4684/microcvstin-adda-
estuarv-sample-application-note-520011.pdf Reproduced with permission. Except for Abraxis' solutions labeled as
seawater, EPA has removed references to "brackish" and "seawater" which typically are defined as having different
cut points than 3.5 ppt for salinity.
5 Additional correspondence between EPA and Abraxis notes that this step leaves the resin in the column.
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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 the tube to prevent
aspiration of water back into the column.
f. Place the column into an appropriately labeled 4 mL glass vial.
2. Clean up the sample as follows:
a. Add 1 mL of the sample to a clean, appropriately labeled 4 mL glass vial. Add 50
|aL of Microcystins-ADDA Seawater Sample Treatment Solution. Vortex.
b. Add 375 |aL 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 |aL 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 |aL 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 (4.6.3).
4.6.3 Kit Preparation
The technician prepares the kits using the following instructions:
1. Check the expiration date on the kit box and verify that it has not expired. If the kit has
expired, discard and select a kit that is still within its marked shelf life. (Instead of
discarding the kit, consider clearly labelling it as expired and keeping it for training
activities.)
2. Verify that each kit contains all the required contents:
¦ Microtiter plate
¦ Standards (6) referenced in this procedure as follows with the associated
concentration:
o SO: 0 |ag/L
o SI: 0.15 |ag/L
o S2: 0.40 |ag/L,
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o S3: 1.0 |ag/L
o S4: 2.0 |ag/L
o S5: 5.0 |ag/L
¦ Kit Control (KC): 0.75 |ag/L
¦ Antibody solution
¦ Anti-Sheep-HRP Conjugate
¦ Wash Solution 5X Concentrate
¦ Color Solution
¦ Stop Solution
¦ Diluent
¦ Foil bag with 12 microtiter plate strips
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 place in the foil bag, ziplocked closed, and store in the
refrigerator (4-8° C).
6. Prepare a negative control (NC) using distilled water.
7. The standards, controls, antibody solution, enzyme conjugate, color solution, and stop
solutions are ready to use and do not require any further dilutions.
8. 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.
9. Handle the stop solution containing diluted H2SO4 with care.
4.6.4 Insertion of Contents into Wells
This section describes the steps for placing the different solutions into the 96 wells. Because of
the potential for cross contamination using a shaker table, the following steps specify manual
shaking of the kits instead mechanized shaking.
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.
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2. Turn on the computer so that it can control and access the plate reader.
3. Print the template (Figure 4.1) to use as reference when loading the standards, controls,
and samples as described in the next step. Templates contain rows, labeled with a
marking pen, of strips of 8 wells that snap into the blank frame. (If the laboratory wishes
to use a different template, provide a copy to the EPA HQ Laboratory Review
Coordinator for approval prior to first use. (See Section 2.2 of the manual for contact
information.)
1
2
3
4
5
6
7
8
9
10
11
12
A
SO
S4
PI
P5
P9
P13
P17
P21
P25
P29
P33
P37
B
SO
S4
D1
D5
D9
D13
D17
D21
D25
D29
D33
D37
C
SI
S5
P2
P6
P10
P14
P18
P22
P26
P30
P34
P38
D
SI
S5
D2
D6
D10
D14
D18
D22
D26
D30
D34
D38
E
S2
KC
P3
P7
Pll
P15
P19
P23
P27
P31
P35
P39
F
S2
KC
D3
D7
Dll
D15
D19
D23
D27
D31
D35
D39
G
S3
NC
P4
P8
P12
P16
P20
P24
P28
P32
P36
P40
H
S3
NC
D4
D8
D12
D16
D20
D24
D28
D32
D36
D40
Figure 4.1 Microcystin: sample template
Key: S0-S5 = Standards; KC = Control supplied with Kit (i.e., Kit Control);
NC = Negative Control (Laboratory Reagent Blank); P = Primary aliquot for each unknown
sample collected by field crew; D= "DUPLICATE" aliquot for each matching unknown Primary
sample
4. Using the 100-|aL pipette, add 50 |aL, each, of the standards, controls, and samples to the
appropriate wells in the plate. Place all six standards (0.00, 0.15, 0.40, 1.00, 2.0 and 5.0
|ag/L), the kit control (0.75 |aL), and negative control, in pairs, starting in the well in the
upper left-hand corner of the kit as shown in Figure 4.1. Verify that the software displays
the same template or make any necessary corrections. Laboratories with access to an
auto-pipetter may use said machinery after proper documentation of set up, training and
calibration has been provided and approved by EPA HQ Laboratory Review Coordinator
prior to first use. (See Section 2.2 of the manual for contact information).
5. Add 50 |_iL 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.
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8. Place the plate in a dimly lit area (as defined in Section 4.3.1) for 90 minutes.
9. After 90 minutes, carefully remove the Parafilm.
10. Empty the contents of the plate into the sink, pat inverted plate dry on a stack of paper
towels, and then wash the wells of the plate three times with 250 |aL of washing solution
using the multi-channel pipette. After adding the washing solution each time, empty the
solution into the sink and use the paper towels as before.
11. Add 100 |j.L of enzyme conjugate solution to all wells using the multi-channel pipettor.
12. Cover the wells with Parafilm.
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.
14. Place the strip holder in a dimly lit area for 30 minutes.
15. After 30 minutes, remove the Parafilm, decant, and rinse the wells three times again
with 250 |j.L of washing solution as described in step 10.
16. Add 100 |j.L 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.
17. Cover the wells with Parafilm.
18. 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.
19. Place the plate in a dimly lit area for 20 minutes.
20. After 20 minutes, remove the Parafilm and add 50 |aL 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.
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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, R, of less than 0.99
ii. Standards S0-S5 must have decreasing absorbance values. First, calculate
the average values for each standard. That is, if Aj is the absorbance
average for Si, then the absorbance averages must be:
Ao > Ai > A2 > A3 > A4 >As
iii. The average absorbance of the standard SO less than 0.8 (i.e., Ao< 0.8).
iv. Two or more negative control sample results report detectable
concentrations of microcystins (i.e., values > 0.1 |-ig/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.
v. Results for control samples of outside the acceptable range of 0.75 +/-
0.185 |-ig/L. That is, results must be between 0.565 |ag/L and 0.935 |-ig/L.
b. If either, or both, of the following situations 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 4.6.5.
ii. The %CV > 15% between the duplicate absorbance values for a sample.
24. If the sample has a salinity of 3.5 ppt or greater, then convert the results by multiplying
by 1.75. If the assay was non-detected, then the sample-specific detection limit is 0.175
l-ig/L. The reporting limit is 0.263 |-ig/L. The calibration range is 0.263 |ag/L to 8.75 |-ig/L.
25. Record the results, even if the data failed the quality control requirements in #23b, for
each well in EPA's data template (see Table 4.2 for required elements). The required
entries are for the following columns:
a. TYPE indicates the sample type using one of the following codes: S0-S5 for
standards; KC or NC for controls; and or "D" for unknown sample.
b. CONC contains the numeric concentration value. Two special cases:
i. Non-detected concentrations: If the sample is non-detected, provide the
result within CONC column, record the data as 'ND' in the DATA FLAG
column and provide the sample-specific detection limit (0.1 |ag/L it the
sample is undiluted with salinity less than 3.5 ppt) in the method
detection limit column (MDL). See step 24 for reporting values for
samples with salinity greater than or equal to 3.5 ppt. See Section 4.6.5
for calculating the sample-specific detection limit for a diluted sample.
ii. If the result shows that it is "HIGH," this indicates that the sample value is
outside of the calibration range and must be diluted and re-run using
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another analytical run. Leave the CONC column blank and record 'HI' in
the DATA FLAG column.6
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
|ag/L (for undiluted samples with salinity less than 3.5 ppt; see step 24 for
samples with salinity greater than or equal to 3.5 ppt);
iii. HI if the concentration value registers as HIGH (exceeds the calibration
range).
iv. QCF if there is a QC failure per step 23 above. The QCF code must be used
for all failures to facilitate data analysis.
v. H if the sample did not meet the holding time and was not analyzed
within 90 days of collection.
vi. Q for any other quality issue (describe in COMMENTS)
d. DILUTION FACTOR is only required if the sample was diluted.
e. DUPAVG and DUPCV are required for duplicate samples and control samples
(use all three values if the controls are used in triplicate).
Table 4.2 Microcystins: Required Data Elements
STAGE FIELD FORMAT DESCRIPTION
LOGIN
LAB ID
Character
Name or abbreviation for QC laboratory
DATE RECEIVED
MMDDYY
Date sample was received by lab
SITE ID
Character
NCCA site ID code as recorded on
sample label or tracking form (blank if
standard or control)
VISIT_NO
Numeric
sequential visits to site (1 or 2) (blank if
standard or control)
SAMPLEJD
Numeric
6-digit Sample ID number as recorded
on sample jar or tracking form (blank if
standard or control)
DATE COL
MMDDYY
Date sample was collected (blank if
standard or control)
CONDITION_CODE
Character
Sample condition upon arrival at the
laboratory (blank if standard or control)
Flag
Definition
Blank or N
Not a sample
(blank, standard,
or control)
6 EPA compares the microcystin data values to 8 |ig/L. which is the magnitude of the EPA criteria for recreational
waterbodies in Recommended Human Health Recreational Ambient Water Quality Criteria or Swimming Advisories
for Microcystins and Cylindrospermopsin. 2019. EPA 822-R-19-001. Retrieved June 5, 2019.
https://www.epa.gov/sites/production/files/2019-05/documents/lili-rec-criteria-habs-document-2019.pdf
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OK
ANALYSIS
Sample is in good
condition
Sample container
is cracked
Sample or
container is
leaking
ML
NF
Sample label is
missing
Sample is not
frozen
COND_ COMMENT
Character
Comments about the condition of the
sample. If the condition code='W' then
provide the temperature
BATCHJD
Numeric
Batch identification code, assigned by
lab
TECHNICIAN
Character
Name or initials of technician
performing the procedure
DATE_ANALYZED
MMDDYY
Date when samples are inserted into
the wells per Section 4.6.4
KIT_EXPIRE_DATE
MMDDYY
Expiration date on kit box
KITJD
Character
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.
SAM_CODE
Character
Type of solution being tested in the well
Flag Definition
KC Kit control
NC Negative control
SO, SI, S2, S3, Standard
S4,S5
QC Quality control
sample
U Sample of
unknown
concentration
LOCATION
Character
Location of well in the kit (e.g., B5
would be the fifth well from the left in
the second row B)
PRIM_DUP
Text
Regular samples are listed as "P" for
Primary/first aliquot or "D" for second
aliquot (see Figure 4.1)
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.
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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
for a sample with salinity <3.5 ppt which
hasn't been diluted.
(Sample-specific detection limit is 0.175
Hg/L for samples with
salinity >3.5 ppt using the ADDA ELISA
test kit).
UNITS
Text
The units of the concentration of the
CONC column.
MDL
Numeric
Minimum detection limit in same units
as the CONC column
RL
Numeric
Reporting Limit in same unit as the
CONC column
ABSORBANCE
Numeric
Absorbance value
DILUTION FACTO
Numeric
10, 100, etc for number of times the
R
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 forTYPE=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 for
TYPE=U, KC, NC, or S. 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 for any
value below the reporting limit.
OA FLAG
Character
Data qualifier codes associated with
(if appropriate)
specific identifications of voucher
samples. These codes provide more
information than 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
H Sample did not
| meet the holding
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LAB COMMENT
Character
HI
QCF
Q
time and was not
analyzed within 90
days.
Result indicated a
high concentration
(i.e., outside
calibration range)
Concentration
above detection
but below
reporting limit
QC failure
Other quality
concerns not
identified above
Explanation for data flag(s) (if needed)
or other comments.
4.6.5 Dilutions (if needed)
Dilutions if needed are prepared as follows (using clean glass tubes):
1. 1:10 dilution
a. Add 900 |aL 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 |aL from the sample into the vial. (To provide more accurate dilutions
and less chance of contaminating the diluent, add the diluent to the vial before
the sample.)
c. Mix by vortexing.
d. Multiply final concentration and Abraxis' method detection limit by 10 to obtain
the sample-specific detection limit. For example, for a sample with salinity less
than 3.5 ppt, Abraxis' detection limit is 0.1 |ag/L and the sample-specific
detection would be 1.0 |ag/L for a 1:10 dilution.
2. 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 |aL from the sample and
add to the water (or diluent) in the appropriate labeled vial. Vortex.
c. Multiply the final concentration and Abraxis' method detection limit by 100 to
obtain the sample-specific detection limit. For example, for a sample with salinity
less than 3.5 ppt, Abraxis' method detection limit is 0.1 |ag/L and the sample-
specific detection limit would be 10 |ag/L for a 1:100 dilution.
3. Other dilutions can be calculated in the same manner as #1 and #2 if needed.
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4.7 Quality Measures
This section describes the quality assurance and quality control measures used to ensure that
the data will meet NCCA's requirements.
4.7.1 Assistance Visits
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 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. EPA will develop, review and approve the checklist prior to conducting an
assistance visit.
4.7.2 QC Samples
The External QC Coordinator will instruct the QC contractor to provide one or two identical sets
of freshwater and/or seawater performance test samples to all participating laboratories. If the
laboratory will assay both freshwater and seawater samples, then it will receive both sets (i.e.,
freshwater and seawater). Each set will contain five samples to test the expected range of
concentrations in the NCCA samples.
For the contract laboratory, the QC contractor will provide the first set to be run with the first
set of samples and a second set to be run at the midpoint of the assigned samples. If available,
a third set will be run with the final batch of samples. Because most state laboratories will have
relatively few samples that can be analyzed using a single kit, the QC contractor will send only
one set to each state laboratory.
Each laboratory will run the QC samples following the same procedures used for the other
samples. The External QC Coordinator will compare the results and assess patterns in the data
(e.g., one laboratory being consistently higher or lower than all others). 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.
4.7.3 Summary of QA/QC Requirements
Table 4.3 provides a summary of the quality control requirements described in Sections 4.5 and
4.6.
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Table 4.3 Microcystins: Sample analysis quality control activities and objectives
QUALITY DESCRIPTION AND REQUIREMENTS
CORRECTIVE ACTION
CONTROL
ACTIVITY
Kit - Shelf Life
Is within its expiration date listed on kit box.
If kit has expired, then discard or
Clearly label as expired and 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:
Standard curve must have a correlation
coefficient of >0.99; Average absorbance
value, Ao, for SO must be >0.80; and Standards
S0-S5 must have decreasing average
absorbance values. That is, if A, is the average
of the absorbance values for Si, then the
absorbance average values must be: A0> 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
calibration provides acceptable
results. At its discretion, the lab
may consult with EPA for guidance
on persistent difficulties with
calibration.
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, the
average must be between 0.565 ng/L and
0.935 ng/L.
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:
All concentration values must be < 0.15 ng/L
(i.e., the reporting limit; and one or more
concentration results must be nondetectable
(i.e., <0.10 ng/L)
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 using different start
dates and/or start times to
distinguish between the runs.
Report the data for both duplicate
results using Quality Control
Failure flag "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 (which will have
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been flagged with "QCF"). 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%).
If a result registers as "HIGH", then
record the result with a data flag
of "HI." If one or both duplicates
register as 'HIGH,' then the sample
must be diluted and re-run. No
samples are to be run more than
twice. If samples are re-run, do
not enter concentration
information of the first run.
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.
4.8 Sample and Record Retention
The laboratory shall retain:
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.
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.
Results 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/L for undiluted samples
with salinity<3.5 ppt; < 8.75 ng/Lfor undiluted
samples with salinity >3.5 ppt), then the
requirement is met.
External Quality
Control Sample
External QC Coordinator, supported by QC
contractor, provides 1-2 sets of identical
samples to all laboratories and compares
results.
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4.9 References
Eurofins Technologies, "Microcystins-ADDA SAES ELISA (Microtiter Plate): Product No.
520011SAES" Retrieved on March 12, 2020 from https://www.eurofins-
technologies.com/microcystins-nodularins-adda-saes-elisa-96-tests.html
Eurofins Technologies, "Microcystins-ADDA ELISA (Microtiter Plate): Product No. 520011."
Retrieved on March 12, 2020 from https://www.eurofins-technologies.com/microcystins-
nodularins-adda-epa-etv-epa-method-546-elisa-96-tests.html
Eurofins Technologies, "Microcystins in Brackish Water or Seawater Sample Preparation"
Undated. Retrieved on March 12, 2020 from https://abraxis.eurofins-
tech nologies.com/med ia/4684/microcystin-adda-estua ry-sample-a pplication-note-520011. pdf.
Loftin, K.A., et al., "Comparison of Two Cell Lysis Procedures for Recovery of Microcystins in
Water Samples from Silver Lake in Dover, Delaware, with Microcystin Producing Cyanobacterial
Accumulations," in USGS Open-File Report 2008 -1341. 2008. Retrieved April 2013 from
http://pubs.usgs.gov/of/2008/1341/pdf/of20Q8 1341.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
Kamp, L. (Eurofins Technologies- formerly Abraxis) "Re: question about instructions for
brackish water or seawater"; Email to M. Smith (EPA). June 23, 2015.
Recommended Human Health Recreational Ambient Water Quality Criteria or Swimming
Advisories for Microcystins and Cylindrospermopsin. 2019. EPA 822-R-19-001. Retrieved June 5,
2019.
https://www.epa.gov/sites/production/files/2019-05/documents/hh-rec-criteria-habs-
document-2019.pdf
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5.0 BENTHIC MACRO IN VE RTE B RATES
This chapter describes the steps for identifying benthic macroinvertebrate organisms in
samples collected in estuarine coastal waters and the nearshore Great Lakes during the 2020
NCCA. Field crews preserve samples in the field with formalin and ship them to a central
holding facility or directly to the laboratory. This procedure requires the laboratory to fully sort
and identify all organisms in the sample. Subsampling procedures are not authorized for any
samples collected for the NCCA program.
5.1 Summary of Method
The procedure describes the steps for picking and identifying organisms from sediment
samples. This section provides a summary of the procedure and quality control measures.
The sorter evenly distributes each sample across a tray(s) and then picks all organisms from the
sample. During the identification step, a taxonomist identifies all organisms to the target
taxonomic levels for the survey and discards materials that do not meet the identification
criteria. For each species or lowest practical taxonomic level, the taxonomist includes at least
one representative organism in the laboratory's reference collection for NCCA 2020.
As part of the quality control measures, a second taxonomist will re-identify a subset (usually
10%) of the samples to quantify enumeration and taxonomic precision, or consistency, as
percent difference in enumeration (PDE) and percent taxonomic disagreement (PTD), to help
target corrective actions, and ultimately to help minimize problems during data analysis.
5.2 Health and Safety Warnings
In addition to the laboratory's requirements, persons using this procedure must abide by the
following health and safety procedures:
1. Wear proper personal protection clothing and equipment (e.g. lab coat, protective
eyewear / goggles).
2. When working with potential hazardous chemicals (e.g. Rose Bengal) or biological
agents (benthic organisms and sediments), avoid inhalation (e.g., use a fume hood or
other appropriate ventilation when necessary), skin contact, eye contact, or ingestion. If
skin contact occurs, remove clothing immediately and wash / rinse thoroughly. Wash
the affected skin areas thoroughly with large amounts of soap and water.
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5.3 Definitions and Required Resources (Laboratory, Personnel, and Equipment)
This section provides definitions, required experience and resources necessary to follow the
procedure for preparing, sorting, and identifying benthic macroinvertebrate organisms in
samples.
5.3.1 Definitions
The procedure uses the following throughout the document:
AphialD: A stable and globally unique identifier that the World Register of Marine Species
(WoRMS) couples with each scientific name to serve as the "common denominator" for
accessing information. AphialDs are preferred for marine samples, but Taxonomic Serial
Number (TSN) may be used for low salinity species lacking AphialDs. When either of these are
unavailable, secondary sources are acceptable.
Dissecting microscope: Microscope configured to allow low magnification of three-dimensional
objects that are larger or thicker than the compound microscope can accommodate.
Distinct taxa: Data analysts use the number of distinct (i.e., unique) taxa within a given sample
to evaluate the richness associated with the sample location. The distinctness attribute is
assessed sample by sample, and not across all samples. To facilitate the data analyses, the
database includes an additional variable ("flag") that is used for the first identification of a
particular taxon in a sample. Section 5.6 provides the steps used to identify which taxa are
flagged.
Elutriate: Circulate water over the sample in order to wash away the lighter or finer particles of
the detritus.
Good quality digital photograph: Good quality means that other taxonomists can readily
identify the taxon from one or multiple photographs and the library can readily locate the
photographs. To ensure that the photographs meet these objectives, the image must be:
• Taken through the microscope at a high enough resolution so that the key diagnostic
features are distinguishable and clear. Include all features that would be necessary for
an experienced taxonomist to identify the specimen, this may require multiple
photographs and at different magnifications.
• Positioned so that it includes:
o Only one taxon in the photo. If necessary, the laboratory may edit (e.g.,
crop) the digital photograph and save the file with a new filename as
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specified below. Both the original and edited files must be included in the
digital library.
o A scale bar or measurements in an appropriate location to indicate the
size of the specimen,
o One specimen that lies flat on the surface instead of tilted (to the extent
practicable).
• Saved using a format that preserves the image in the highest resolution possible.
• Saved with a filename that is consistent within the digital library and shall include
the following elements in the order listed below:
o NCCA2020
o Laboratory name (or abbreviation)
o Sample number
o Taxa name
o Magnification (if applicable, otherwise indicate no magnification as "lx")
o Date (format YYYYMMDD) that the photograph was taken,
o Appendage of "e" if the photograph was edited (e.g., cropped).
For example, on September 8, 2020, laboratory ABC identified the specimen in sample 1234 to
be a Capitella capitata and took a digital photograph at a resolution of 40x and then cropped
the photograph to eliminate extraneous material. The filenames of the original and edited
photographs would be: NCCA2020_ABC_1234_ capitella capitata_40x_20200908.gif and
NCCA2020_ABC_1234_ capitella capitata_40x_20200908e.gif.
Inorganic material: Material that is not capable of further decay (e.g., gravel, sand, silt)
Integrated Taxonomic Information System (ITIS): Database with standardized, reliable
information on species nomenclature and their hierarchical taxonomic classification used for
Great Lakes taxa and low salinity estuarine taxa.
NARS: National Aquatic Resource Surveys. The National Coastal Condition Assessment (NCCA) is
part of the NARS program.
NARS Information Management (IM) System: The IM system established to support all
surveys, including NCCA, in the NARS program. The NARS IM system is used to track the
samples from field collection to the laboratory.
NCCA: National Coastal Condition Assessment. The samples are collected during the field stage
of NCCA.
Organic material: Material derived from living organisms that is capable of further decay (e.g.,
leaves, sticks, algae).
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Percent sorting efficiency (PSE): Number of organisms recovered by sorter (A) compared to
the combined (total) number of recoveries by the sorter (A) and independent sorter (B) for a
sample (sorter B sorts through pickate and counts only organisms missed by Sorter A).
A
PSE =
A + B
¦ x 100
(1)
Percent disagreement in enumeration (PDE): measure of taxonomic precision comparing the
number of organisms, ni, counted in a sample by the primary taxonomist with the number of
organisms, r)2, counted by the internal or external QC taxonomist.
PDE =
-xlOO
(2)
Percent taxonomic disagreement (PTD): measure of taxonomic precision comparing the
number of agreements (positive comparisons, compp0s) of the primary taxonomist and internal
or external QC taxonomists. In the following equation, N is the total number of organisms in the
larger of the two counts.
comPpos
PTD =
1 —
N
xlOO
(3)
Pickate: This is the remaining material left from the tray after the sorter has removed all
benthic macroinvertebrates. This could include small stones, sticks or leaves, etc.
Primary laboratory: The laboratory that 1) sorts the sample; and 2) provides the first
identification of benthic macroinvertebrates in the sample.
Secondary laboratory: The laboratory selected by the External QC Coordinator. It provides an
independent identification of the benthic macroinvertebrates in the sample. The secondary
laboratory must provide QC taxonomists who did not participate in the original identifications
for the sample.
Target taxonomic levels: Target taxonomic levels for the NCCA is typically species (lowest
practical level). NCCA excludes meiofauna (due to being smaller than 0.5 mm) from
identifications. Additional exceptions include Oligochaeta (Class) and Chironomidae (Family) in
samples from marine, polyhaline and mesohaline regions ONLY.
Taxonomic Bench Sheet: Form used by the laboratory to record information about the sample
during the identification procedure.
Taxonomic Serial Number (TSN): stable and unique identifier that the IT IS, Encyclopedia of
Life, and/or Catalogue of Life couples with each scientific name to serve as the "common
denominator" for accessing information. ITIS numbers are preferred for Great Lakes taxa and
low salinity estuarine taxa without AphialDs, but when they are not available, secondary
sources are acceptable.
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World Register of Marine Species (WoRMS): a database with standardized and reliable
information on species nomenclature and their hierarchical taxonomic information used for
estuarine taxa.
5.3.2 Laboratory
The procedure may be used by any laboratory that demonstrates competency in analytical
work and quality procedures as documented by any one or more of the following:
1. Analytical work: To demonstrate its expertise, the laboratory shall provide EPA with one
or more of the following:
a. Memorandum that identifies the relevant services that the laboratory provided
for the National Aquatic Resource Surveys in the past five years.
b. Memorandum describing experience with analyses that are the same or similar
to the requirements of this method.
c. Dated copy of relevant Accreditation or Certification (NELAC, ISO, state, etc.) for
the laboratory and/or its experts who will perform and/or oversee the analyses.
The accreditation must be for the entirety of analysis that the laboratory will be
performing.
d. Memorandum that describes the laboratory's participation in round robin
studies and/or performance studies.
e. Report of findings from an on-site technical assessment or audit.
2. Quality procedures.
a. To demonstrate its expertise in quality assurance and quality control procedures,
the laboratory shall provide EPA with copies of the quality-related documents
relevant to the procedure. Examples include QMPs, QAPPs, and applicable
Standard Operating Procedures (SOPs).
b. To demonstrate its ongoing commitment, the person in charge of quality issues
for the laboratory shall sign the NCCA 2020 QAPP Certification Page.
3. Reporting standardized data. To demonstrate its expertise, the laboratory shall provide
EPA with a memorandum that confirms that the laboratory has a computerized
Laboratory Information Management System (LIMS) routinely used to track samples and
record laboratory results. The memorandum also shall confirm that the laboratory will
use LIMS to record and report results from the procedure.
5.3.3 Personnel
The procedure may be used by any person who has received training in processing and
identification of benthic macroinvertebrates. For purposes of this procedure, EPA assumes that
the following personnel are responsible for performing specific duties:
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Internal Taxonomy QC Officer provides oversight of daily operations, sample processing,
monitors QC activities at the laboratory to determine conformance, and conducts performance
and systems audits of the procedures. The laboratory must retain, and make available to EPA
upon request, documentation of the qualifications for the Internal Taxonomy QC Officer. The
Internal Taxonomy QC Officer is an experienced taxonomist who meets the following
requirements:
1. Demonstrated an initial enumeration and identification proficiency (as measured by
PDE<5% and PTD<15%).
2. Maintains enumeration and identification proficiency in periodic QC checks (i.e., 1 in 10
samples with a minimum of one sample checked).
External QC Taxonomists are selected by the External QC Coordinator (after consultation with
EPA experts) and have demonstrated expertise and experience to be used as a quasi "gold
standard" for taxonomic evaluations.
Taxonomists are trained, and have considerable experience, in identifying benthic
macroinvertebrates. It is also important that the taxonomist maintains contact with other
taxonomists through professional societies and other interactions, and keeps up with the
pertinent literature, since systematics and species identifications change over time.
Taxonomists identifying marine taxa should have experience with marine and estuarine fauna,
while those identifying Great Lakes fauna should be familiar with those fauna. EPA prefers, but
does not require, that the freshwater taxonomists are certified by the Society of Freshwater
Science (SFS). Each laboratory must submit the resume or CVfor the taxonomists who identify
benthic macroinvertebrates for the NCCA samples to the EPA Project QC Officer.
Sorters are laboratory technicians who have basic training in laboratory procedures. An
"experienced" sorter is one that has achieved greater than or equal to 90% sorting efficiency in
five consecutive samples.
5.3.4 Equipment/Materials
The procedure requires the following equipment and materials for sample preparation, sorting,
and taxonomic identifications.
5.3.4.1 Sample Preparation and Sorting Equipment/Materials
• U.S. 35 sieve (500 |am)
• Round buckets
• Standardized, possibly, gridded screen (40 Mesh (380-|am openings, T304 stainless steel
wire, 34GA (0.010"))
• 6-cm scoop
• White plastic or enamel pan (6" x 9") for sorting
• Teaspoon
• Permanent ink pen (e.g Pigma Micron® pen)
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• Dropper
• Fine-tipped forceps (watchmaker type, straight and curved)
• Vials with caps or stoppers
• Sample labels for vials
• 70-80% ethanol
• Stereo zoom microscope (6-10X magnification)
5.3.4.2 Taxonomy Identification Equipment/Materials
• Stereo dissecting microscope with fiber optics light source (50-60X magnification)
• Compound microscope (10, 40, and 100X objectives, with phase-contrast capability)
• Digital camera with high resolution capability mounted on a microscope
• Petri dishes
• Microscope slides (1" x 3" flat, precleaned)
• Cover slips (appropriately sized)
• CMCP-10 (or other appropriate mounting medium)
• Permanent ink pen (e.g., Pigma Micron® pen)
• Dropper
• Fine-tipped forceps (watchmaker type, straight and curved)
• Vials with caps or stoppers
• Sample labels for vials
• 70 - 80% non-denatured ethanol in plastic wash bottle
• Taxonomic Bench Sheet (Attachment 4.1 provides an example)
• Hand tally counter
5.4 Sample Receipt
Because EPA initiates tracking procedures designed to recover any missing shipment, the
laboratory personnel should start the following login steps within 24 clock hours of receiving a
delivery.
1. Record receipt of samples in the NARS IM system (within 24 clock hours) and the
laboratory's Information Management System (LIMS). Assign the appropriate
chronological bench number to each sample. 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 (see Section 2.2 for contact information).
2. Inspect each jar THE SAME DAY THEY ARE RECEIVED:
a. Add 70-80% EtOH to the jar, if necessary (i.e., to cover the contents completely).
b. Verify that the site identification and sample number on the label also appear on the
chain of custody form in the shipment.
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c. Notify the EPA HQ Laboratory Review Coordinator (see contact information in
Section 2.2) if any jars were broken and/or there are discrepancies between the
custody form and jars.
3. Store the sample containers at room temperature until sorting begins. If the sample will
be stored for a long time before sorting, replace the formalin with ethanol for better
preservation of the organisms.
4. Maintain the chain-of-custody form with the samples; it will be needed if the samples
are transported to any other location (e.g., for taxonomic identification, external QC
evaluation).
5. Verify that the login information includes the required data elements in Table 5.1. After
completing all required elements, provide the information to the data entry personnel.
Table 5.1 Benthic Macroinvertebrates Login: Required Data Elements
FIELD
FORMAT
DESCRIPTION
LAB_NAME
Character
Name of lab
LABJD (optional)
Character
Lab sample ID
DATE_RECEIVED
MMDDYY
Date sample was received by lab
SITEJD
Character
NCCA site identification code as used on sample label
VISIT_NO
Numeric
Sequential visits to site (1 or 2, if specified on label)
SAMPLE J D
Numeric
Sample number as used on field sheet (on sample label)
DATE_COL
Date
Date sample was taken
SALINITY
Numeric
Salinity: Value is provided on the sample label
CONDITION_CODE
Character
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
NP
Not enough preservative used
Q
Other quality concerns, not identified above
(explain in COND_COMMENT)
COND_COMMENT
Character
Explanation for Q FLAG (if needed)
5.5 Sample Preparation and Picking Organisms
This section describes the steps for the sorter in preparing the sample and picking organisms.
1. Remove the lid from the sample container and remove the internal sample label.
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2. Carefully decant the formalin from the sample container by pouring the fluid through a sieve
(U.S. 35) into a separate container. Inspect the mesh of the sieve for any organisms and
return any organisms found to the sample container so they can be included in the sample
sort process.
3. Remove sieved organisms from the sample container and place into a sorting tray.
4. Sort all samples under a minimum of 6x (maximum of lOx) dissecting microscope. Remove
the macroinvertebrates from the detritus with forceps. In general, do not remove (i.e., do
not include in counts):
o Empty snail or bivalve shells
o Organisms of water surface-dwelling or strict water column2 arthropod
taxa,
o Meiofauna,
o Incidentally-collected terrestrial taxa,
o Fragments such as legs, antennae, gills, wings, or tails,
¦ For Oligochaeta, attempt to remove only whole organisms or
fragments that include the head. In other words, do not remove
fragments without the head.
In case of uncertainties, place the organism in the sort vial for the taxonomist to make the
final determination.
5. Place picked organisms of the same type into a single set of jars and vials containing 70-80%
ethanol.
6. This QC step is performed if: 1) the sorter (sorter A) has not reached 90% proficiency in 5
consecutive samples (referred to as the "proficiency QC check" below); or 2) this sample is
the 1 in 10 sample QC check for experienced sorters (referred to as the "periodic QC check"
below). For this step, a second sorter (sorter B):
o Performs QC checks using the same power microscope as the sorter;
o Extracts any missed organisms found in the pickate from Sorter A and
places them into the sample vial, or other suitable sample vial;
o Notes the number of organisms missed; and
o Adds that number to the final count of the sample,
o Calculates the PSE for the sample (see Section 5.3.1 for definition;
equation 1). If the PSE is:
¦ <90% and the sample is the:
• Proficiency QC check, a second sorter must check the next
5 samples until the original sorter has PSE>90% for 5
consecutive samples.
2Strict water column taxa are those that do not have at least one life stage that is benthic (i.e.,
bottom-dwelling).
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• Periodic QC check, then a second sorter examines the
original sorter's samples since the last QC check for missed
organisms. The original sorter must again demonstrate
proficiency by achieving a PSE>90% in 5 consecutive
samples.
¦ >90% and the sample is the:
• Proficiency QC check, the sample counts towards the 1 in 5
consecutive samples used to establish proficiency.
• Periodic QC check, no corrective action is required.
o Records the results from the QC step. The laboratory must record the
results from all QC steps, even if they exceed the frequency required by
this step. The laboratory must provide the sorter QC results to EPA upon
request.
7. Remove the remaining material left on the sorting pan (i.e. material such as sticks and
organic debris) and place it in a separate container with preservative (70-80% ethanol).
Label the container "Pickate," on both internal and external labels.
8. Label the vials and jars of sorted organisms and material using permanent ink (e.g., using a
Pigma Micron® pen). Internal sample labels should be made of cotton rag paper or an
acceptable substitute.
9. Retain the vials and materials for the time period specified in Section 5.8.
10. Thoroughly clean all sample preparation and sorting equipment and make sure all
equipment is free of organisms prior to sorting the next sample.
5.6 Taxonomic Identification
The taxonomist performs the following steps in identifying the benthic macroinvertebrate
organisms:
1. Upon receipt of a set of sample vials from the sorter:
a. Compare all site identification number and sample identification numbers on the
form with those entered on the labels of samples and resolve any discrepancies
with the sorter.
b. Determine if any vials are broken. For any broken vial, attempt to recover as
much of the sample as possible. Describe the damage in the LAB_COMMENT
field in the database.
c. Maintain the chain-of-custody form with the sample vials; it will be needed to
return/store them.
2. Empty one sample vial at a time into a small Petri dish. Add 70-80% ethanol to keep the
organisms covered. Remove the internal sample label and complete the top portion of a
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Taxonomic Bench Sheet (for an example, see Attachment 5.1), using the information
from the label. Depending on the type of organisms, select the appropriate step:
a. For all Chironomidae organisms, extract the organisms from the Petri dish.
i. Prepare slide mounts using CMCP-10 (or CMC-9, CMC-10, or other media)
and applying a coverslip. All organisms must be visible, which generally
means a maximum of 10-20 organisms per slide. Label the slides with the
sample identification number and lab tracking number (per internal
SOPs).
ii. If the laboratory prefers to use another method than slide mounting, the
EPA External QC Coordinator will grant a waiver if the following applies:
1) The request is for a laboratory located at a single location. In
other words, EPA would not consider a request for combined
locations of a prime contract laboratory and its subcontract
laboratories. Instead, a separate waiver request must be
submitted for each of the individual prime and subcontractor
laboratories, and EPA would evaluate and grant (or deny) a waiver
individually, based upon each individual laboratory's
qualifications.
2) The request for a waiver must identify and describe a minimum of
three studies, for each of which, the external QC evaluation
demonstrated that the laboratory met or exceeded the NCCA QC
requirements (i.e., PDE<5% and PTD<15%) for its Chironomidae
organisms.
3) The laboratory agrees to mount the organisms on slides if it fails
one of the periodic (NCCA) external QC evaluations, as follows:
a. It must mount all Chironomidae organisms in samples
processed since the previous external QC evaluation (i.e.,
for which it met the PDE and PTD requirements).
b. It must continue to mount all Chironomidae organisms for
the unprocessed samples.
b. For all other organisms, remove similar organisms to other dishes (keep these
covered with 70-80% ethanol).
3. View the sample to ensure that all necessary diagnostic characters have been observed,
according to the taxonomic key or other literature using:
a. A stereo dissecting microscope for organisms in dishes.
b. A compound microscope for slides of Chironomidae and Oligochaeta organisms
4. Identify organisms to the lowest practical taxonomic level (species is the target for all
organisms with the exception of meiofauna, (which are not counted in the NCCA, due to
being smaller than 0.5 mm). Additional exceptions include Oligochaeta (Class) and
Chironomidae (Family) in samples from marine, polyhaline and mesohaline regions
ONLY. If a laboratory or individual taxonomist is having trouble reaching species for a
taxonomic group (not for an individual organism which might be damaged or otherwise
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difficult to identify), the lab must contact the NCCA project lead for guidance. Add any
necessary data qualifiers (see list provided with Required Data Elements in Table 5.2).
a. Enter the AphialD for estuarine taxa (when available) or the Taxonomic Serial
Number (TSN) for Great Lakes and low salinity estuarine taxa as it appears in the
column "Unique Identifier" of the taxa list provided by EPA.
b. Note whether the identification of a group of organisms is distinct (Distinct=Y/N)
from other organisms in the same sample as follows:
i. If the organisms can be identified to the target level, then Distinct="Y."
ii. If an organism cannot be identified to the target level then assign values
as follows:
1) If at least some of the organisms in the sample can be identified
to the target level, then:
a. Distinct="Y" for organisms identified at the target level;
and
b. Distinct="N" for organisms that were identified at a higher
taxonomic level (e.g., family) that may contain a target
level taxa already identified in a given sample (e.g., genus).
c. For example: If some organisms from a sample are
identified to Macoma, but other organisms in the sample
could only be identified to Tellinidae (Family) and/or
Veneroida (Order), then Macoma would be distinct, but
Tellinidae and/or Veneroida would not be Distinct.
2) If none of the organisms in the sample could be identified at the
target level, then:
a. Distinct="Y" for organisms identified at the lowest
taxonomic level (e.g., family); and
b. Distinct="N" for organisms identified at a higher level (e.g.,
order).
c. For example: If a taxonomist can identify a number of
Veneroida (Order) families, but a number of the organisms
could not be taken past Veneroida, then the individual
families would be distinct, but the order would not be
distinct.
iii. If the target taxonomic level cannot be achieved due to immature or
damaged organisms this should be noted in the data file in the QA_FLAG
field (e.g., QA_FLAG=IM). Table 5.2 provides other codes for the
QA_FLAG field.
iv. If damaged organisms can be identified, they are counted ONLY if the:
1) Fragment includes the head, and, in the case of arthropods, the
thorax;
2) Oligochaetes have a sufficient number of segments in the head;
3) Mollusk shell (bivalve or gastropod) is occupied by an organism;
4) Organism is the sole representative of a taxon in the sample.
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v. If a unique taxon is determined for which the appropriate taxonomic
level is not available in the literature and there are other taxa in that
taxonomic level:
1) Provide good quality digital photographs of the organism to
outside experts for identification; and
2) Include the tentative identification in the database with a data
qualifier code of QA_FLAG='UN' so that these organisms can be
distinguished from other organisms in the data analysis.
3) When the outside expert identifies the organism, update the
database with the correct identification.
Record the identifications. For example, using the taxonomic bench sheet in Attachment 5.1,
record the identification in the Column labeled "taxon." Enter the number of larvae, pupae, and
adults, or total count (e.g. mollusks), if appropriate life history column does not apply, of each
taxon under the appropriate columns.
5. Compare taxa names from the taxa list provided by EPA to the names used for the
identifications. Check the non-matches for the following common problems and correct
them. Examples of problematic taxa name entries include:
a. Abbreviations
b. Extra information identifiers (e.g., sp., spp., nr., cf., genus 1, w/ hair chaetae)
c. Extra character (e.g., "?", "Acentrella Pturbida", blank space)
d. The word "probably" or "prob" (e.g., "Microcylloepus prob. similis")
e. Double names (e.g., Callibaetis callibaetis)
f. Common misspellings
g. Tribes/subfamilies/subgenus sometimes may not appear
h. Species with incorrect genus (Hydatopsyche betteni)
i. Split level taxonomy (e.g., Cricotopus/Orthocladius)
Invalid name (e.g., taxonomic change, synonym; Sphaeriidae vs. Pisiidae)
6. Complete the identification by entering the totals for each developmental stage and the
total number of each taxon in the cells at the bottom of the sheet. Cross-check to be
sure the totals were summed correctly.
7. Provide the data to the Internal Taxonomic Officer for another review to confirm that
the identifications use the same nomenclature as the taxa list provided by EPA and the
laboratory's reference collection.
8. Make two copies of the bench sheet or computer file used to record the identifications.
They are distributed as follows: 1) the project file; and 2) EPA's External QC Coordinator.
9. Prepare a list of primary and secondary technical literature used in completing the
identifications. Provide complete citations in bibliographic format, including authors'
names, date of publication, title of document, name of journal or publisher, volume and
page numbers, or ISBN number, as appropriate. These citations will be kept on file with
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the Internal Taxonomic QC Officer, who will periodically review the reference collection
to ensure that it is complete.
10. Verify that the reference collection contains at least one organism that represents each
genus (or lowest taxonomic level) identified from all sample. For any missing references,
choose an appropriate organism(s) from the sample to represent a taxon name in the
master taxa list:
a. Place the physical specimen in the reference library.
b. Place two labels in the sample container to identify: organisms placed in the
reference collection, and those in the non-reference organisms.
c. Obtain a good quality representative digital photographs of the specimen (see
instructions in Section 5.3.1).
11. If the Internal Taxonomy QC Officer selects the sample for a QC check, the Internal
Taxonomy QC Officer re-counts and re-identifies the organisms in the sample following
the same steps above for the original taxonomist. One in 10 of the taxonomist's samples
must be checked. The Internal Taxonomy QC Officer records the independent
verifications on a bench sheet or computer file. The Internal Taxonomy QC Officer will
also supply a list of taxa that were found to be problematic during their QC sorting
check, which can be submitted in an Excel or Word document format. (If the Internal
Taxonomy QC Officer performs the QC check more frequently, then all QC data must be
submitted.)
12. Carefully return the rest of the organisms to the original sample vial, fill with 70-80%
ethanol, and cap tightly.
13. Re-package the samples and slide-mounted organisms carefully, and sign and date the
chain-of-custody form. Return or store the samples according to laboratory protocols
and requirements in Section 5.8.
14. Verify that all required data elements in Table 5.2 have been recorded by the
taxonomist and Internal Taxonomy QC Officer. If the results were recorded on paper,
provide the Taxonomic Bench Sheet to the data entry personnel.
Table 5.2 Benthic Macroinvertebrates Taxonomic Identification: Required Data Elements
FIELD
FORMAT
DESCRIPTION
LAB_NAME
Character
Name of lab
LABJD (optional)
Character
Lab sample ID
DATE_RECEIVED
MMDDYY
Date sample was received by lab
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SITE J D
Character
NCCA site identification code as used on sample
label
VISIT_NO
Numeric
Sequential visits to site (1 or 2, if specified on
label)
SAMPLE J D
Numeric
Sample number as used on field sheet (on sample
label)
DATE_COL
MMDDYY
Date sample was taken
SALINITY
Numeric
Salinity in psu of the water from which the sample
was collected (from label)
CONDITION_CODE
Character
Condition of sample upon arrival in lab, from Table
5.1 in LOM
COND_COMMENT
Character
Explanation of condition code (if needed). "Q"
condition code always requires a comment.
DATE_TAXON
MMDDYY
Date that the taxonomist started identifying
organisms in the sample
ANALYST_NAME
Character
Name of taxonomist or Internal Taxonomy QC
Officer (if record provides results of QC check)
QC_VERIFICATION
Character
Y if the record provides the results from the QC
check
PHYLUM
Character
Taxonomic phylum
CLASS
Character
Taxonomic class
ORDER
Character
Taxonomic order
FAMILY
Character
Taxonomic family
SUBFAMILY
Character
Taxonomic subfamily
TRIBE
Character
Taxonomic tribe
GENUS_GROUP
Character
Taxonomic genus group (e.g., thienemannimyia)
GENUS
Character
Taxonomic genus
SPECIES
Character
Taxonomic species
WORMS APHIA ID
Numeric
World Register of Marine Resources Database ID
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ITIS TSN
Numeric
LAB_TIN (OPTIONAL)
TAX A NAME
ABUNDANCE LARVAE
ABUNDANCE PUPAE
ABUNDANCE ADULT
Character
Character
Numeric
Numeric
Integrated Taxonomic Information System
Taxonomic Serial Number (for Great Lakes
samples and low salinity marine samples)
Lab taxa ID number
Unique taxon name in the taxa list provided by
EPA
Number of individual larvae or immature bugs
Number of individual pupae
Numeric
Number of individual adults
ABUNDANCE TOTAL
Numeric
Total number of individuals
DISTINCT
CITATION
QA_FLAG (if appropriate)
Character
Character
Character
Distinct taxa in sample (y/n) (See description in
Section 5.6)
Citation for reference used to identify organism, if
taxon not present in taxa list provided by EPA
database
QA/QC flag (lab may use its own flags, if defined in
QA_COMMENTS field or provided to NARS IM
team)
Flag
DD
IM
IN
NP
NT
UN
Definition
Damaged Organism, poor condition or
fragments
Immature
Indeterminate (explain in QA_COMMENT
field)
Not enough preservative used
Not able to meet target level for
identification (may be used with other
codes, or explain in QA_COMMENTS
field)
Sample shipping problem (explain in
QA_COMMENTS field)
Unknown. Identification is tentative.
Organism has been sent to expert
taxonomist for definitive identification.
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Q
Other quality concerns, not identified
above
QA_COMMENT
Character
Explanation for QA FLAG (if needed)
LAB_COMMENT
Character
General laboratory analysis comments
5.7 Data Entry
Table 5.1 and Table 5.2 identify the required data elements that the sorting and taxonomic
laboratories must provide to EPA, preferably in EPA's data template, available separately from
EPA. In addition, the laboratory must provide the resume or CVfor each taxonomist who
identifies benthic macroinvertebrates for the NCCA samples. The resume or CV for each
taxonomist is submitted once to EPA's External QC Coordinator.
5.8 Sample and Record Retention
The laboratory shall retain:
1. The sample materials, including vials, slides, and sorting residuals, for a minimum of
three years from the date the EPA makes the data public. During this time, the
laboratory shall store the materials in a cool location away from sunlight. The laboratory
shall periodically check the sample materials for degradation and refill jars and vials with
70-80% ethanol if necessary.
a. Sample collection permits (i.e., for some samples collected in waters under the
jurisdiction of the National Park Service) may require samples to be returned to
the permitting entity. In this case, the EPA Headquarters Project Management
Team will contact the laboratory to arrange for the samples to be returned after
QC reconciliation has been completed.
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.
5.9 External Taxonomic Quality Control
EPA requires that all NCCA laboratories ("primary laboratories") participate in the External
Taxonomic Quality Control Evaluation. Each taxonomist must participate in the QC evaluation,
even if the taxonomist is under subcontract with, or consulting for, another firm.
In contrast to the internal QC evaluation in Section 5.6 that verify adherence to the procedures
and ensures in-laboratory consistency between taxonomists, the purpose of the external QC
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evaluation is to ensure taxonomic consistency among laboratories and taxonomists. To achieve
this objective, EPA compares the primary laboratory results to those from a second laboratory,
considered a quasi "gold standard" for taxonomic evaluations.
The External QC Coordinator, who is an EPA staff member, 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 NCCA. The QC contractor is responsible for complying with
instructions from the External QC Coordinator; obtaining and managing the secondary
laboratory; coordinating and paying for shipments of the QC samples between locations;
comparing sample identifications by different laboratories; facilitating reconciliation
teleconferences; and preparing brief summary reports.
The External QC Coordinator will arrange for the QC contractor to conduct a minimum of two
QC evaluations. To the extent practicable, the External QC Coordinator and QC contractor will
schedule batch evaluations at regularly throughout the project period.
Each QC evaluation consists of the following steps:
1. In consultation with the QC contractor, the External QC Coordinator determines an
appropriate time to conduct the evaluation based upon the total number of samples
assigned to the laboratory, the delivery schedule, processing schedule, and the
following constraints:
a. Availability of samples from other laboratories. For example, if three state
laboratories are each processing less than 30 samples, the External QC
Coordinator might combine their samples into one batch for the QC evaluation.
b. If a primary laboratory is responsible for processing 100 samples or more for the
NCCA, the External QC Coordinator will split their samples into several batches
(e.g., each 50 to 100 samples) so that EPA can evaluate and correct performance
on an ongoing basis.
2. The External QC Coordinator provides the QC contractor with a list of laboratories and
processed samples. Sample identification includes the site identification code, sample
number, and taxonomist who performed the identifications.
3. The QC contractor randomly selects 10% of the samples from each NCCA laboratory,
subject to the following constraints:
a. If the primary laboratory received fewer than 30 samples, then the QC
contractor randomly selects three samples for the evaluation.
b. For each taxonomist identified on the list, the QC contractor ensures that the
selection includes one or more of his/her samples.
c. The External QC Coordinator may elect to provide an initial evaluation of the
national laboratory by selecting a small batch from the samples that the
laboratory completed in the first 2-3 months.
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4. The QC contractor provides a list of the QC samples, and instructions, to the External QC
Coordinator and each primary laboratory participating in the evaluation. Although the
External QC Coordinator and QC contractor may tailor the instructions for the
participating taxonomists' preferences, the instructions are likely to specify the
following:
a. Pack and ship the QC samples to the central holding facility designated by the QC
contractor. Instructions are likely to require that the:
i. Shipments contain chain-of-custody documentation for all slides and
containers.
ii. Containers (e.g., slides, vials) include the site identification code and
sample number.
iii. Containers cannot be marked in any way that might identify the
taxonomic classification for any organism.
iv. The number of taxa in a vial or container should be based on practical
considerations (e.g., size of animals and amount of ethanol needed for
preservation, amount of ethanol allowed in a single shipment to meet
DOT shipping requirements).
b. Track the QC samples using forms provided by the QC contractor.
c. Email a spreadsheet with the data for the QC samples to the External QC
Coordinator. (EPA requires that all labs use its spreadsheet template for
recording the taxonomic data.)
5. The QC contractor reviews the condition of the QC samples (e.g., verifies that the
containers do not identify taxon for any organism) and ships the samples to the
secondary laboratory along with instructions and the EPA template for reporting data.
6. Within 24 hours of receipt, the secondary laboratory:
a. Notifies the QC contractor that it has received the samples;
b. Faxes or emails any additional receipt records, including discrepancies, within 24
hours; and
c. Completes any other instructions from the QC contractor.
7. The secondary laboratory:
a. Re-identifies and re-counts following the procedures in the Method, except does
not:
i. Develop a reference library.
ii. Photograph organisms unless the taxa are identified for reconciliation
discussion.
iii. Perform any internal QC checks.
b. Records the required data elements in Section 5.7
c. Enters the data using EPA's spreadsheet template for the taxonomic data.
d. Emails the completed spreadsheet to the QC contractor.
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8. The QC contractor compares the original taxonomic results (i.e., data) generated by the
primary laboratory to the taxonomic results generated by the secondary laboratory for
each sample. As part of this evaluation, the QC contractor calculates PDE and PTD using
the equations in Section 5.3.1 and compares their values to the QC requirements in the
Section 5.10.
9. If any samples exceed the PDE or PTD limits in Section 5.10, the QC contractor consults
with the External QC Coordinator to determine if reconciliation calls are necessary to
resolve differences. The External QC Coordinator may decide that a reconciliation call is
unnecessary if there appears to be an obvious explanation for differences, few samples
are affected, or other reasons.
10. The QC contractor schedules and facilitates reconciliation teleconferences with EPA and
the laboratories.
a. In preparation for the teleconferences:
i. The QC contractor instructs the secondary laboratory to photograph
representative specimens for each taxon identified for discussion.
ii. The QC contractor provides the participants with a spreadsheet that
includes:
1. List of samples and taxon identifications for discussion;
2. Relevant data from the primary and secondary laboratories; and
3. PDE and PTD values.
iii. The primary and secondary laboratories provide participants with the
relevant reference (or citation) and photograph for each taxonomic
identification for the discussion.
iv. The QC contractor emails a meeting announcement for a convenient time
for all participants. The email identifies instructions for accessing the
External QC Coordinator's toll-free teleconference line.
b. Within a week after the teleconference, the QC contractor sends an email to the
External QC Coordinator and other teleconference participants that summarizes:
i. Agreements to use common nomenclature for discrepancies;
ii. Commitments to reevaluate identifications by reexamining samples;
iii. Application of changes that are appropriate for all samples, not just the
QC samples (e.g., common nomenclature)
iv. Items that will not be resolved for some reason (e.g., sample degraded
during shipment).
11. After completing the reconciliation calls, the participants complete the following steps:
a. Secondary laboratory:
i. Reexamines samples as deemed necessary during the reconciliation call
ii. Updates its database with changes to:
1. QC samples per reexamination and other items in the QC
contractor email; and
2. Non-QC samples as appropriate (e.g., nomenclature changes
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apply to all samples, not just QC samples),
iii. Provides database to QC contractor.
b. QC contractor confirms that the secondary laboratory (i.e., its subcontractor)
completed its assignments before allowing the secondary laboratory to move to
the next step.
c. Secondary laboratory stores its original records, including laboratory notebooks
and the reference library, for a minimum of 10 years from the date that EPA
publishes the final report.
d. Secondary laboratory and QC contractor follow steps 4 and 5 above to return the
samples to the primary laboratory.
e. After receiving the samples (and tracking per step 4), the primary laboratory:
i. Reexamines samples as deemed necessary during the reconciliation call;
ii. Updates its database with changes to:
1. QC samples per reexamination and other items in the QC
contractor email; and
2. Non-QC samples as appropriate (e.g., nomenclature changes
apply to all samples, not just QC samples)
iii. Provides the revised database to the External QC Coordinator (not the QC
contractor). It also confirms that it has completed all relevant items
identified in the QC contractor's email summary of the teleconferences
(from Step 10.b).
f. QC contractor provides EPA with a report or memorandum that:
i. Identifies the participating laboratories, with the following information
about each laboratory:
1. Laboratory name
2. Address
3. Contact person (name, telephone, and email)
ii. Quantifies the taxonomic precision (PDE and PTD) as they were prior to
the reconciliation call;
iii. Assesses data acceptability;
iv. Highlights taxonomic problem areas;
v. Identifies any discrepancies for which the External QC Coordinator
determined that a reconciliation teleconference was not necessary;
vi. Identifies primary and secondary laboratory commitments to change its
identifications or provide additional review of any organisms; and
vii. Provides recommendations for improving precision for other samples not
included in the QC evaluation.
12. After review, the External QC Coordinator:
a. Submits the report, and draft technical direction with next steps for the
laboratory, to the EPA staff managing or coordinating with the primary
laboratory.
b. Determines if significant differences within the batch of QC samples warrant re-
identification of samples by the primary laboratory and a second QC evaluation
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by the secondary laboratory. If deemed necessary, EPA will instruct the primary
laboratory to include the samples for review with the next batch of QC samples.
As an additional verification on the generation of the data, EPA may conduct assistance visits at
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. The
objective of the visit would be to:
• Confirm the laboratory is properly implementing the steps in the method.
• Assist with questions from laboratory personnel.
• Suggest corrections if any errors are made.
5.10 Quality Assurance/Quality Control (QA/QC)
Equations for percent sorting efficiency (PSE), percent disagreement in enumeration (PDE) and
percent taxonomic disagreement (PTD) are listed in Section 5.3.1. Table 5.3 and Table 5.4
outline the data quality objectives and describe the laboratory quality control measures for
benthic macroinvertebrates.
Table 5.3 Benthic Macroinvertebrates: Measurement Data Quality Objectives
VARIABLE OR MEASUREMENT
PRECISION
ACCURACY
Sort and Pick
90%a
90%a
Identification
85% b
95%c
NA = not applicable; aAs measured by PSE; bAs measured by (100%-PTD); cAs measured by (100%-PDE)
Table 5.4 Benthic Macroinvertebrates: Laboratory Quality Control
CHECK OR
SAMPLE
DESCRIPTION
FREQUENCY
ACCEPTANCE CRITERIA
CORRECTIVE ACTION
SAMPLE PROCESSING AND SORTING
Sample pickate
examined by
another
sorter
10% of all
(minimum of
1) completed
per sorter
PSE > 90%
If < 90%, examine all
residuals of samples by
that sorter and retrain
sorter
IDENTIFICATION
Duplicate
identification by
Internal
Taxonomy
QC Officer
1 in 10
samples
per
taxonomist,
PTD <15%
If PTD >15%, reidentify all
samples completed by that
taxonomist since last
meeting the acceptance
criteria, focusing on taxa
of
concern
Independent
identification by
outside, expert,
taxonomist
All uncertain
taxa
Uncertain
identifications to be
confirmed by expert
in particular taxa
Record both tentative and
independent IDs
External QC
10% of all
PDE < 5%
If PDE > 5%, implement
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samples
PTD < 15%
recommended corrective
completed per
actions.
laboratory
If PTD > 15%,implement
Recommended corrective
actions.
Use of
For all
All keys and references
If a lab proposes to use
widely/commonly
identifications
used by
other references, the lab
accepted
each lab must be on
must obtain prior
taxonomic
bibliography prepared by
permission from External
references by all
one or
QC Officer before
NCCA labs
more additional NCCA
labs or in
the taxa list provided by
EPA.
This requirement
demonstrates the general
acceptance of the
references by
the scientific community.
submitting the data with
the identifications based
upon the references.
Prepare reference
Each new
Complete reference
Internal Taxonomy QC
collection
taxon per
collection to be
Officer periodically
laboratory
maintained by each
individual laboratory
reviews data and
reference collection to
ensure reference
collection is complete and
identifications are
accurate
DATA VALIDATION
Taxonomic
All data sheets
Taxa known to occur for
Second or third
"reasonableness"
coastal
identification by
checks
waters or Great Lakes.
expert in that taxon
5.11 References
Epler, J.H. 2001. Identification manual for the larval chironomidae (Diptera) of North and South
Carolina. A guide to the taxonomy of the midges of the southeastern United States, including
Florida. Special Publication SJ2001-SP13. North Carolina Department of Environment and
Natural Resources, Raleigh, NC, and St. Johns River Water Management District, Palatka, FL.
526 pp.
Merritt, R.W., K.W. Cummins, and M.B. Berg (editors). 2008. An Introduction to the Aquatic
Insects of North America, 4rd edition. Kendall/Hunt Publishing Company, Dubuque, Iowa.
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(4):621-631.
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USEPA. 2012. National Rivers and Streams Assessment 2013-2014: Laboratory Operations
Manual. EPA-841-B-12-010. U.S. Environmental Protection Agency, Office of Water,
Washington, DC.
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Attachment 5.1: Benthic Macroinvertebrates: Taxonomy Bench Sheet (example)
Laboratory Information
Sample Information
Project ID
Sample ID
Station Name
Site ID
Station Location
Date Collected
Station Number
Field Crew ID
Taxonomist Name
Date 1st Organism Identified in Sample: QC Check? Y / N
Alpha ID
(Use # in
Unique
Identifier
from taxa
list provided
by EPA)
TSN
(Use # in
Unique
Identifier
from taxa
list provided
by EPA)
Taxon
Distinct
(Y/N)
Counts of Organisms in the
Taxon:
Cumulative
Number of
Organisms in
Sample
Data
Qualifier
(Codes in
Table 5.2)
Total
(any
stage)
Larvae
Pupae
Adults
Comments:
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6.0 WHOLE BODY FISH PROCESSING AND CONTAMINANT ANALYSIS
This chapter describes fish processing and analysis requirements for whole body fish samples.
The purpose is to determine concentrations of contaminants in fish samples collected in the
2020 NCCA and related studies. The laboratory shall perform analysis to determine the lipid
content, concentrations of metals, pesticides, and PCBs found in fish within estuarine waters
and nearshore Great Lakes.
At each sampling site, the FOM instructs the crews to collect five fish of the same species (or 10
collector sea urchins in Hawaii only) and similar size for each sample. The fish specimens are
shipped to the analytical laboratory on dry ice.
6.1 Summary of the Procedure
This chapter describes the processing and contaminant analysis of whole fish samples collected
for EPA's 2020 NCCA. To ensure consistent preparation across all fish samples and to avoid
sample contamination, it is important that all NCCA participating laboratories adhere to the fish
procedures described in Section 6.5. The procedure is an adaption of instructions developed for
fish tissue preparation for the National Rivers and Streams Assessment. As described in Section
6.6 the laboratory may choose to use any method that meets EPA's specifications for
contamination measurements unless contractually bound to use specific methods (note,
alternate methods must still meet the measurement quality objectives required in the QAPP).
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).
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.
4. When operating grinding equipment, the laboratory personnel must exercise
caution.
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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:
Method 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."
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.
Fish Composite: Each composite consists of all parts of the fish including the head, skin, internal
organs, muscle, and bones. For sea urchins, it includes only the gonad tissue because it is
essentially the only tissue present. Unless otherwise specified, references to "fish" include "sea
urchins." With the exception of sea urchins, NCCA does not provide support for analyses of any
other invertebrates such as crustacean (e.g., lobster, crabs).
NARS: National Aquatic Resource Surveys. The National Coastal Condition Assessment (NCCA) is
part of the NARS program.
NARS Information Management System (NARS IM): The IM system established to support all
surveys, including NCCA, in the NARS program. The NARS IM system is used to track the
samples from field collection to the laboratory.
NCCA: National Coastal Condition Assessment. Freshwater and marine samples will be collected
during the field stage of NCCA.
Non-routine sample: A non-routine sample is any sample that does not meet the definition of a
routine sample. EPA will provide instructions for the use, if necessary, of the non-routine
samples. Non-routine includes most species not listed in Appendix B with the exception of
species listed in the Endangered Species Act, cartilaginous fishes, and invertebrates of any type
(with the exception of collector sea urchins). These instructions also may include discarding
some of the fish in the composite sample based on size before proceeding with homogenizing.
For non-routine composites, the laboratory homogenizes only the designated specimens, i.e.,
those that EPA identifies by specimen number.
Percent Recovery: Recovery is measured by comparing the concentrations of a sample split into
two aliquots; and one aliquot is spiked with a known concentration value. Cs is the
concentration measured in the spiked aliquot; C is the concentration measured in the un-spiked
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aliquot; and s is the known concentration amount for the spike. The following equation is used
to calculate the percent recovery (%Rs)\
%Rs =
Cs- C
x 100
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, S, as follows:
5 =
n
n
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:
5
RSD = -= X 100
C
Reporting Limit: A reporting limit is the point at which the measured value of the analyte can
be reported with confidence.
Routine sample: A routine composite sample consists of individual adult fish of a single species
that meet EPA's length requirement (Length of smallest fish in the composite must be at least
75% of the length of the longest fish), and sufficient number of fish to meet target mass of 300
grams. The laboratory homogenizes the fish to prepare one composite sample. The species
must be one of the target species identified in Appendix B of this LOM.
Sample-Specific Detection Limit: Most samples will have a sample-specific detection equal to
the method detection limit. For diluted samples, the sample-specific detection limit will be the
product of the method 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.
TOCOR: Task Order Contracting Officer's Representative is EPA's contact person for
laboratories under contract to EPA.
Quality Control Check Sample (QCCS) is a sample prepared from an independent standard at a
concentration within the calibration range. A QCCS is intended as an independent check of
technique, methodology, and standards and should be run with every batch.
6.3.2 General Requirements for Laboratories
Competency: To demonstrate its competency, the laboratory shall provide analyte and matrix
specific information to EPA. In addition to documentation of achieving the method detection
limits, accuracy, and precision targets (as specified by the NCCA QAPP) for the required analytes
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in fish samples, 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 fish-related analyses, membership in professional societies, and
experience with analyses that are the same or similar to the requirements of this
method.
Quality assurance and quality control requirements.
The organization shall provide EPA with copies of the quality-related documents relevant to the
procedure. Examples include QMPs, QAPPs, and applicable Standard Operating Procedures
(SOPs).
To demonstrate its ongoing commitment to maintaining data quality, the person in charge of
quality issues for the organization shall sign the NCCA QAPP Certification Page.
6.3.3 Equipment/Materials
The procedures require the following equipment and information:
o Scale (Electronic balance)
o Powder-free nitrile gloves
o Tape measure
o 5% nitric acid
o Deionized water (Dl water)
o Grinding equipment
o Glass containers
o Jars
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. The laboratory must inspect the samples promptly
on receipt. As samples arrive, the laboratory must:
1. Log the samples into the National Aquatic Resource Survey Information Management
system (NARS-IM) within 24 clock hours. Alternatively, for shipments with many
samples, the laboratory may email a spreadsheet with the sample login and sample
condition information to NARSIM (see Chapter 2 for contact information).
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"
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and record the reading. Record the condition and temperature of the sample in the
database using the codes in Table 6.1.
3. Compare the information on the label on each individual fish specimen to the sample
tracking form for each composite and verify that each specimen was included in the
shipment and is properly wrapped and labeled. The crew labels each fish specimen
using the sample identification code and appends a specimen identification code. For
example, if the sample number is "NCCA20-1111," then the crew might label specimen
"A" as "NCCA20-1111. A." Record the number of fish in each sample.
4. Weigh each sample (i.e., all fish specimens collectively), record the weight in the
database, and confirm that the sample meets the weight requirements of 300 grams (g)
for a routine sample. If the sample weight is less than the required minimum, contact
EPA for instructions, which are likely to involve preparing fewer aliquots for possibly
fewer types of analyses than originally intended (e.g., perhaps EPA might eliminate the
pesticides analysis for the sample).
5. Verify that all required data elements, per Table 6.1, have been recorded. If any
elements are missing, then enter them into the database.
6. Transfer the samples to the freezer for long-term storage. Except during processing and
analysis stages, the samples must be stored frozen to less than or equal -20 °C.
7. Notify the EPA immediately about any problems involving sample integrity, conformity,
or inconsistencies as soon as possible following sample receipt and inspection.
Table 6.1 Whole Body Fish Login: Required Data Elements
FIELD TYPE DESCRIPTION
SITE ID
Character
Site identification code
SAMPLE ID
Character
Sample number
DATE COL
MMDDYY
Date that the field crew collected the sample
ARRIVAL_TEMP
Numeric
Temperature of sample upon arrival at the laboratory
(fish should be frozen).
NUMBER FISH
Numeric
Number of fish in the sample
SAMPLE WT
Numeric
Total weight of sample (all fish)
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 wrapping 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
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COND COMMENT
Character
Explanation for Q Flag (if needed)
6.5 Whole Fish Preparation and Homogenization Procedures
This section describes the whole fish preparation and homogenization procedures. As described
in Section 6.5.1, if a laboratory determines that a sample is non-routine, the laboratory
contacts the EPA HQ NCCA Laboratory Review Coordinator (Section 2.2 provides contact
information) for additional instructions before continuing with the compositing and
homogenization procedures in Section 6.5.2. Section 6.5.3 describes rigorous equipment
cleaning and rinsate collection steps used before the compositing and homogenization steps in
Section 6.5.4.
6.5.1 Sample Classification: Routine or Non-Routine
Each sample is either a "routine" composite sample, or a "non-routine" composite sample,
based on the definitions provided in Section 6.3.1. If instructions are unclear for the handling
and/or processing of any composite fish sample (e.g. samples collected from incorrect sampling
location, unnecessary duplicate sample, inappropriate fish species, etc.), the laboratory shall
contact EPA for clarification before proceeding with further activities involving the sample. The
laboratory is expected to maintain the integrity of the sample (e.g., frozen) until EPA
determines next activities involving the fish sample in question.
6.5.2 Fish Examination and Preparation
This section describes the steps for fish examination and preparation.
1. Put on powder-free nitrile gloves (if not already gloved) before unpacking individual fish
specimens. For sea urchins, wear thick rubber gloves to provide protection from the
urchin spines. As samples are unpacked and unwrapped, inspect each fish carefully for
any damage (e.g., tears in the skin or punctures in the gut). Document any damage in
comments per Table 6.2.
2. The field crews measured the total length of each fish specimen in the field and
recorded those lengths on the sample tracking form. Because of the importance of
length measurements, EPA requires laboratories to perform a second series of
measurements of the length for each fish. Because it may be difficult to reproduce the
field measurements of fish length when the specimens are still partially frozen, begin
processing the specimens in the following steps:
a. Lay them out in order by specimen number (e.g., the portion of the sample ID
after the decimal point)
b. Allow them to partially thaw to the point that each specimen can be laid
relatively flat.
c. Using the length data on the sample tracking form (or the relative length order
data in the fish sample processing instructions spreadsheet), confirm that the
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specimen ID for the longest specimen recorded on the tracking form is the same
as the specimen ID on the label of the longest specimen. Repeat this relative
length comparison for each of the other specimen IDs to ensure that the length
orders based on the recorded lengths in the sample tracking form are consistent
with the specimen IDs on the individual fish labels. This check is important for
confirming that the field crews attached the correct label to each fish in the
composite sample.
d. Record the required data elements per Table 6.2 for the length of each species.
8. Weigh each fish to the nearest gram (wet weight) prior to any sample processing. In the
database, record the required weight data elements per Table 6.2 for each specimen.
9. Identify and record the species of each fish specimen. Confirm that the species is one of
the target species listed in Appendix B of this LOM.
10. Determine if the sample is routine or non-routine (per classification definitions in
Section 6.5.1) and record its classification and any applicable fish code from Table 6.3.
Return any non-routine sample to the freezer and contact the EPA HQ NCCA Laboratory
Review Coordinator for processing instructions (see Chapter 2 for contact information).
11. Verify that all required data elements, per Table 6.2 and Table 6.3, have been recorded.
Enter any missing elements into the database.
12. Rinse each fish with deionized water and remove any adhering slime as a precautionary
measure to mitigate possible contamination from sample handling in the field. Use
HDPE wash bottles for rinsing fish and for cleaning homogenization equipment and
utensils. Do NOT use Teflon® wash bottles for these procedures.
13. Return to freezer for storage until ready to homogenize the sample. If the laboratory
intends to proceed directly to homogenization, then allow the sample to partially thaw
while cleaning the equipment as described in the next section.
Table 6.2 Whole Body Fish: Data Elements for Each Fish Specimen
FIELD
TYPE
DESCRIPTION
SITE J D
Character
Site identification code
SAMPLEJD
Character
Sample number
SPECIMENJD
Character
Identification code assigned to a single fish
SPECIES
Character
Species of fish
FISH_WT
Numeric
Weight of fish
WT_UNIT
Character
Units offish weight (kg, lb)
FISH_LEN
Numeric
Length of fish
LENJJNIT
Character
Units offish length (cm, in)
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COMMENT
Character
Comment about condition of fish or other
observations
Table 6.3 Whole Body Fish: Data Elements from Examination of Each Sample
FIELD TYPE DESCRIPTION
SITE ID
Character
Site identification code
SAMPLE ID
Character
Sample number
SAMPLE CLASS
Character
Sample classification: Routine or Non-routine
FISH_CODE
Character
Codes describing any deviations from the FOM criteria for
fish collection for each sample
Flag
Definition
SP
Not all specimens are of the same species
LE
Not all specimen's lengths are within 75% of
longest fish
NS
Specimen number is fewer than minimum of 5
or greater than 20 maxima
WT
Mass does not meet minimum of 300 grams
LL
Longest fish exceeds 400 mm maximum length
LS
Shortest fish below 100 mm minimum length
Q
Other quality concerns, not identified above
6.5.3 Equipment Cleaning and Rinsate Collection
This section describes the rigorous cleaning required to protect against cross-contamination of
samples. To verify that the cleaning procedures are effective, EPA requires the collection of
rinsate samples as described below.
1. Before processing any sample, thoroughly clean all of the homogenization equipment.
Disassemble the homogenization equipment (i.e., blender, grinder, or other device) and
thoroughly clean all surfaces and parts that contact the sample. Similarly, clean all
knives, cutting boards, and other utensils used. The cleaning steps are as follows:
a. Wash with a detergent solution (phosphate- and scent-free) and warm tap water
b. Rinse three times with warm tap water
c. Rinse three times with deionized (Dl) water
d. Rinse with acetone
e. Rinse three times with Dl water
f. Rinse with (not soak in) 5% nitric acid
g. Rinse three times with Dl water
h. Allow the components to air dry
i. Reassemble the homogenization equipment
2. Once per batch (i.e., once per maximum of 20 samples), collect rinsate samples for use
in assessing any equipment contamination. To minimize the number of project samples
that might be affected by cross contamination, collect the normal rinsate samples on
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the first day that samples in a batch of 20 are processed. Ideally (not required), the
laboratory will vary the point at which the rinsates are collected on that first day over
the course of the project (e.g., between the 1st and 2nd samples for one batch, the 2nd
and 3rd samples for another batch, etc.). Prior to reassembling the homogenization
equipment, use the following steps to prepare enough rinsate samples for the relevant
QA/QC activities:
a. Prepare each hexane rinsate sample by pouring a 100-mL portion of pesticide-
grade hexane over all parts of homogenization equipment, including the cutting
boards and knives, and collect it in a clean glass container. Place an additional
100-mL aliquot of clean hexane in a similar glass container for use as a solvent
blank. Allow the solvent to evaporate from the equipment. Per QA/QC
requirements, the laboratory will analyze the rinsate and solvent blank for the
Polychlorinated biphenyls (PCBs), and pesticides selected for NCCA analysis.
b. Once the hexane has evaporated, prepare each Dl water rinsate using 250 mL of
Dl water. Collect the Dl water rinsate in a clean glass or HDPE container. Place a
second aliquot of Dl water in a separate similar clean container for use as a
blank. Acidify these two samples to pH < 2 with nitric acid. Per QA/QC
requirements, the laboratory will analyze the rinsate and blank samples for
metals and mercury.
c. Store the rinsates and blanks at a cold, not freezing, temperature (<6 9C).
6.5.4 Compositing and Homogenization Procedure
This section describes the steps for a "batch" homogenization method that uses the entire
homogenized volume of all fish specimens to prepare the composite. In contrast to an
"individual" method that would combine equal weights of tissue from each specimen, the batch
homogenization method uses the complete specimens regardless of each individual specimen's
proportion to one another. The steps are as follows:
1. Change gloves between samples. The technician may use the same gloves in handling all
fish within a given sample.
2. Partially thaw samples for ease of grinding during homogenization.
3. For sea urchins, prepare the sea urchin for compositing by cracking open the shell of
each sea urchin in the sample. From all of the sea urchins in the sample, extract and
composite only the gonad tissue. (The gonad tissue is essentially the only tissue present
in sea urchins.)
4. Process each sample using a size-appropriate homogenization apparatus (e.g.,
automatic grinder or high-speed blender). If difficulties arise with the samples sticking to
equipment, try the following:
a. Chill the grinder briefly with a few small pieces or pellets of dry ice.
b. Add pellets of dry ice to the specimens as they enter the grinder.
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5. Mix the specimens thoroughly until completely homogenized as evidenced by a final
composite sample of soupy composition with uniform color and texture. Visible chunks
or pieces of skin, bone, or tissue (e.g., liver tissue has red bits) will hinder extraction and
digestion and, therefore, are NOT acceptable.
6. Grind the sample a second time, using the same grinding equipment. It is not necessary
to clean the grinding equipment between grinding cycles of the same sample. This
second grinding should proceed more quickly. The final sample must have a soupy
composition with uniform color and texture. If there are obvious differences in color or
texture, grind the entire sample a third time.
7. Prepare the sample aliquots for each type of analysis (e.g., mercury, PCBs) and place any
remaining sample materials in a separate jar. Table 6.4 provides target mass weights
needed for each type of analysis. When filling jars, leave sufficient space, at least 20%,
at the top of each jar to allow for expansion of the tissue as it freezes. Jars filled beyond
80% capacity may break when freezing. Wipe off the outside of the jars to remove any
residue or moisture. Label each container and place inside one heavy-weight food-
grade self-sealing plastic freezer bag to avoid sample loss due to breakage. Freeze the
tissue aliquots at -20 9C and maintain samples in the freezer until analysis.
8. For one sample in every batch (same batch as specified for the rinsate samples collected
in Section 6.5.3), the laboratory conducts triplicate analyses of the lipid content to
confirm that the grinding has resulted in a homogeneous sample. As with the collection
of rinsate samples, the laboratory performs the homogeneity testing on the first day on
which samples in a batch of 20 are processed. However, the sample chosen for
homogeneity testing must be one that yields enough tissue mass to support the added
mass needed for triplicate lipid aliquots (15 to 30 g).
a. The laboratory selects one sample processed on the first day of every batch that
will provide well over 300 g of total tissue mass.
b. From that sample, place three 5- to 10-g aliquots in clean glass or plastic
containers of suitable size and label as appropriate.
c. Calculate the mean lipid content (in percent), the standard deviation (SD), and
the relative standard deviation (RSD) as follows:
^ (% lipids);
mean % lipids =
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SD =
3
X (% lipids ; -mean lipids) ~
i-l
RSD = ^-
mean
d. If the RSD of the triplicate results is:
¦ Less than or equal to the QC criterion, then the homogenization effort is
judged to be sufficient for all samples in that QC batch.
¦ Otherwise, corrective action consists of regrinding all of the aliquots from
each composite sample in the affected batch until meeting the QC
criterion. This may entail retrieving all sample aliquots (see Table 6.4
from the freezer, allowing them to partially thaw, homogenizing them
again, determining new lipids results, and performing a new
homogenization QC determination. New sample containers are required
for storing any rehomogenized samples. Also, follow the steps in Section
6.5.3 for cleaning the equipment between each composite sample in
rehomogenizing the samples.
e. For this sample analyzed in triplicate, record the lipid content measured in the
first analysis.
9. Before homogenizing the next sample, clean the grinding equipment and all other
sample preparation equipment using the procedures described in Section 6.5.3.
Table 6.4 Whole Body Fish: Initial Aliquot Requirements
ANALYSIS
TARGET
SAMPLE JAR REQUIREMENTS
MASS
Mercury
5 -10 g
50-mL HDPE straight-sided jar with foil-lined lid, or conical HDPE tube
with snap top
Metals other
than mercury
5 -10 g
50-mL HDPE straight-sided jar with foil-lined lid, or conical HDPE tube
with snap top
PCBs
30 -35 g
125-mL straight-sided amber or clear glass jar with
PTFE-lined lid
Pesticides
30 -35 g
125-mL straight-sided amber or clear glass jar with
PTFE-lined lid
Lipids
10 -15 g
Laboratory's choice, as this aliquot will be used in-house to
determine the lipid content of the sample
6.6 Contaminant Analysis: Requirements
The laboratory shall perform analysis of the homogenized composites to determine the lipid
content, concentrations of metals, mercury, pesticides, and PCBs. With the exception of sea
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urchins, NCCA does not provide support for analyses of any other invertebrates such as
crustaceans (e.g., lobster, crabs).
After preparing the fish composites as described in Section 6.5, laboratories may choose to use
any analysis method, including those in Table 6.5, that measures contaminants to the levels of
the method detection limits identified in Table 6.6. In addition, the method must meet the
target precision of 30% and the target accuracy as follows:
• Metals: 20%
• Organics (PCBs and pesticides): 35%
The laboratory must store the fish samples frozen at a maximum of -20° C and complete the
analyses within one year.7
Table 6.5 Whole Body Fish: Analytical Methods
ANALYSIS
EXTRACTION
METHODS THAT MEET THE QA/QC
REQUIREMENTS (ANY METHOD THAT
MEETS THE QA/QC REQUIREMENTS IS
ACCEPTABLE)
Metals (except
Any method using microwave
EPA Method 6020A9
Mercury)
assisted digestion8
Mercury
EPA Method 24510
PCBs and Pesticides
EPA Method 3540C11
EPA Method 827012
Percent Lipids
Any method using hexane
EPA Method 9071B13
Table 6.6 Whole Body Fish: Lipids and Required Contaminants
TYPE
UNITS
PARAMETERS
CAS
PCB
MAX
MDL
Target
TARGET
NUMBER
NUMBER
CONC
TARGET**
ACCURACY
PRECISION
7 NCCA allows for a 1-year holding time because of the sheer volume of sample collected in a short amount of time.
Generally, EPA recommends different holding times, see for example Appendix J "Recommended procedures for
preparing whole fish composite homogenate samples" in Guidance for Assessing Chemical Contaminant Data for
Use in Fish Advisories, Volume 1 (Fish Sampling and Analysis), 3rd Edition, 2000. EPA #823-13-00-007. Retrieved
May 22, 2019 from https://www.epa.gov/sites/production/files/2015-06/documents/volumel.pdf
8 For example, see Method 3051A "Microwave Assisted Acid Digestion of Sediments, Sludges, Soils, and Oils,"
retrieved May 22, 2019 from https://www. epa.gov/sites/production/files/2015-12/documents/3051 a.pdf
9 For example. Method 6020A "Inductively Coupled Plasma-Mass Spectrometry" retrieved May 22, 2019 from
http://www.epa.gov/epawaste/hazard/testmethods/sw846/pdfs/6020a.pdf.
10 For example. Method 245.7 "Mercury in Water by Cold Vapor Atomic Fluorescence Spectrometry, Revision 2.0"
(EPA-821-R-05-001, February 2005), retrieved May 22, 2019 from
https://www.nemi.gov/methods/method summary/9629/
11 For example, see Method 3540C "Soxhlet Extraction" retrieved March 12, 2020 from
https://www.epa.gov/sites/production/files/2015-12/documents/3540c.pdf
12 For example, Method 8270D "Semivolatile Organic Compounds by Gas Chromatography/Mass Spectrometry
(GC/MS)" retrieved March 12, 2020 from https://19ianuarv2017snapshot.epa.gov/sites/production/files/2015-
07/documents/epa-8270d.pdf
13 Method 907 IB "n-Hexane Extractable Material (HEM) for Sludge, Sediment, And Solid Samples," retrieved May
22, 2019 from https://www.epa.gov/sites/production/files/2015- 12/documents/907lb.pdf
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(2010 &
2015)
LIPID
% Wet
Weight
% LIPID
MOISTURE
%
Moisture
% MOISTURE
46.1
Mg/wet g
Aluminum
7429-90-
5
1100
10
20
30
Mg/wet g
Iron
7439-89-
6
1730
50
20
30
Mg/wet g
Lead
7439-92-
1
707
0.1
20
30
Mg/wet g
Mercury
7439-97-
6
1190
0.01
20
30
Mg/wet g
Nickel
7440-02-
0
22.798
0.5
20
30
Mg/wet g
Silver
7440-22-
4
11.5
0.3
20
30
—I
E*
Mg/wet g
Tin
7440-31-
5
184
0.05
20
30
LU
Mg/wet g
Arsenic
7440-38-
2
63.6
2
20
30
Mg/wet g
Cadmium
7440-43-
9
22.377
0.2
20
30
Mg/wet g
Chromium
7440-47-
3
28.495
0.1
20
30
Mg/wet g
Copper
7440-50-
8
212
5
20
30
Mg/wet g
Vanadium
7440-62-
2
24.923
1
20
30
Mg/wet g
Zinc
7440-66-
6
694.607
50
20
30
Mg/wet g
Selenium
7782-49-
2
24.774
1
20
30
ng/wet g
2,2',3,3',4,4',5,5',6,6'-
Decachlorobiphenyl
2051-24-
3
209
76
2
35
30
ng/wet g
2,3',4,4',5-
Pentachlorobiphenyl
31508-
00-6
118
458.7
2
35
30
ng/wet g
2,3',4,4'-
Tetrachlorobiphenyl
32598-
10-0
66
207.3
2
35
30
PCB
ng/wet g
3,3',4,4'-
Tetrachlorobiphenyl
32598-
13-3
77
95.2
2
35
30
ng/wet g
2,3,3',4,4'-
Pentachlorobiphenyl
32598-
14-4
105
121.4
2
35
30
ng/wet g
2,4'-Dichlorobiphenyl
34883-
43-7
8
60.3
2
35
30
ng/wet g
2,2',4,4',5,5'-
Hexachlorobiphenyl
35065-
27-1
153
621.6
2
35
30
ng/wet g
2,2',3,4,4',5'-
Hexachlorobiphenyl
35065-
28-2
138
402.3
2
35
30
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LU
Q.
ng/wet g
2,2',3,4,4',5,5'-
35065-
180
362.1
2
35
30
Heptachlorobiphenyl
| 29-3
ng/wet g
2,2',3,3',4,4',5-
| 35565.
170
154
| 2
| 35
30
Heptachlorobiphenyl
| 30-6
ng/wet g
2,2',5,5'-
35693-
52
471
2
35
30
Tetrachlorobiphenyl
99-3
ng/wet g
2,2',5-
37680-
18
113.1
2
1 35
1 30
Trichlorobiphenyl
65-2
ng/wet g
2,2',4,5,5'-
37680-
101
393.5
2
35
30
Pentachlorobiphenyl
73-2
ng/wet g
2,3,3',4,6'-
38380-
110
291.4
| 2
35
30
Pentachlorobiphenyl
03-9
ng/wet g
2,2',3,3',4,4'-
38380-
128
86.2
2
35
30
Hexachlorobiphenyl
07-3
ng/wet g
2,2',3,3',4,4',5,5',6-
| 401.86-
206
84.5
2
35
1 30
Nonachlorobiphenyl
| 72-9
ng/wet g
2,2',3,5'-
41464-
44
104.2
2
35
30
Tetrachlorobiphenyl
39-5
ng/wet g
2,2',3,4',5,5',6-
1 52663-
187
243
2
35
30
Heptachlorobiphenyl
| 68-0
ng/wet g
2,2',3,3',4,4',5,6-
| 52663-
195
83.8
| 2
35
30
Octachlorobiphenyl
| 78-2
ng/wet g
3,3',4,4',5-
1 57465-
1 126
87.2
2
35
30
Pentachlorobiphenyl
| 28-8
ng/wet g
2,4,4'-
7012-37-
| 28
339.3
| 2
35
30
Trichlorobiphenyl
5
ng/wet g
Heptachlor Epoxide
1024-57-
3
636.9
| 2
| 35
30
ng/wet g
Endosulfan Sulfate
1031-07-
8
503
2
35
1 30
ng/wet g
Hexachlorobenzene
118-74-1
401.7
2
35
30
ng/wet g
Mirex
| 2385-85-
| 5
357.6
2
35
30
ng/wet g
Oxychlordane
26880-
| 478.3
1 2
1 35
30
10
48-8
10
>-
ng/wet g
Aldrin
309-00-2
431.8
2
3
30
ng/wet g
Alpha-BHC
319-84-6
566.6
2
3
1 3"
<
ng/wet g
Beta-BHC
319-85-7
800.9
2
3
3'
+-»
c
ng/wet g
Delta-BHC
319-86-8
435.9
2
3
3'
ru
c
ng/wet g
Endosulfan II
| 33213-
| 462.1
2
35
| 30
E
I 65-9
ru
ng/wet g
2,4'-DDE
3424-82-
1 522.6
1 2
| 35
30
c
6
U
ng/wet g
Trans-Nonachlor
39765-
523.9
| 2
35
30
"D
80-5
<
ng/wet g
ng/wet g
4,4'-DDT
50-29-3
Alpha-Chlordane
5103-71-
9
ng/wet g Cis-Nonachlor
5103-73-
1
ng/wet g | 2,4'-DDD
ng/wet g Endrin Ketone
53-19-0
53494-
70-5
ng/wet g Gamma-Chlordane
ng/wet g
ng/wet g
Lindane
Dieldrin
5566-34-
7
58-89-9
60-57-1
754.8
566.3
384.8
668.3
644.9
593.7
559
557.3
35
35
35
35
35~
iif
35
30
30
30
30
30
30
30
30
00
c
10
10
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ng/wet g
Endrin
72-20-8
1735.2
2
35
30
ng/wet g
4,4'-DDD
72-54-8
666.9
2
35
30
ng/wet g
4,4'-DDE
72-55-9
1080
2
35
30
ng/wet g
Endrin Aldehyde
7421-93-
4
406.8
2
35
30
ng/wet g
Heptachlor
76-44-8
799.2
2
35
30
ng/wet g
2,4'-DDT
789-02-6
670
2
35
30
ng/wet g
Endosulfan 1
959-98-8
11943.4
2
35
30
** In the event for sample dilution is necessary to overcome the matrix effect, please notify EPA Laboratory Coordinator
6.7 Data Entry
Table 6.1, Table 6.2, Table 6.3, and Table 6.7 identify the required data elements that
laboratories must provide to EPA, preferably in EPA's data template, available separately from
EPA.
Table 6.7 Whole Body Fish: Data Elements for Each Sample
FIELD TYPE DESCRIPTION
SITE ID
Character
Site identification code or type of QC sample (e.g., LAB BLANK)
SAMPLE ID
Character
Sample number, LCS, BLANK, MS, or Rinsate
REPEAT
Numeric
Duplicate or Triplicate (otherwise blank)
DATE COL
MMDDYY
Date that the field crew collected the sample
ARRIVAL_TEMP
Numeric
Temperature of sample upon arrival at the laboratory (fish should be
frozen).
NUMBER FISH
Numeric
Number offish in the sample
SAMPLE WT
Numeric
Total weight of sample (all fish)
SAMPLE CLASS
Character
Sample classification: Routine or Non-routine
PER MOIST
Numeric
Moisture percentage offish
PERJ.IPID
Numeric
Lipid percentage based on lab proposed data quality objectives based or
standard operating procedures.
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 wrapping is cracked
L
Sample or wrapping is leaking
ML
Sample label is missing
NF
Sample is not at proper temperature
COND COMMENT
Character
Explanation for Q FLAG (if needed)
FISH_CODE
Character
Codes describing any deviations from the criteria for
fish collection for each sample
Flag
Definition
SP
Not all specimens are of the same species
LE
Not all specimen's lengths are within 75%
of longest fish
NS
Specimen number is fewer than minimum
of 5 or greater than 20 maxima
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PARAMETER
CAS_NO
LAB
METHOD
ANALYST
REVIEWER
INSTRUMENT
Character
DATE_PREPARED MMDDYY
DATE_ANALYSIS MMDDYY
QC_BATCH_LOT Character
HOLDING_TIME Y/N
MATRIX Character
MDL* N umeric
LRL N umeric
DILUTION N umeric
RECOVERY N umeric
RESULT N umeric
REASON Character
RESULT_QUAL Character
UNIT Character
QC_CODE Character
COMMENT Character
WT
LL
LS
Q
Mass does not meet minimum of 300 grams
Longest fish exceeds 400 mm maximum length
Shortest fish below 100 mm minimum length
Other quality concerns, not identified above
Character Analyte name
Character
Character
Character
CAS Registry number corresponding to the analyte
Laboratory name (abbreviation)
Laboratory method used
Character Last name or initials of person who performed the analysis
Character Last name or initials of the person who provided a separate
independent review of the data
Identification of instrument used for the analysis - provide
enough information to identify the particular instrument in the laboratory
Date that the sample homogenization started
Date that the sample analysis started
Unique laboratory quality control lot numbers assigned to the
batch of samples. The lot number must associate each batch of
field samples to the appropriate rinsates, laboratory control sample,
matrix spike, laboratory duplicate, and method blank samples.
Analysis performed within holding time
Fish
Lab method detection limit (based upon lab's historical data)
Lab reporting limit (based upon lab's historical data)
Dilution of sample (blank or 1 if no dilution)
Only for appropriate QC samples
Concentration value
Reason for qualification in RESULT_QUAL (usually blank)
Data qualifier (usually blank)
Unit of measurement for RESULT, MDL, and RL
Apply laboratory defined QC codes and describe in the comments field.
Provide set of laboratory codes as part of the case narrative
Explain situation that created QC code, or any unusual aspects of the
analysis
* In the event for sample dilution is necessary to overcome the matrix effect, please notify EPA Laboratory
Coordinator
6.8 Quality Measures
This section describes the quality assurance and quality control measures used to ensure that
the data will meet NCCA's requirements.
6.8.1 Assistance Visits
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 EPA and the laboratories. If EPA decides to conduct an assistance visit, a
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qualified EPA scientist or contractor will administer a checklist based upon the steps described
in this chapter.
6.8.2 Summary of QA/QC 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 Whole Body Fish: Quality Control Activities
QUALITY
DESCRIPTION AND REQUIREMENTS
CORRECTIVE ACTION
CONTROL
ACTIVITY
Demonstrate
competency for
analyzing fish
samples with the
required
methods
Demonstration of competency with fish
samples in achieving the method detection
limits, accuracy, and precision targets
EPA will not approve any
laboratory for NCCA sample
processing if the laboratory cannot
demonstrate competency. In other
words, EPA will select another
laboratory that can demonstrate
competency for its NCCA samples.
Check condition
of sample when
it arrives.
Sample issues, such as punctures or rips in
wrapping; missing label; temperature;
adherence to holding time requirements;
sufficient volume for test. All samples should
arrive at the laboratory in a frozen state.
Assign appropriate condition code
identified in Table 6.1.
Store sample
appropriately.
While stored at
the laboratory,
the sample must
be kept at a
maximum
temperature of
-20° C.
Check the temperature of the freezer per
laboratory's standard operating procedures.
Record temperature of sample
upon arrival at the laboratory. If at
any other time, samples are
warmer than required, note
temperature and duration in
comment field.
Determine if all
fish meet the
criteria
Evaluate if the sample contains fish of the
same species and are similar in size (within
75%) and provides enough material to run the
analysis.
Contact the EPA HQ NCCA
Laboratory Review Coordinator*
for a decision on fish selection
and/or chemical analysis.
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Analyze sample
within holding
time
The test must be completed within the holding
time (i.e., 28 days for mercury; 6 months for
other metals; and 1 year for all others). If the
original test fails, then the retest also must be
conducted within the holding time.
Perform test 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.
Perform once at
the start of each
batch to evaluate
the labeled
compound
recovery (LCR)in
a Laboratory
Control Sample
(LCS). This tests
the performance
of the equipment
Control limits for recovery cannot exceed
100±20%.
First, prepare and analyze one
additional LCS. If the second blank
meets the requirement, then no
further action is required. If the
second LCS fails, then determine
and correct the problem before
proceeding with any sample
analyses.
Perform once at Control limits cannot exceed the laboratory
the start of each reporting level (LRL)
batch to evaluate
the entire
extraction and
analysis process
using a Method
Blank
First, prepare and analyze one
additional blank. If the second
blank meets the requirement,
then no further action is required.
If the second blank fails, then
determine and correct the
problem (e.g., homogenization,
reagent contamination,
instrument calibration, or
contamination introduced during
filtration) before proceeding with
any sample analyses. Reestablish
statistical control by analyzing
three blank samples. Report
values of all blanks analyzed.
Check calibration
immediately
before and
immediately
after the sample
batch is run
(abbreviated as
QCCS for quality
control check
sample)
Results must be ±10% of each other or as If calibration fails before analysis,
specified in method criteria recalibrate and reanalyze QCCS
until it passes. If check fails after
all samples in the batch have been
analyzed, verify the QCCS reading.
If the QCCS reading fails a second
time, then reanalyze all samples in
the batch and report both sets of
results. For the first run, include a
data qualifier that indicates that
the QCCS reading taken
immediately following the first run
failed. For the second run, include
a data qualifier that indicates that
it is the second set and whether
the QCCS reading immediately
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following that second run
passed. No sample is to be
analyzed more than twice.
Evaluate rinsates
for first sample
in each batch.
This evaluation is
a surrogate for
assessing cross-
contamination.
Results must be below the LRL.
If first rinsate is above LRL, analyze
rinsatefrom a second sample. If
second rinsate sample also has
results above the LRL, then assign
a data qualifier to all samples in
the batch for the parameters with
results above the LRL in the
rinsates. Also, improve procedures
for cleaning all surfaces, knives,
and homogenization equipment
between samples.
Compare lipids in
triplicate for the
first sample in
each batch. This
evaluation is a
surrogate for
assessing
homogenization
Substitute the LRL for any value below the LRL
before calculating the RSD. If the RSD of the
triplicate results is <20%, then the
homogenization effort is judged to be
sufficient for all samples in the batch.
If the RSD could not be achieved,
then regrind all samples in the
batch one or more times as
described in Section 6.5.
Compare results
of one laboratory
duplicate sample
or matrix spike
duplicate sample
for each batch
Results must be within the target precision If both results are below LRL, then
goal in Table 6.6 (30% for all analytes). conclude that the test has passed.
Otherwise, prepare and analyze a
split from different sample in the
batch. If the second result is within
the target precision goal (see
Table 6.6) of the original sample,
then report the data and findings
for both QC samples. However, if
the two results differ by more
than the target precision goal,
review precision of QCCS
measurements for batch; check
preparation of split sample; etc.
and report evaluation and findings
in the case narrative. Consult with
the EPA HQ NCCA Laboratory
Review Coordinator* to determine
if reanalysis of the entire batch (at
the laboratory's expense) is
necessary. If no reanalysis is
necessary, report and quantify all
samples in batch. If reanalysis is
necessary, then report all QC
sample and the 2nd analysis of the
batch. If the second set also is
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Compare results
of one matrix
spike sample per
batch to evaluate
performance in
matrix
Evaluate performance after the first 3 batches.
Ideally, control limits for recovery will not
exceed the target accuracy goal (Table 6.6),
but this may not be realistic for all parameters
with this matrix.
unacceptable, then assign a data
code to each sample in the batch.
If both results are below LRL, then
conclude that the test has passed
for the batch. Otherwise, if any
results are not within the target
accuracy goal for the 3 batches,
within 2 working days, contact the
EPA HQ NCCA Laboratory Review
Coordinator* to discuss method
performance and potential
improvements. Continue to
perform the test for every batch.
Report the results from the
original analysis, the matrix spike,
matrix spike duplicate, and
%recovery.
Maintain the
required MDL
identified in the
Section 6.6
Evaluate for each sample
If MDL could not be achieved, then
provide dilution factor or QC code
and explanation in the comment
field.
Use consistent
units for QC
samples and field
samples
Verify that all units are provided in wet weight
units and consistently within each indicator
type as follows:
Metals in ng/g or ppm.
PCBs and pesticides in ng/g or ng/L.
If dry units are reported for any
sample (QC or field), reanalyze the
sample and report only the
reanalysis results. If it is not
possible to provide the results in
wet units, then assign a QC code
and describe the reason for dry
units in the comments field of the
database.
Maintain
completeness
Completeness objective is 95% for all
parameters.
Contact EPA HQ NCCA Laboratory
| Review Coordinator* immediately
if issues affect laboratory's ability
to meet completeness objective.
Section 2.2 provides contact information for the EPA HQ NCCA Laboratory Review
to EPA must contact the Task Order's Contracting Officer's Representative (TOCOR)
Coordinator.
Coordinator. Laboratories under contract
instead of the Laboratory Review
6.9 Sample and Record Retention
The laboratory shall retain:
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.
6.10 References
All references are from U.S. Environmental Protection Agency:
Guidance for Assessing Chemical Contaminant Data for Use in Fish Advisories, Volume 1 (Fish
Sampling and Analysis), 3rd Edition, 2000. Appendix J "Recommended procedures for preparing
whole fish composite homogenate samples". EPA #823-B-00-007. Retrieved March 12, 2020
from https://nepis.epa ¦gov/Exe/ZvPDF.cgi/200030MP.PDF?Dockev=20003QM P. PDF
Method 245.7 "Mercury in Water by Cold Vapor Atomic Fluorescence Spectrometry, Revision
2.0" (EPA-821-R-05-001, February 2005), retrieved March 12, 2020 from
https://nepis.epa.gov/Exe/ZyPDF.cgi/P1008IY8.PDF?Dockev=P1008IY8.PDF.
Method 3051A "Microwave Assisted Acid Digestion of Sediments, Sludges, Soils, and Oils,"
retrieved March 12, 2020 from https://www.epa.gov/sites/production/files/2015-
12/documents/3051a.pdf.
Method 6020A "Inductively Coupled Plasma-Mass Spectrometry" retrieved March 12, 2020
from https://19ianuarv2017snapshot.epa.gov/sites/production/files/2015-07/documents/epa-
6020a.pdf
Method 8270D "Semivolatile Organic Compounds by Gas Chromatography/Mass Spectrometry
(GC/MS) retrieved March 12, 2020 from
https://19ianuarv2017snapshot.epa.gov/sites/production/files/2015-
12/documents/8270d.pdf.
Method 9071B "n-Hexane Extractable Material (HEM) for Sludge, Sediment, And Solid
Samples," retrieved from https://www.epa.gov/sites/production/files/2015-
12/documents/9071b.pdf.
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7.0 SEDIMENT CONTAMINANT, GRAIN SIZE, AND TOC ANALYSES
This chapter describes the analysis requirements for sediment samples. The purpose is to
determine concentrations of contaminants, grain size, and total organic carbon (TOC) in
sediment samples collected in the 2020 NCCA and related studies. The laboratory shall perform
analysis to determine the moisture content, concentrations of metals, mercury, pesticides,
PAHs, and PCBs found in sediments in coastal waters and Great Lakes.
At each sampling site, the FOM instructs the crews to collect surficial sediment samples. The
field crew then ships the samples on wet ice to either its own state laboratory or EPA's batching
laboratory. Once the samples arrive, the laboratory will refrigerate the grain size samples and
freeze the TOC contaminant analysis samples.
7.1 Summary of the Procedure
This chapter describes the contaminant, grain size, and TOC determination of sediment samples
collected for EPA's 2020 NCCA. As described in Section 7.4, unless otherwise contractually
bound by other requirements, the laboratory may choose to use any method that meets EPA's
specifications for contaminant and grain size analyses.
7.2 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).
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.
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 procedure uses the following terms:
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Certified Reference Materials (CRM) are materials of various matrices for which
analytical information has been determined and certified by a recognized authority to provide a
quantitative assessment of the accuracy of an analytical method. CRMs provide evidence that
the laboratory preparation and analysis produces results that are comparable to those obtained
by an independent organization.
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.
Grain Size Classifications are broken down into three categories:
• Sand (0.0625 mm < 2.0 mm);
• Silt (0.0039 mm < 0.0625 mm);
• Clay (< 0.0039 mm)
Method Detection Limit the lowest level of analyte that can be distinguished from zero with 99
percent confidence based on a single measurement (Glaser et al., 1981) 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."
NARS: National Aquatic Resource Surveys. The National Coastal Condition Assessment (NCCA) is
part of the NARS program.
NARS Information Management System (NARS IM): The IM system established to support all
surveys, including NCCA, in the NARS program. The NARS IM system is used to track the
samples from field collection to the laboratory.
NCCA: National Coastal Condition Assessment. Freshwater (Great Lakes) and estuarine samples
will be collected during the field stage of NCCA.
Percent Recovery: Recovery is measured by comparing the concentrations of a sample split into
two aliquots; and one aliquot is spiked with a known concentration value. Cs is the
concentration measured in the spiked aliquot; C is the concentration measured in the unspiked
part aliquot; and s is the known concentration amount for the spike. The following equation is
used to calculate the percent recovery (Rs):
Relative Percent Difference (RPD): Relative percent difference compares the matrix spike (S)
and the matrix spike duplicate (D) using the following equation:
%Rs =
cs- c
x 100
s
RPD =
\S-D\
x 100
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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 detection limit. For diluted samples, the sample-specific detection limit will be the
product of the method 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.
TOC: Total Organic Carbon
TOCOR: Task Order Contracting Officer's Representative is EPA's contact person for
laboratories under contract to EPA.
7.3.2 General Requirements for Laboratories
Competency. To demonstrate its competency, the laboratory shall provide analyte and matrix
specific information to EPA. 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, showing competency for both
estuarine and Great Lake samples.
• Documentation detailing the competency of the organization, including professional
certifications for sediment-related analyses, membership in professional societies, and
experience with analyses for estuarine and Great Lakes sediments that are the same or
similar to the requirements of this method.
• Demonstration of competency with sediment samples from estuarine and freshwater
environments in achieving the method detection limits, accuracy, and precision targets.
Quality assurance and quality control requirements.
The organization shall provide EPA with copies of the quality-related documents relevant to the
procedure. Examples include QMPs, QAPPs, and applicable Standard Operating Procedures
(SOPs).
To demonstrate its ongoing commitment to maintaining data quality the person in charge of
quality issues for the organization shall sign the NCCA QAPP Certification Page.
7.3.3 Equipment/Materials
The analytical methods, selected by the laboratory, specify the required equipment.
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7.3.4 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. The laboratory must inspect the samples promptly
on receipt. As samples arrive, the laboratory must:
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 (see Section 2.2 for contact information).
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 from 21 9C (i.e., room
temperature) down to -20 9C or lower (i.e., the expected temperature of frozen
samples), or an infra-red (IR) temperature "gun" and record the reading. Field crews
ship sediment samples on wet ice; the batch laboratory freezes the organic
contaminants [or chemical] (SEDO) and TOC (SEDC) samples and ships with dry ice.
Record the condition and temperature of the sample in the database using the codes in
Table 7.1.
3. Verify that all required data elements, per Table 7.1, have been recorded. If any
elements are missing, then enter them into the database.
4. Transfer the chemical and TOC samples to the freezer for long-term storage. Except
during processing and analysis stages, the samples must be stored frozen to less than or
equal -20 °C.
5. Notify the EPA immediately about any problems involving sample integrity, conformity,
or inconsistencies as soon as possible following sample receipt and inspection.
Table 7.1: Sediment Chemistry, Grain Size, and TOC Login: Required Data Elements
FIELD
TYPE DESCRIPTION
SITE J D
Character
Site identification code
SAMPLEJD
Character
Sample number
DATE_COL
MMDDYY
Date that the field crew collected the sample
ANALYSIS_TYPE
Character
Contaminant (SEDO), TOC(SEDC), or Grain Size (SEDG)
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
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C
Sample container is cracked
L
Sample or container is leaking
ML
Sample label is missing
Q
Other quality concerns, not identified
above
COND_COMMENT
Character
Explanation for Q FLAG (if needed)
7.4 Laboratory Analysis: Requirements
The laboratory shall perform analysis of the sediment samples to determine the moisture
content, grain size, and concentrations of TOC, metals, mercury, pesticides, PAHs, and PCBs.
Table 7.2 identifies the storage requirements. Laboratories may choose to use any analysis
method, including those in Table 7.2, which measures the parameters to the levels of the
method detection limits identified in Table 7.3. In addition, the contaminant analysis method
must meet the precision and accuracy targets of 30% and 20%, respectively. For each batch of
contaminant samples, precision is assessed using the RPD between the matrix spike (MS) and
the matrix spike duplicate (MSD); and accuracy by the average percent recovery (%Rs) between
the matrix spike and matrix spike duplicate. Section 7.3.1 provides the equations used to
calculate the RPD and %Rs. The precision and accuracy targets for each batch of TOC are both
10% and determined by the RPD of one sample and its duplicate (for precision) and the analysis
of Certified Reference Material (CRM; for accuracy). The grain size target precision is 10% as
determined using a Laboratory Control Sample (LCS) (accuracy is not applicable).
Table 7.2 Sediment Chemistry, Grain Size, and TOC: Analytical Methods
STORAGE
REQUIREMENTS
TYPE
METHODS THAT MEET THE QA/QC
REQUIREMENTS (ANY METHOD
THAT MEETS THE QA/QC
REQUIREMENTS IS
ACCEPTABLE)
Freeze samples with
maximum of -20° C
Metals (except Mercury)
Extraction: EPA Method 3051A
Analysis: EPA Method 6020A14
Mercury
EPA Method 245.715
PCBs, Pesticides, PAHs
Extraction: EPA Method 3540C
14 For example, see:
• Method 3051A "Microwave Assisted Acid Digestion of Sediments, Sludges, Soils, And Oils" retrieved
November 13, 2018 from https://www.epa.gov/sites/production/files/2015-12/documents/3051a.pdf;
and
• Method 6020A "Inductively Coupled Plasma-Mass Spectrometry" retrieved April 28, 2018 from
https://www.epa.gov/sites/production/files/2015-07/documents/epa-6Q20a.pdf.
15 For example, see Method 245.7 "Mercury in Water by Cold Vapor Atomic Fluorescence Spectrometry, Revision
2.0" (EPA-821-R-05-001, February 2005), retrieved March 13, 2019 from
https://www.nemi.gov/methods/method summary/9629/.
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Analysis: EPA Method 8270D16
TOC
USEPA Method 9060
Refrigerate at 4° C
(do not freeze)
Grain Size
Any method that reports the
determination as percent silt, sand
and clay and meets QA/QC
requirements
Table 7.3 Sediment Chemistry, Grain Size, and TOC: Required Parameters
TYPE
UNITS
CAS
PARAMETER
PCB NUMBER
MAX
MDL
TARGET
TARGET
NUMBER
(WHERE
APPLICABLE)
CONC
BASED
UPON
2010 AND
2015
DATA
TARGET*
ACCURACY
PRECISION
% silt,
% sand,
% clay
Grain Size
0.05%
10% (LCS)
mg/kg
or%
Total Organic
Carbon
54.5
0.01%
10%
10%
dry
7429-90-5
Aluminum
162000
1500
20
30
weight
7440-36-0
Antimony
38.1
0.2
20
30
Mg/g,
7440-38-2
Arsenic
147.61
1.5
20
30
(ppm)
7440-43-9
Cadmium
9.9
0.05
20
30
7440-47-3
Chromium
1078.78
5
20
30
7440-50-8
Copper
2290
5
20
30
7439-89-6
Iron
169000
500
20
30
£
7439-92-1
Lead
461
1
20
30
LU
7439-96-5
Manganese
6587.02
1
20
30
7439-97-6
Mercury
3.12
0.01
20
30
7440-02-0
Nickel
360.17
1
20
30
7782-49-2
Selenium
121.019
0.1
20
30
7440-22-4
Silver
35.34
0.3
20
30
7440-31-5
Tin
258
0.1
20
30
7440-62-2
Vanadium
4734
1
20
30
7440-66-6
Zinc
1750
2
20
30
>CB
dry
weight
ng/g,
(ppb)
2051-24-3
2,2',3,3',4,4',5,5'
,6,6'-
Decachlorobiph
enyl
209
22.4
1
20
30
34883-43-
7
2,4'-
Dichlorobipheny
1
8
10.7
1
20
30
16 For example, see:
• Method 3540C "Soxhlet Extraction" retrieved November 8, 2018 from
https://www.epa.gov/sites/production/files/2015-12/documents/3540c.pdf; and
• Method 8270D "Semivolatile Organic Compounds by Gas Chromatography/Mass Spectrometry (GC/MS)
retrieved March 13, 2019 from https://www.epa.gov/sites/production/files/2015-07/documents/epa-
8270d.pdf.
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35065-30-
6
2,2',3,3',4,4',5-
Heptachlorobip
henyl
170
115.4
20
30
52663-68-
0
35065-29-
3
38380-07-
3
35065-28-
2
35065-27-
1
40186-72-
9
52663-78-
2
32598-14-
4
_____
2
31508-00-
6
38380-03-
9
57465-28-
41464-39-
5
32598-13-
3
35693-99-
3
32598-10-
0
37680-65-
2
7012-37-5
2,2',3,4',5,5',6-
Heptachlorobip
henyl
2,2',3,4',5,5',6-
Heptachlorobip
henyl
2,2',3,3',4,4'-
Hexachlorobiph
enyl
2,2',3,4,4',5'-
Hexachlorobiph
enyl
2,2',4,4',5,5'-
Hexachlorobiph
enyl
,6-
Nonachlorobiph
enyl
2,2',3,3',4,4',5,6-
Octachlorobiphe
nyl
2,3,3',4,4'-
Pentachlorobiph
enyl
"2^75^1
Pentachlorobiph
enyl
187
180
128
138
153
206
195
105
101
56.8
249.4
61.3
362
168.7
75.5
40
78.2
256
2,3,4,4',5-
Pentachlorobiph
enyl
118
201
2,3,3',4,6'-
Pentachlorobiph
enyl
3,3',4,4',5-
Pentachlorobiph
enyl
2,2',3,5'-
Tetrachlorobiph
enyl
3,3',4,4'-
Tetrachlorobiph
enyl
110
126
44
77
249
3.5
54.3
2,2',5,5'-
Tetrachlorobiph
enyl
52
123
2,3',4,4'-
Tetrachlorobiph
enyl
Trichlorobiphen
yi
2,4,4'-
Trichlorobiphen
yi
66
18
28
36.6
18.4
39.5
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
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dry
| 53-19-0
2,4'-DDD
| 10.2 | 1
| 20
| 30
weight
3424-82-6
2,4'-DDE
6.4 1
20
3
ng/g,
789-02-6
2,4'-DDT
114.1 1
20
3
(ppb)
72-54-8
4,4'-DDD
100.6 1
20
3
72-55-9
4,4'-DDE
29.5 1
20
3
50-29-3
4,4'-DDT
59.3 1
20
3
309-00-2
Aldrin
1 13.3 1
20
3
319-84-6
Alpha-BHC
| #N/A 1 1
1 20
3
319-85-7
Beta-BHC
| 510.4 1 1
1 20
3
319-86-8
Delta-BHC
| 7.2 1
1 20
3
5103-71-9
Alpha-
| 3.7 | i
1 20
30
Chlordane
5566-34-7
Gamma-
5.1 1
20
30
Chlordane
60-57-1
Dieldrin
1 2.3 1
1 20
1 30
959-98-8
Endosulfan 1
| #N/A 1 1
1 20
3'
33213-65-
Endosulfan II
| 212 | I
| 20
30
LU
Q.
<
Q.
dry
weight
ng/g,
(ppb)
39765-80-
5
83-32-9
208-96-8
120-12-7
56-55-3
205-99-2
207-08-9
191-24-2
50-32-8
192-97-2
92-52-4
218-01-9
132-65-0
1031-07-8
72-20-8
7421-93-4
53494-70-
5
76-44-8
1024-57-3
Endosulfan
Sulfate
Endrin
118-74-1
58-89-9
2385-85-5
5103-73-1
26880-48-
Endrin Aldehyde
Endrin Ketone
Heptachlor
Heptachlor
Epoxide
Hexachlorobenz
ene
Lindane
Mirex
Cis-Nonachlor
Oxychlordane
Trans-Nonachlor
Acenaphthene
Acenaphthylene
Anthracene
Benz(a)anthrace
ne
Benzo(b)fluoran
thene
Benzo(k)fluoran
thene
Benzo(g,h,i)pery
lene
Benzo(a)pyrene
Benzo(e)pyrene
Bi phenyl
Chrysene
Dibenz(a,h)anth
racene
Dibenzothiophe
ne
8.1
13.2
#N/A
#N/A
5.3
3.5
173.7
163.3
9.1
1.9
13.4
3.6
1437.9
1530
4343
#N/A
11125.6
8530.9
#N/A
10158.6
#N/A
#N/A
13600
_____
1000
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
10
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581-42-0
2,6-
Dimethylnaphth
alene
283.7
1
20
30
206-44-0
Fluoranthene
38970
1
20
30
86-73-7
Fluorene
1594.8
1
20
30
193-39-5
lndeno(l,2,3-
c,d)pyrene
6615.5
1
20
30
90-12-0
1-
Methylnaphthal
ene
487
1
20
30
91-57-6
2-
Methylnaphthal
ene
430.5
1
20
30
832-69-9
1-
Methylphenant
hrene
903
1
20
30
91-20-3
Naphthalene
694
1
20
30
198-55-0
Perylene
2157
1
20
30
85-01-8
Phenanthrene
20000
1
20
30
129-00-0
Pyrene
30000
1
20
30
2245-38-7
2,3,5-
Trimethylnapht
halene
411
1
20
30
* In the event for sample dilution is necessary to overcome the matrix effect, please notify EPA Laboratory
Coordinator
7.5 Data Entry
Table 7.4 identifies the required data elements that laboratories must provide to EPA,
preferably in EPA's data template, available separately from EPA. If the laboratory applies its
own QC codes, the data transmittal must define the codes.
Table 7.4 Sediment Chemistry, Grain Size, and TOC: Data Elements for Each Sample
FIELD
TYPE
DESCRIPTION
SITE J D
Character
Site identification code or type of QC
sample (e.g., LAB BLANK)
SAMPLE J D
Character
Sample identification code
VISIT_NO
Numeric
Sequential Visits to site (1 or 2)
SAM_CODE
Character
REGULAR, DUP LCS, Blank, MS or CRM
DATE_COL
MMDDYY
Date that the field crew collected the
sample
SAMPLE_TYPE
Character
Metals (except Mercury); Mercury; PCBs,
Pesticides, PAHs; TOC; GRAIN SIZE
ARRIVAL_TEMP
Numeric
Temperature of sample upon arrival at the
laboratory
REPEAT
Numeric
Identifies a duplicate run of a sample: 1 or
2. Do not enter a number if no duplicate
of sample is analyzed.
CONDITION_CODE
Character
Condition codes describing the condition
of the sample upon arrival at the
laboratory; leave blank for control
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COND_COMMENT
PARAMETER
CAS NO
LAB NAME
METHOD
ANALYST
REVIEWER
INSTRUMENT
HOLDING TIME
DATE ANALYZED
QC_BATCH_LOT
MATRIX
MDL*
RL
MOISTURE
MOIST UNIT
Character
Character
Character
Character
Character
Character
Character
Character
Y/N
MMDDYY
Character
Character
Numeric
Numeric
Numeric
Character
Flag
OK
C
L
ML
VT
VR
Q
Definition
Sample is in good condition
, Sample container is cracked
i Sample or container is leaking
j Sample label is missing
i Volume not sufficient for
i testing
j Volume not sufficient for a
j retest, if required
Other quality concerns, not
identified above
Explanation for Q FLAG (if needed)
Analyte name
CAS Registry number
Laboratory name (abbreviation)
Laboratory method used
Last name or initials of person who
performed the analysis
Last name or initials of the person who
provided a separate independent review
of the data
Identification of instrument used for the
analysis - provide enough information to
identify the particular instrument in the
laboratory
Analysis performed within holding time
Date that the analysis started
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
(LCS), matrix spike (MS), laboratory
duplicate, method blank (BLANK), and
CRM samples.
Sediment (Water also is a permissible
value if the laboratory analyzes a very
liquid sediment sample as water)
Lab method detection limit (based upon
lab's historical data)
Actual Reporting limit (based on the
unique matrix of sediment for each batch
of samples)
Moisture in the sample (value used by lab
to convert wet units to dry)
Unit used to report moisture (% or mg/kg)
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DILUTION
Numeric
Dilution of sample (blank or 1 if no
dilution)
RECOVERY
Numeric
Only for appropriate QC samples
RESULT
Numeric
Concentration value
RESULT_UNIT
Character
Unit of measurement for RESULT, MDL,
and RL
QC_CODE
Character
Apply laboratory defined QC codes and
describe in the comments field. Provide
set of laboratory codes as part of the case
narrative
COMMENT
Character
Explain situation that created QC code, or
any unusual aspects of the analysis
* In the event for sample dilution is necessary to overcome the matrix effect, please notify EPA Laboratory
Coordinator
7.6 Quality Measures
This section describes the quality assurance and quality control measures used to ensure that
the data will meet NCCA's requirements.
7.7 Assistance Visits
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 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.
7.7.1 Summary of QA/QC 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, matrix
spike duplicate 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 given a unique sample identification. Table 7.5 provides a
summary of the quality control requirements.
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samples to meet the
performance measures
Check condition of
sample when it arrives.
Store sample
appropriately. While
stored at the laboratory,
the sample must be kept
at a temperature <-20° C
except jars for grain
analyses are refrigerated
at 4°C.
Perform once at the start
of each batch to
evaluate the labeled
compound recovery
(LCR) in a Laboratory
Control Sample (LCS).
This tests the
performance of the
equipment.
Perform once at the start
of each batch to
evaluate the entire
extraction and analysis
process using a Method
Blank.
limits, accuracy, and precision
targets.
Sample issues such as cracked
container; missing label;
sufficient volume for test.
Check the temperature of the
refrigerator/freezer and
refrigerator per laboratory's
standard operating procedures.
Analyze sample within
holding time.
The test must be completed
within the holding time of 1 year.
If the original test fails, then the
retest also must be conducted
within the holding time.
Control limits for recovery
cannot exceed 100±20%.
Control limits cannot exceed the
laboratory reporting level (LRL).
competency. In other words, EPA will
select another laboratory that can
demonstrate competency for its
NCCA samples.
Assign appropriate condition code
identified in Table 7.4.
Record temperature of sample upon
arrival at the laboratory. If at any
other time, samples are warmer than
required, note temperature and
duration in comment field.
Data analyst will consider
temperature deviations in evaluating
the data. He/she will flag the
deviations and determine whether
the data appear to be affected and/or
the data should be excluded from the
analyses.
Perform test 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.
First, prepare and analyze one
additional LCS. If the second blank
meets the requirement, then no
further action is required. If the
second LCS fails, then determine and
correct the problem before
proceeding with any sample analyses.
First, prepare and analyze one
additional blank. If the second blank
meets the requirement, then no
further action is required. If the
second blank fails, then determine
and correct the problem (e.g.,
contamination, instrument
calibration) before proceeding with
any sample analyses. Reestablish
statistical control by analyzing three
blank samples. Report values of all
blanks analyzed.
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Check calibration
immediately before and
immediately after the
sample batch
(abbreviated as QCCS for
quality control check
sample).
Compare results of one
laboratory duplicate
sample (for TOC) or
matrix spike duplicate
sample (for
contaminants) for each
batch (not required for
grain size).
Compare results of one
matrix spike sample per
batch to evaluate
performance in matrix
(not required for TOC
and grain size).
Results must be ±10% of each
other or as specified in method
criteria.
Results must be within the target
precision goal in Table 7.3.
Evaluate performance after the
first 3 batches; and then every
subsequent batch. Ideally,
control limits for recovery will
not exceed the target accuracy
goal, but this may not be realistic
for all parameters with this
matrix.
If calibration fails before analysis,
recalibrate and reanalyze QCCS until
it passes. If check fails after all
samples the batch have been
analyzed, verify the QCCS reading. If
the QCCS reading fails a second time,
then reanalyze all samples in the
batch and report only the set of
results associated with the acceptable
QCCS reading. Also report all QCCS
readings for the batch.
If both results are below LRL, then
conclude that the test has passed.
Otherwise, prepare and analyze a
split from different sample in the
batch. If the second result is within
the target precision goal (see Section
7.5) of the original sample, then
report the data and findings for both
QC samples. However, if the two
results differ by more than the target
precision goal, review precision of
QCCS measurements for batch; check
preparation of split sample; etc. and
report evaluation and findings in the
case narrative. Consult with the EPA
HQ NCCA Laboratory Review
Coordinator to determine if reanalysis
of the entire batch (at the
laboratory's expense) is necessary. If
no reanalysis is necessary, report and
quantify all samples in batch. If
reanalysis is necessary, then report all
QC sample and the 2nd analysis of the
batch. If the second set also is
unacceptable, then assign a data code
to each sample in the batch.
If both the original and duplicate
results are below LRL, then conclude
that the test has passed for the batch.
Otherwise, if any results are not
within the target accuracy goal for
the first 3 batches, within 2 working
days, contact the EPA HQ NCCA
Laboratory Review Coordinator to
discuss method performance and
potential improvements. After
achieving acceptable results or EPA's
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permission to continue, perform the
test for every subsequent batch. For
each batch, report the results from
the original analysis and its duplicate
and their RPD for TOC; the matrix
spike, matrix spike duplicate, RPD and
%recovery for contaminants.
Compare results of TOC
Certified Reference
Material once per each
batch
Value must be within 10% of the If value is outside the acceptable
certified value. range, analyze a second CRM. If the
second CRM also is measured outside
the acceptable range, then determine
and correct the problem (e.g.,
contamination, instrument
calibration) before reanalyzing all
samples in the batch.
Maintain the required
MDL identified in Section
7.4
Evaluate for each sample If MDL could not be achieved, then
provide dilution factor or QC code
and explanation in the comment field.
Participate in External
Quality Control
Evaluate QC samples provided by Based upon the evaluation, the
the External QC Coordinator. 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.
Maintain completeness
Completeness objective is 95% Contact EPA HQ NCCA Laboratory
for all parameters. Review Coordinator immediately if
issues affect laboratory's ability to
meet completeness objective.
*Chapter 2.0 provides contact information for the EPA HQ NCCA 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.
7.8 Sample and Record Retention
The laboratory shall retain:
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
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used in the contaminant and TOC analyses and refrigerate those used for grain size. The
laboratory shall periodically check the sample materials for degradation.
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
All references are from U.S. Environmental Protection Agency:
Method 245.7 "Mercury in Water by Cold Vapor Atomic Fluorescence Spectrometry, Revision
2.0" (EPA-821-R-05-001, February 2005), retrieved March 13, 2019 from
https://www.nemi.gov/methods/method summary/9629/.
Method 3051A "Microwave Assisted Acid Digestion of Sediments, Sludges, Soils, And Oils"
retrieved November 13, 2018 from https://www.epa.gov/sites/production/files/2015-
12/documents/3051a.pdf
Method 6020A "Inductively Coupled Plasma-Mass Spectrometry" retrieved March 12, 2020
from
https://www.nemi.gov/methods/method summary/9186/#:~:text=Method%206020%20descri
bes%20the%20multi.gas%20into%20the%20plasma%20torch.
Method 8270D "Semivolatile Organic Compounds by Gas Chromatography/Mass Spectrometry
(GC/MS) retrieved March 12, 2020 from
https://19ianuarv2017snapshot.epa.gov/sites/production/files/2015-07/documents/epa-
8270d.pdf
SW- 846 Method 3540C "Soxhlet Extraction" retrieved November 8, 2018 from
https://www.epa.gov/sites/production/files/2015-12/documents/3540c.pdf
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8.0 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 and chlorophyll a in water quality
samples collected in the 2020 NCCA and related studies. The laboratory shall perform analysis
to determine levels of ammonia (NH3), nitrite (NO2) (optional), nitrate plus nitrite (NO3 plus
NO2), total nitrogen (TN), total phosphorus (TP) and ortho-phosphate (PO4) (also called soluble
reactive phosphorus (SRP) or dissolved inorganic phosphorus (DIP)), pH, conductivity (required
for Great Lakes, optional for estuaries) and chlorophyll a found in coastal waters and Great
Lakes. In addition, the laboratory shall measure chloride (CI) and sulfate (SO4) levels in Great
Lakes samples, and measure salinity in estuarine samples (optional).
8.1 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).
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.
8.2 Definitions and Required Resources (Personnel, Laboratories, and Equipment)
This section provides definitions and required resources for using the procedure.
8.2.1 Definitions
The procedure uses the following terms:
CI: Chloride
Method 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."
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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.
NARS: National Aquatic Resource Surveys. The National Coastal Condition Assessment (NCCA) is
part of the NARS program.
NARS Information Management System (NARS IM): The IM system established to support all
surveys, including NCCA, in the NARS program. The NARS IM system is used to track the
samples from field collection to the laboratory.
NCCA: National Coastal Condition Assessment. Freshwater and coastal samples will be collected
during the field stage of NCCA.
NH3: Ammonia
NO2: Nitrite
NO3: Nitrate
NOb Plus N02: Nitrate plus 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):
%Rs =
Cs- C
x 100
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, S, as follows:
5 =
n
n
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:
5
RSD = -= X 100
C
Reporting Limit: A reporting limit is the point at which the measured value of the analyte can
be reported with confidence.
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Sample-Specific Detection Limit: Most samples will have a sample-specific detection equal to
the method detection limit. For diluted samples, the sample-specific detection limit will be the
product of the method detection limit and the dilution factor. Typical values for the dilution
factors will be 10 or 100.
SO4: Sulfate.
Spiked Sample: See Percent Recovery definition for purpose of spiked samples.
SRP: Soluble Reactive Phosphorus (also called orthophosphate or dissolved inorganic
phosphate)
TN: Total nitrogen
TP: Total phosphorus
8.2.2 General Requirements for Laboratories
Expertise. To demonstrate its competency/expertise, the laboratory shall provide EPA with
performance data demonstrating their proficiencies in analyzing water quality samples. In
addition, the laboratory must provide one or more of the following:
• 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.
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 QMPs, Laboratory Quality Assurance Manuals, 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 NCCA QAPP Certification Page.
8.2.3 Equipment/Materials
The analytical method, selected by the laboratory, identifies the necessary equipment.
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8.3 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. For each sampled site, the lab will receive the
following samples on wet ice:
• One 250 ml amber bottle labeled 'CHEM' for water chemistry analyses
• A filter in a 50 ml tube for chlorophyll a labeled 'CHLA'
Additionally, as a separate batch shipment the lab will receive 250 ml bottles labeled 'NUTS' for
dissolved nutrients analyses (either from the crews or from an EPA batching laboratory). Crews
and the batch lab will maintain these samples frozen but will ship overnight on wet ice.
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 (see Chapter 2 for contact information).
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 8.1.
3. Verify that all required data elements, per Table 8.1, 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 8.4
and then are stored in a refrigerator at 4° C.
b. Chlorophyll-o 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 °C.
c. Dissolved nutrient samples are prepared following the requirements in Section
8.4 and then are stored in a refrigerator at 4 °C.
5. Notify the EPA immediately about any problems involving sample integrity, conformity,
or inconsistencies as soon as possible following sample receipt and inspection.
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Table 8.1 Water Chemistry Login: Required Data Elements
FIELD
TYPE
DESCRIPTION
SITE ID
Character
Site identification code
SAMPLE ID
Character
Sample number
DATE COL
MMDDYY
Date that the field crew collected the sample
ANALYSIS_TYPE
Character
Water Chemistry or Chlorophyll a or Nutrients
ARRIVAL_TEMP
Numeric
Temperature of sample upon arrival at the laboratory
(CHEM, CHLA and NUTS sample 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
COND_COMMENT
Character
Explanation for Q FLAG (if needed)
8.4 Preparation of Water Chemistry Aliquots
Figure 8.1 presents the sample preparation processing steps for the water chemistry indicators,
including filtering and acidifying.
For the dissolved nutrient (NUTS) sample, the laboratory technician:
1. Thaws the frozen sample.
2. Splits the sample into two aliquots as shown in Figure 8.1.
3. Adds ultra-pure acid (H2SO4, depending on the analytes, see Table 8.2) to one of the two
aliquots. Caps the bottle tightly and inverts the bottle several times to mix.
4. Stores all aliquots in a refrigerator at 4 °C.
For the unfiltered, water chemistry (CHEM) sample, the laboratory technician
1. Thaws the frozen sample.
2. Splits the sample into two aliquots as shown in Figure 8.1.
3. Adds ultra-pure acid (H2SO4,) to one aliquot of the unfiltered, CHEM sample. Caps the
bottle tightly and inverts the bottle several times to mix.
4. Stores all aliquots in a refrigerator at 4 °C.
If the dissolved nutrient sample is compromised in some way, the laboratory technician will
filter a new sample from the water chem (CHEM) sample as follows:
1. Uses 0.4 |am pore size polycarbonate filters for all filtration.
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2. Rinses 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. Places 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. After all filtered and unfiltered aliquots are collected, adds ultra-pure acid (H2SO4,
depending on the analyte, see Table 8.2) to the sample in the aliquot container. Cap
tightly and invert the bottle several times to mix.
5. Stores all aliquots in a refrigerator at 4 °C.
Figure 8.1 Water Chemistry and Dissolved Nutrient Samples: Receipt and Holding Times
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Table 8.2 Water chemistry: acid preservatives added for various indicators
PRESERVATIVES
H2S04USED FOR:
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INDICATORS NH4
TOTAL N
TOTAL P
N02 plus N03
8.5 Water Chemistry and Chlorophyll a Analysis: Requirements
The laboratory shall perform the following analyses on all samples:
• ammonia (NH3) • ortho-phosphate (PO4)
• nitrate plus nitrite (NO3 plus NO2) • total phosphorus (TP), and
• nitrite (NO2) (optional) • chlorophyll a
• total nitrogen (TN), • pH
The laboratory shall perform the following analyses on only the samples indicated:
• conductivity (Great Lakes-required) • chloride (CI) (Great Lakes samples only)
• conductivity (estuaries-optional) • sulfate (SO4) (Great Lakes samples only)
• salinity (estuarine samples only,
optional)
As an alternative to specifying laboratory methods for sample analysis, NCCA uses a
performance-based approach that defines a set of laboratory method performance
requirements for data quality as shown in Table 8.3. Method performance requirements for
this project identify the method detection limit, precision, and accuracy objectives for each
parameter. NCCA 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
reporting limit value for each analyte to double the long-term method detection limit (LT-MDL),
following guidance presented in Oblinger, Childress et al. (USGS, 1999) and EPA document 821-
R-16-006 (EPA, 2016).
NCCA 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 lower, midrange and higher pH levels.
For duplicate samples, NCCA estimates the precision as the pooled standard deviation
(calculated as the root-mean square) of all samples at the lower concentration range, and as
the pooled percent relative standard deviation of all samples at the higher concentration range.
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
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the lower concentration range measured across sample batches, and as the percent difference
at the higher concentration range.
Table 8.4 summarizes the analytical methods used at the NCCA central laboratory (EPA ORD-
Corvallis). Other 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 8.3. Appendix G: Laboratory Remote
Evaluation Forms identifies the information that the laboratory should provide to the NCCA
Laboratory Review Coordinator to use in determining whether the laboratories meet the
necessary requirements.
Table 8.3 Water Chemistry and Chlorophyll-cr: Laboratory Method Performance Requirements
PARAMETER
UNITS
POTENTIAL
METHOD
TRANSITIO
PRECISION
ACCURACY
RANGE
DETECTION
N
OBJECTIVE
OBJECTIVE
OF
LIMIT
VALUE3
4
5
SAMPLES1
OBJECTIVE2
Ammonia (NH3)
mg
N/L
Oto 17
0.01 marine
(0.7 neq/L)
0.02
freshwater
0.10
± 0.01 or
±10%
± 0.01 or
±10%
Chloride (CI)
mg
Cl/L
0 to 5,000
0.20 (6
Heq/L)
1
± 0.10 or
±10%
± 0.10 or
±10%
Conductivity*
US/cm
at
25°C
1-66,000
1.0
20
±2 or ±10%
±2 or ± 5%
Nitrate-Nitrite
(NO3-NO2)
mg
N/L
0 to 360
(as nitrate)
0.01 marine
0.02
freshwater
0.10
± 0.01 or
±10%
± 0.01 or
±10%
Salinity*
psu
0-40
pH
(Laboratory)
Std
Units
3.5-10
N/A
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
Total Nitrogen
(TN)
mg
N/L
0.1 to 90
0.01
0.10
± 0.01 or
±10%
± 0.01 or
±10%
Total
Phosphorus
(TP) and
Ortho-
Phosphate
mg P/L
Oto 22
(as TP)
0.002
0.02
±0.002
Or ±10%
± 0.002
Or ±10%
Nitrate (N03)*
mg
N/L
0. to 360
0.01 marine
(10.1 neq/L)
0.03
freshwater
0.1
± 0.01 or
±5%
± 0.01 or
±5%
Sulfate (S04)
mg/L
0 to 5000
0.5
freshwater
2.5
±0.25 or
±10%
±0.25 or
±10%
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(10.4 ueq/L)
Chlorophyll-a
|a,g/L in
Extract
0.7 to
11,000
1.5
15
± 1.5 or
±10%
± 1.5 or
±10%
1 Estimated from samples analyzed 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 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. For pH precision, the looser criteria applies to mid-range
samples. For NCCA, that is less of a concern than the ability to measure more acidic or basic samples
accurately and precisely.
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.
* Parameter reporting is preferred, but not required.
Table 8.4 Water Chemistry and Chlorophyll-a: Analytical Methods Used by Central
Laboratory, EPA ORD-Corvallis)
ANALYTE SUMMARY OF METHOD17 REFERENCES18 WRS SOP19
Nitrate+Nitrite, as N
Nitrate as N*
Ion Chromatography
(freshwater samples)
OR
FIA automated colorimetric
(cadmium reduction for
brackish samples)
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)
Ammonia, as N
FIA automated colorimetric
(salicylate,
dichloroisocyanurate)
Lachat 10-107-06-
3-D
WRS 30A.4
(April 2011)
17 FIA=Flow injection analysis. AAS=Atomic Absorption Spectrometry
18 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.
19 WRS= Willamette Research Station. References are to laboratory SOP being used at central laboratory. Available
upon request from the EPA HQ Laboratory Review Coordinator.
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Total nitrogen
(TN)
Persulfate Digestion; FIA
Automated Colorimetric
Analysis (Cadmium
Reduction, sulfanilamide)
EPA 353.2
(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) and ortho-
Phosphate
Persulfate Digestion;
Automated Colorimetric
Analysis (molybdate,
ascorbic acid)
APHA 4500-P-E
USGS 1-4650-03
U.S. EPA (1987)
Lachat 115-01-1-B
(modified)
WRS 34A.5
(April 2011)
Chloride, Sulfate
Ion Chromatography (Great
Lakes samples only)
SW-846 9056A;
APHA 4110B
WRS 40A.5
(May 2011)
Chlorophyll-a
(Chl-a)
Extraction 90% acetone
analysis by fluorometry
EPA 445.0, EPA
446.0
WRS 71A.3
(April 2011)
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)
Specific
conductance @
25°C*
Electrolytic, Man-Tech
TitraSip automated analysis
OR manual analysis,
electrolytic
EPA 120.6
WRS 16A.0
(April 2011)
WRS 11A.4
(April 2011)
*Analyte is preferred, but not required.
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8.6 Data Entry
Table 8.5 identifies the required data elements that laboratories must provide to EPA,
preferably in EPA's data template, available separately from EPA.
Table 8.5 Water Chemistry and Chlorophyll-cr: Data Elements for Each Sample
VARIABLE TYPE DESCRIPTION
SITE ID
Character
Site identification code or type of QC sample (e.g., LAB BLANK)
SAMPLE
Character
Sample number, LCS, QCCS, Blank, MS, or CRM
ANALYSIS TYPE
Character
Contaminant
REPEAT
Numeric
Duplicate
DATE COL
MMDDYY
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
COND 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 LCS, MS, laboratory duplicate, BLANK, and
CRM sampl
es.
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)
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RESULT
Numeric
Concentration value
REASON
Character
Reason for qualification in RESULT_QUAL (usually blank)
RESULT_QUAL
Character
Data qualifier (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 codes as part of the case narrative
COMMENT
Character
Explain situation that created QC code, or any unusual aspects
of the analysis
8.7 Quality Measures
This section describes the quality assurance and quality control measures used to ensure that
the data will meet NCCA'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 8.6 provides a
summary of the quality control requirements.
Table 8.6 Water Chemistry and Chlorophyll-cr: Quality control activities for water quality
samples
QC SAMPLE INDICATORS DESCRIPTION FREQUENCY
TYPE AND
DESCRIPTION
ACCEPTANCE CORRECTIVE
CRITERIA ACTION
Demonstrate
All
Demonstratio
Once
See Appendix
EPA will not
competency
n of past
G
approve any
for analyzing
experience
laboratory for
water
with water
NCCA sample
samples to
samples in
processing if
meet the
achieving the
the laboratory
performance
method
cannot
measures
detection
limits
demonstrate
competency. In
other words,
EPA will select
another
laboratory that
can
demonstrate
competency
for its NCCA
samples.
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Check
All
Sample issues
Once
No sample
Lab
condition of
such as
issues or
determines if
sample when
cracked
determination
the sample can
it arrives
container;
that sample
be analyzed or
missing label;
can still be
has been too
temperature;
analyzed
severely
adherence to
compromised
holding time
(e.g.,
requirements;
contamination)
sufficient
. Assign
volume for
appropriate
test.
condition code
identified in
Table 8.1.
Store sample
All
Check the
Record
While stored
If at any time
appropriate!
temperature
temperature of
at the
samples are
y
of the
sample upon
laboratory,
warmer than
refrigerator
arrival at the
the sample
required, note
per
laboratory. Check
must be kept
temperature
laboratory's
temperature of
at a maximum
and duration
standard
the
temperature
(either from
operating
refrigerator/freeze
of 4° C (for
the continuous
procedures.
r where samples
aliquots
temperature
are stored at least
except
log or from the
daily if using a
chlorophyll a)
last manual
continuous
and -20° Cfor
reading) in
temperature
the
comment
logger and twice
chlorophyll a
field. Lab will
daily (once at
sample.
still perform
beginning of the
test. EPA
day and once at
expects that
the end) not using
the laboratory
a continuous
will exercise
logger.
every effort to
maintain
samples at the
correct
temperature.
Analyze
All
The test must
Perform test in
sample
be completed
all cases but
within
within the
note reason
holding time
holding time
for performing
specified in
test outside
the analytical
holding time.
method.
EPA expects
that the
laboratory will
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exercise every
effort to
perform tests
before the
holding time
expires.
Analyze
All
Once per day prior
Control limits
Prepare and
Laboratory/
to sample analysis
< MDL
analyze new
Reagent
blank.
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.
Analyze
All dissolved
ASTM Type II
Prepare once per
Measured
Measure
Filtration
analytes
reagent water
week and archive.
concentration
archived
Blank
processed
Prepare filter
s
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Sample
(QCCS)
Analyze
Calibration
QCCS
All
Before and after
sample analyses
Analyze
Laboratory
Duplicate
Sample
All
Analyze
Standard
Reference
Material
(SRM)
When
available for
a particular
indicator
One per batch
One analysis in a
minimum of five
separate batches
confidence
interval)
±10% or
method
criteria
Control limits
< precision
objective
affected
samples for
possible re-
analysis.
Manufacturer
s certified
range
Repeat QCCS
analysis.
Recalibrate
and
analyze QCCS.
Reanalyze all
routine
samples
(including PE
and field
replicate
samples)
analyzed since
the last
acceptable
QCCS
measurement.
If results are
below LRL:
Prepare and
analyze split
from different
sample
(volume
permitting).
Review
precision of
QCCS
measurements
for batch.
Check
preparation of
split sample.
Qualify all
samples in
batch for
possible
reanalysis.
Analyze
standard in
next batch to
confirm
suspected
inaccuracy.
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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
Only
One per batch
Control limits
Select two
Matrix
prepared
for recovery
additional
Spike
when
cannot
samples and
Samples
samples
exceed
prepare
with
100±20%
fortified
potential for
subsamples.
matrix
Reanalyze all
interference
suspected
s are
samples in
encountered
batch by the
method of
standard
additions.
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Prepare three
subsamples
(unfortified,
fortified with
solution
approximately
equal to the
endogenous
concentration,
and fortified
with solution
approximately
twice the
endogenous
concentration).
Use
All
Verify that all
Data reporting
For each
If it is not
consistent
units are
indicator, all
possible to
units for QC
provided
field and QC
provide the
samples and
consistently
samples are
results in
field samples
within each
reported with
consistent
indicator.
the same
units, then
measurement
assign a QC
units
code and
describe the
reason for
different units
in the
comments
field of the
database.
Maintain
All
Determine
Data reporting
Completeness
Contact EPA
completenes
completeness
objective is
HQ NCCA
s
95% for all
Laboratory
indicators
Review
(useable with
Coordinator
or without
immediately if
flags).
issues affect
laboratory's
ability to meet
completeness
objective.
Deliver data
All
Compare
Data Reporting
All fields are
Correct data
using format
electronic
correctly filled
entry errors
required by
data
in, using text,
prior to
EPA
deliverable
numbers and
sending data
format to
characters
deliverable to
template
according to
EPA.
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the EPA data
template
8.8 Sample and Record Retention
The laboratory shall retain:
1. The sample materials for a minimum of one year after collection. During this time, the
laboratory shall store the materials cold (e.g., 4 ° C) and in darkness. The lab shall retain
the sample materials from the one-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.
8.9 References
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 November
4, 2019 from https://nepis.epa.gov/Exe/ZyPDF.cgi/30003K0S.PDF?Dockey=30003K0S.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.
USEPA. December 2016. EPA 821-R-16-006 "Definition and Procedure for the Determination of
the Method Detection Limit, Revision 2", retrieved March 12, 2020 from
https://www.epa.gov/sites/production/files/2016-12/documents/mdl-procedure rev2 12-13-
2016.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 Water Quality Laboratory." Open-File Report: 99-193 by Childress, Oblinger, et a!.,
retrieved November 4, 2019 from http://pubs.usgs.gov/of/1999/0193/report.pdf.
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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.
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9.0 SEDIMENT TOXICITY TESTING
This chapter describes the analysis requirements for sediment toxicity testing. The purpose is to
assess the toxicity of sediment samples collected in the 2020 NCCA and related studies.
At each sampling site, the FOM instructs the crews to collect surficial sediment samples. The
field crew then ships the samples on wet ice to the laboratory. If EPA uses a batching
laboratory, it will refrigerate the samples, before shipping on wet ice to the analysis laboratory.
9.1 Summary of the Procedure
This chapter describes toxicity testing of sediment samples collected for EPA's 2020 National
Coastal Condition Assessment (NCCA).
9.2 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).
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.
9.3 Definitions and Required Resources (Personnel, Laboratories, and Equipment)
This section provides definitions and required resources for using the procedure.
9.3.1 Definitions
The procedure uses the following terms:
Replicates are defined as two or more aliquots of the same sample which are analyzed
separately using identical procedures. The results are used to evaluate the precision of the
laboratory analyses.
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NARS: National Aquatic Resource Surveys. The National Coastal Condition Assessment (NCCA) is
part of the NARS program.
NARS Information Management System (NARS IM): The IM system established to support all
surveys, including NCCA, in the NARS program. The IM system is used to track the samples from
field collection to the laboratory.
NCCA: National Coastal Condition Assessment. Freshwater and coastal samples will be collected
during the field stage of NCCA.
CNTRL_CORR_SURV: Average percentage of organisms that survived in the replicate test
chambers divided by the corresponding batch control average percent survival.
MEAN_NSURV: Average number of organisms that survived in the test chambers for each set of
replicates.
MEAN_PSURV: Average percentage of organisms that survived in the test chambers for each
set of replicates.
NUM_SURVIVAL: The number of organisms that survived in each replicate test chamber.
PER_SURVIVAL: The percentage of organisms that survived in each replicate test chamber.
9.3.2 General Requirements for Laboratories
Expertise. To demonstrate its expertise, the laboratory shall provide EPA with performance
data demonstrating their proficiencies in analyzing sediment toxicity samples. In addition, the
laboratory must provide one or more of the following:
• 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 sediment-related analyses, membership in professional societies, and
experience with analyses that are the same or similar to the requirements of this
method.
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 QMPs, 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 NCCA QAPP Certification Page.
Preparation for the work
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To demonstrate its preparation for the work, the laboratory shall provide documentation that it
has complied with the following control analyses prior to the start of any work.
1. The laboratory shall ensure that the water source for the overlying water has been
demonstrated to support survival, growth, and reproduction of the test organisms. The
laboratory shall provide information on how the laboratory maintains the quality of the
water used for the tests.
2. The laboratory shall ensure that the clean sediment is appropriate for the control tests.
The laboratory shall provide information about the sediment chemistry analysis and
explanation of how the control sediment was selected. The laboratory shall supply
results of grain size, percent moisture and percent TOC analyses for all control sediment
sources at the beginning of the project and before any changes to a different control
sediment source. To the extent possible control sediment source changes should be
kept to a minimum. Please notify the NCCA Project Manager and Quality Assurance
Coordinator if a change in control sediment source may be necessary.
3. The laboratory shall ensure that the organisms are healthy for the tests. The laboratory
shall provide the source of the organisms; historic information about the culturing; and
procedures for evaluating the condition and age of the organism and water quality upon
arrival. If the laboratory intends to purchase the organisms (i.e., instead of in-house
culturing), identify the commercial source; its shipping arrangements (e.g., test
organisms are shipped in well-oxygenated water in insulated containers to maintain
temperature during shipment); and evaluation upon arrival at the laboratory (e.g.,
measuring temperature and dissolved oxygen of the water in the shipping containers to
determine if the organisms might have been subjected to low dissolved oxygen or
temperature fluctuations).
4. The laboratory shall complete a "non-toxicant" test of each new chamber before using
the chamber for NCCA samples. A "new" chamber is one that the laboratory has not
previously used for any sediment toxicity testing for any client (e.g., replacement
glassware). Ideally, although EPA is not requiring it, the laboratory will test freshwater
and estuarine samples in wholly separate chambers.
Test requirements: The test chambers contain control sediment (sometimes
called the negative control) and clean overlying water for the amphipod species
to be tested. Survival of the test organisms will demonstrate whether facilities,
water, control sediment, and handling techniques are adequate to achieve
acceptable species-specific control survival. For the test to be acceptable,
survival at 10 days must equal or exceed the survival requirements in QA/QC
specifications in Section 9.7.
9.3.3 Equipment/Materials
The analytical method, selected by the laboratory, identifies the necessary equipment.
9.4 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
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within 24 clock hours of receiving a delivery. The laboratory must inspect the samples promptly
on receipt. As samples arrive, the laboratory must:
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 (see Chapter 2 for contact information).
2. Check that each shipping container has arrived undamaged. Check the temperature of
one of the samples in the cooler using a thermometer that measures temperatures
between 0 °C (refrigerated samples are typically 4 °C) and 30 °C (ambient room
temperature is typically less than 26 °C), or an infra-red (IR) temperature "gun" and
record the reading. Field crews and the batching laboratory will ship sediment samples
on wet ice. Record the condition and temperature of the sample in the database using
the codes in Table 9.1.
3. Verify that all required data elements, per Table 9.1, have been recorded. If any
elements are missing, then enter them into the database.
4. Transfer the samples to the refrigerator until ready for toxicity testing. Except during
processing and analysis stages, the samples must be stored at 4 °C.
5. Notify the EPA immediately about any problems involving sample integrity, conformity,
or inconsistencies as soon as possible following sample receipt and inspection.
Table 9.1 Sediment Toxicity Login: Required Data Elements
FIELD FORMAT DESCRIPTION
LAB NAME
Character
Name or abbreviation for laboratory
TYPE
Character
Control or NCCA Sample
DATE COL
MMDDYY
Date Sample was collected by the crew in the field (from sample
label)
DATE RECEIVED
MMDDYY
Date sample was received by lab; leave blank for control
SITE ID
Character
NCCA site id as used on sample label; leave blank for control
VISIT NO
Numeric
Sequential visits to site (1 (or blank) or 2); leave blank for control
SAMPLE J D
Numeric
Sample ID as used on field sheet (on sample label); leave blank
for control
DATE COL
MMDDYY
Date sample was collected; leave blank for control
ARRIVAL_TEMP
Numeric
Temperature of sample upon arrival at the laboratory (it should
arrive 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
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c
Sample container is cracked
L
Sample or container is leaking
ML
Sample label is missing
NF
Sample is not at proper temperature
VT
Volume not sufficient for testing (VT)
VR
Volume not sufficient for a retest, if required
HT
Received outside holding time
Q
Other quality concerns not identified above
COND_COMMENT Character Explanation for Q FLAG (if needed)
9.5 Toxicity Testing: Requirements
The laboratory shall perform toxicity testing of sediment samples. Laboratories may choose to
use any analysis method using the required organisms of Hyalella azteca (freshwater) or
Leptocheirus plumulosus (estuarine). The laboratory's method must meet the quality
requirements in Section 9.7, including mean survival of the control's treatments must remain
greater than or equal to 80% and 90%, respectively. It is essential that the contractor require
that all of its laboratory technicians use the same procedures and meet the required quality
elements. At a minimum, the laboratory must:
1. Perform the procedures using the 10-day tests. Possible methods include those
described in the following documents:
a. Estuarine: Test Method 100.4 in EPA 600/R-94/02520 or ASTM E1367-0321
b. Freshwater: Test Method 100.1 in EPA 600/R-99/06422 or ASTM E170623
2. Test the following number of replicates for each sample and control:
a. Estuarine: 5 replicates with 20 organisms per replicate
b. Freshwater: 4 replicates with 10 organisms per replicate
3. Test no more than 10 samples and one control within each batch.
4. Use the following organisms for the tests:
a. Estuarine: Leptocheirus plumulosus
b. Freshwater: Hyalella azteca
20 Chapter 11 in Methods for Assessing the Toxicity of Sediment-associated Contaminants with Estuarine and
Marine Amphipods, June 1994, retrieved May 22, 2019 from
https://nepis.epa.gov/Exe/ZvPDF.cgi/300032A9.PDF?Dockev=300032A9.PDF
21 American Society for Testing and Materials (ASTM). 2008. E1367-03 "Standard Guide for Conducting 10-Day
Static Sediment Toxicity Tests With Marine and Estuarine Amphipods." Annual Book of Standards, Water and
Environmental Technology, Vol. 11.05, West Conshohocken, PA.
22Section 11 in Methods for Measuring the Toxicity and Bioaccumulation of Sediment-associated Contaminants
with Freshwater Invertebrates, Second Edition, March 2000, retrieved May 22, 2019 from
https://nepis.epa.gov/Exe/ZvPDF.cgi/30003SBA.PDF?Dockev=30003SBA.PDF
23 ASTM 2009 E1706. "Standard Test Method for Measuring the Toxicity of Sediment-Associated Contaminants
with Freshwater Invertebrates."
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5. Select organisms for each batch of tests that are:
a. From the same culture;
b. Cultured at the same temperature as will be used for the tests;
c. (optional) EPA would prefer but does not require that the organisms are cultured
in the same water as that used for testing.
6. Use a water source (for the overlying water) demonstrated to support survival, growth,
and reproduction of the test organisms. The laboratory shall provide information on
how the laboratory maintains the quality of the water used for the tests.
a. For estuarine sediments, 175 mL of sediment and 800 mL of overlying seawater
b. For freshwater sediments, lOOmL of sediment and 175mL of overlying
freshwater
7. The laboratory shall ensure that the clean sediment is appropriate for the control tests.
The laboratory shall provide information about the sediment chemistry analysis and
explanation of how the control sediment was selected. The laboratory shall supply
results of grain size, percent moisture and percent TOC analyses for all control sediment
sources at the beginning of the project and before any changes to a different control
sediment source. All tests of the control sediment sources are to be done by the lab and
results are to be submitted to EPA. To the extent possible control sediment source
changes should be kept to a minimum. Please notify the NCCA Project Manager and
Quality Assurance Coordinator if a change in control sediment source may be necessary.
8. Implement the following for exposure/feeding:
a. For estuarine sediments, exposure is static (i.e., water is not renewed), and the
animals are not fed over the 10-day exposure period
b. For freshwater, exposure is renewed (i.e., 2 volumes a day) and the animals are
fed over the 10-day exposure period
9. Use the following procedure for homogenization/sieving: Water above the sediment is
not discarded but is mixed back into the sediment during homogenization. Sediments
should be sieved for estuarine samples (following the 10-day method) and the sieve size
should be noted. For freshwater samples, they should not be sieved to remove
indigenous organisms unless there is a good reason to believe indigenous organisms
may influence the response of the test organism. Large indigenous organisms and large
debris can be removed using forceps.
Additional details are provided in the summary Table 9.2 and Table 9.3.
Table 9.2 Test Conditions for Conducting 10-d Sediment Toxicity Tests for estuarine sediments
PARAMETER
CONDITIONS
1. Test type
Whole sediment toxicity test, static
2. Temperature
25 °Cfor L. plumulosus
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3. Salinity
20%o
4. Light quality
Wide-spectrum fluorescent lights
5. Illuminance
500-1000 lux
6. Photoperiod
24L:0D
7. Test chamber
1 L glass beaker or jar with ~10 cm I.D.
8. Sediment volume
175 mL (2 cm)
9. Overlying water
800 mL
volume
10. Renewal of overlying
None
water
11. Size and life stage of
L. plumulosus: 2-4 mm (no mature males or females)
amphipods
12. Number of organisms
20 per test chamber
per chamber
13. Number of replicate
5 (required)
chambers/ treatment
14. Feeding
None
15. Aeration
Water in each test chamber should be aerated overnight before start of
test and throughout the test aeration at rate that maintains >90%
saturation of dissolved oxygen concentration
16. Overlying water
Clean sea water, natural or reconstituted water
17. Overlying water
Temperature daily; pH, ammonia, salinity, and DO attest start and end.
quality measurements
18. Test duration
10 d
19. Endpoints
Survival
20. Test acceptability
Minimum mean control survival of 90%
Table 9.3 Test Conditions for Conducting 10-d Sediment Toxicity Tests for freshwater
sediments
PARAMETER CONDITIONS
1. Test type
Whole-sediment toxicity test with renewal of overlying water
2. Temperature
23°± 1°C
3. Light quality
Wide-spectrum fluorescent lights
4. Illuminance
100 to 1000 lux
5. Photoperiod
16L:8D
6. Test chamber
300 mL high-form beaker
7. Sediment volume
100 mL
8. Overlying water volume
175 mL
9. Renewal of overlying
2 volume additions/d; continuous or intermittent (e.g., 1 volume
water
addition every 12 h)
Overlying water quality
Estuarine Toxicity Tests: Temperature daily; pH, ammonia,
measurements
conductivity, and DO at test start and end.
Freshwater Toxicity Tests: Temperature and dissolved oxygen daily.
Hardness, alkalinity, conductivity, pH, and ammonia at the beginning
and end of a test.
10. Age of organisms
7- to 14-d old at the start of the test (1- to 2-d range in age)
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11. Number of organisms/
chamber
10
12. Replicate
chambers/treatment
4 required
13.Feeding
YCT food, fed 1.0 mL daily (1800 mg/L stock) to each test chamber.
14. Aeration
None unless DO in overlying water drops below 2.5 mg/L
15. Test duration
10 d
16. Endpoint
Survival
17. Test acceptability
Min. mean control survival of 80%.
9.6 Data Entry
Table 9.4 and Table 9.5 identify the method performance requirements and data elements
describing the test conditions and outcomes for each replicate and batch, respectively.
Laboratories shall provide the data elements to EPA, preferably in EPA's data template, available
separately from EPA. The laboratory shall digitize bench sheets and provide them to the EPA
TOCOR.
Table 9.4 Sediment Toxicity Replicates: Laboratory method performance data deliverable
requirements
FIELD FORMAT DESCRIPTION
LAB NAME
Character
Name or abbreviation for laboratory
SITE ID
Character
Unique SITEJD reported on sample label
TYPE
Character
Control or NCCA Sample
SAMPLE J D
Numeric
Sample id as used on field sheet (on sample labels-
leave blank
for control
VISIT_NUMBER
Numeric
Number of visit (sequential) to site ("1" or "2" are
the only legal
values)
RETEST
Y or blank
Y for yes if the sample is being retested; blank if
original test or
control
CHAMBER ID
Character
Identification code for test chamber
BATCH ID
Character
Identification code for batch
REPLICATE
Numeric
Replicate number: 1-5 for marine; 1-4 for
freshwater
TEST TYPE
Character
Marine or Freshwater
ORGANISM
Character
Leptocheirus plumulosus (marine) or Hyalella azteca
(freshwater)
NUM_SURVIVAL
Numeric
Number of organisms that survived out of 20
(marine) and 10
(freshwater)
PER_SURVIVAL
Numeric
Percentage of organisms that survived in the test
chamber for
the replicate
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REP_COMMENT
Character
Any comments about the test procedures or any
abnormalities
TEMP
Numeric
Daily replicate temperature
PH
Numeric
For each replicate on day 0 and day 10
Nh3
Numeric
For each replicate on day 0 and day 10
Salinity
Numeric
For each estuary replicate on day 0 and day 10
Conductivity
Numeric
For each Great Lakes replicate on day 0 and day 10
DO
Numeric
For each replicate on day 0 and day 10
Table 9.5 Laboratory method performance data deliverable requirements for sediment
toxicity batch summaries
FIELD FORMAT DESCRIPTION
LAB NAME
Character
Name or abbreviation for laboratory
SITE ID
Character
Unique SITEJD reported on sample label
TYPE
Character
Control or NCCA Sample
SAMPLE J D
Numeric
Sample id as used on field sheet (on sample label); leave
blank for control
VISIT_NUMBER
Numeric
Number of sequential visit to site ("1" or "2" are the only
legal values)
DATE COL
MMDDYY
Date sample was collected by crew (from sample label)
DATE RECEIVED
MMDDYY
Date sample was received in lab
ARRIVAL TEMP
Numeric
Temperature of sample upon arrival in lab (in degrees C)
CONDITION_CODE
Character
Code for condition of sample upon arrival at lab (See Table
9.1 for allowable
codes)
CONDITION_COMMENT
Character
Comment explaining condition code (required for code "Q".
Optional for others)
BATCH ID
Character
Identification code for batch
BATCH_SAMPLES
Numeric
Number of NCCA samples in the batch (integer<10) excluding
the control
TEST TYPE
Character
Estuarine or Freshwater
ORGANISM
Character
Leptocheirus plumulosus (marine) or Hyalella azteca
(freshwater)
CONTROL
Character
Source of control sediment (artificial or name of collected
reference sediment)
START_DATE
MMDDYY
Date that the laboratory starts the test procedure for the
batch
END_DATE
MMDDYY
Date that the laboratory ends the test procedure for the
batch
MEAN_NSURV
Numeric
Mean number survival for all replicates of the sample (or
control) calculated
using the NUM_SURVIVAL
MEAN_PSURV
Numeric
Mean percent survival for all replicates of the sample (or
control) calculated
using the PER_SURVIVAL
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CNTRL_CORR_SURV
Numeric
Optional Field: Average percentage of organisms that
survived in the replicate
test chambers divided by the corresponding average control
percent survival
BATCH_PASS
P/F
Indicate if the batch passed (P) or failed (F)the QA/QC
requirements (e.g., mean
control survival achieved required survival rates)
QC_CODE
Character
Laboratory assigned code for QC issues with the sample
QC_DESCRIPTION
Character
Description of conditions associated with the QC_CODE
SURV_COMMENT
Character
Any comments about the test procedures or any
abnormalities
9.7 Quality Measures
This section describes the quality assurance and quality control measures used to ensure that
the data will meet NCCA's requirements.
9.7.1 Assistance Visits
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 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.
9.7.2 Summary of QA/QC 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 10 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 samples. Table 9.6 provides a
summary of the quality control requirements.
Table 9.6 Quality control activities for sediment toxicity samples
ACTIVITY
EVALUATION
CORRECTIVE ACTION
Laboratory demonstrates
competency for conducting
sediment toxicity analyses in
Section 9.5
EPA will review SOPs, lab
certifications, past performance
results, etc. as part of the lab
verification process.
EPA will not approve any
laboratory for NCCA sample
processing if the laboratory
cannot demonstrate
competency. In other words,
EPA will select another
laboratory that can
demonstrate competency for its
NCCA samples.
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Check condition of sample
when it
arrives
Sample issues, such as cracked
or leaking container; missing
label; temperature; adherence
to holding time requirements;
insufficient volume for test.
Assign appropriate condition
code identified in Table 9.1.
Sample storage
All samples: 4°C upon arrival at
the laboratory (temperature
recorded at arrival) and while
stored at the laboratory.
Record temperature upon arrival at
the laboratory. Check temperature
of the refrigerator where samples
are stored at least daily if using a
continuous temperature logger and
twice daily (beginning and end of
day) if the lab does not have a
continuous logger. If refrigerator is
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.
Holding Time
The test must be completed
within 8 weeks after sample
collection. If the original test
fails, then the retest also must
be conducted within the 8
weeks after sample collection.
Perform test 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.
Check that the organisms are
healthy before starting the
test. The laboratory shall
describe the source of the
organisms; historic information
about the culturing; and
procedures for evaluating the
condition and age of the
organism and water quality
upon arrival. If the laboratory
intends to purchase the
organisms (i.e., instead of in-
house culturing), identify the
commercial source; its shipping
arrangements (e.g., test
organisms should be shipped in
well oxygenated water in
insulated containers to
maintain temperature during
Unhealthy organisms may
appear to be discolored, or
otherwise stressed (for
example, greater than 20
percent mortality for the 48
hours before the start of a test).
Don't start test using unhealthy
organisms.
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shipment); and upon arrival at
the laboratory.
Maintain conditions as
required in Section 9.3.
Control survival rates
For a test of a batch of samples
to be considered valid, the
control's mean survival in
Hyalella and Leptocheirus
treatments must remain >80%
and >90%, respectively.
Check conditions (e.g., Note any deviations in
temperature, DO) each test day. comments field (Table 9.1). In
Record conditions in bench extreme cases, conduct a new
sheet or in laboratory database, toxicity test for all samples
affected by the adverse
conditions.
Data template includes a field
to record if a test passed or
failed the control requirements.
If a test fails, retest all samples
in the batch. EPA prefers that
results from failing test be
deprecated from the
spreadsheet and completely
detailed in that narrative
progress reports, while passing
retest results be included in the
spreadsheet. If this is
not possible, the lab shall
clearly
differentiate between the
original
(failing) test results and the
retest
results within the spreadsheet.
If both tests fail, submit data to
EPA for further consideration.
Include comments in the data
template noting any particular
factors that may have caused
the test to fail twice.
*Chapter 2 provides contact information for the EPA HQ NCCA 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.
9.8 Sample and Record Retention
The laboratory shall retain:
1. The lab shall retain the samples until the NCCA 2020 report and data are published or
the lab is notified in writing by the EPA that samples may be disposed of sooner. Until
this time, the laboratory shall refrigerate the sediment samples. The laboratory shall
periodically check the sample materials for degradation.
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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.
9.9 References
American Society for Testing and Materials (ASTM). 2008. E1367-03 "Standard Guide for
Conducting 10-Day Static Sediment Toxicity Tests with Marine and Estuarine Amphipods."
Annual Book of Standards, Water and Environmental Technology, Vol. 11.05, West
Conshohocken, PA.
ASTM. 2009. E1706. "Standard Test Method for Measuring the Toxicity of Sediment-Associated
Contaminants with Freshwater Invertebrates.
United Stated Environmental Protection Agency (USEPA). 1994. Chapter 11 in Methods for
Assessing the Toxicity of Sediment-associated Contaminants with Estuarine and Marine
Amphipods, retrieved on February 11, 2019 from https://www.itrcweb.org/contseds-
bioavailability/References/ma rinemethod.pdf
USEPA. 2000. Section 11 in Methods for Measuring the Toxicity and Bioaccumulation of
Sediment-associated Contaminants with Freshwater Invertebrates, Second Edition, retrieved on
February 11, 2019 from
https://nepis.epa.gov/Exe/ZyPDF.cgi/30003SBA.PDF?Dockev=30003SBA.PDF
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10.0 GREAT LAKES HUMAN HEALTH FISH TISSUE SAMPLES
Laboratory Methods incorporated in EPA OST Manuals/QA
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11.0 MERCURY IN FISH TISSUE PLUGS
11.1 Summary of the Procedure
This procedure is applicable to the analysis of mercury in fish tissue plugs. The method is
performance based. Laboratories may use any method that meets the requirements below to
analyze the fish tissue samples (for example, EPA Method 1631). Example SOPs are provided in
Appendix C: Example SOPs For Mercury In Fish Tissue Plug Analyses of this LOM.
11.2 General Requirements for Laboratories
Competency. To demonstrate its competency, the laboratory shall provide EPA with
performance data demonstrating their proficiencies in analyzing water quality samples. In
addition, the laboratory must provide one or more of the following:
• 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.
Also, the lab must provide a demonstration of past experience with fish tissue samples in
applying the laboratory SOP in achieving the method detection limit.
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 QMPs, 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 NCCA QAPP Certification Page.
11.2.1 Equipment/Materials
The analytical method, selected by the laboratory, identifies the necessary equipment.
11.3 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.
1. Report receipt of samples in the NARS IM sample tracking system (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 (see
Chapter 2 for contact information).
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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 11.1 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. VT\ Volume not sufficient for testing
vi. 1/1/: Sample is warm (>8°), record the temperature in the comment field, and perform
the assay
vii. Q: other quality concerns, not identified above.
c. If any sample is damaged or missing, contact the EPA HQ Laboratory Review
Coordinator to discuss whether the sample can be analyzed. (See contact information in
Chapter 2 of the Manual).
3. Store samples in the freezer until sample preparation begins.
4. Maintain the chain of custody or sample tracking forms with the samples.
Table 11.1 Fish Tissue Plugs Login: Required Data Elements
FIELD FORMAT DESCRIPTION
LAB NAME
text
Name or abbreviation for QC laboratory
DATE RECEIVED
MMDDYY
Date sample was received by lab
SITE ID
text
NCCA 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
VT
Volume or mass not sufficient for
testing (VT)
W
Sample is warm (>8°)
Q
Other quality concerns, not identified
above
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CONDITION
text
Comments about the condition of the sample. If
COMMENT
the condition code='W' then provide the
temperature
11.4 Quality Measures
This section describes the quality assurance and quality control measures used to ensure that
the data will meet NCCA's requirements. Table 11.2 and Table 11.3 provide a summary of the
measurement data quality objectives and quality control requirements.
11.4.1 Assistance Visits
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 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.
Table 11.2 Measurement data quality objectives
VARIABLE OR MEASUREMENT
MDL*
QUANTITATION
LIMIT
Mercury
0.47 ng/g
5.0 ng/g
* In the event for sample dilution is necessary to overcome the matrix effect, please notify EPA Laboratory Coordinator
Table 11.3 Fish Tissue Plugs Quality Control
ACTIVITY EVALUATION/ACCEPTANCE CRITERIA CORRECTIVE ACTION
Demonstrate
competency
For analyzing fish
samples to
meet the performance
measures
Demonstration of experience with fish tissue
samples in applying the laboratory SOP in
achieving the method detection limit
EPA will not approve
any laboratory for
NCCA sample
processing if the
laboratory cannot
demonstrate
competency. In other
words, EPA will select
another laboratory that
can demonstrate
competency for
its NCCA samples.
Check condition of
sample when it arrives.
Sample issues, such as punctures or rips in
wrapping; missing label; temperature;
adherence to holding time requirements;
sufficient volume for test. All samples should
arrive at the laboratory frozen.
Assign an appropriate
condition code.
Store sample
appropriately.
While stored at the
laboratory, the sample
must be kept at a
Check the temperature of the freezer per
laboratory's standard operating procedures.
Record temperature of
sample upon arrival at
the laboratory. If at any
other time, samples
are warmer than
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maximum temperature
of -20° C.
required, note
temperature and
duration in comment
field.
Analyze sample within The test must be completed within the Perform test but note
holding time holding time (i.e., 1 year). If the original test reason for performing
fails, then test outside holding
the retest also must be conducted within the time. EPA expects that
holding time. For plugs taken from frozen the laboratory will
fish, the one year holding time begins when exercise every effort to
the fish is thawed to collect the tissue perform tests before
sample in the lab. the
For the plugs taken from the fish in the field, holding time expires,
the one year holding time begins at the time
of field
collection.
Maintain quality Data meet all QC specifications in the
control selected method/SOP.
specifications from
selected method/SOP
(that meets the
measurement data
quality objectives)
If data do not meet all
QC requirements,
rerun sample or qualify
data. If the lab
believes the data are to
be qualified without
rerunning sample, the
lab must consult
with the EPA Survey
QA Lead before
proceeding.
Maintain the required
MDL
Evaluate for each sample If MDL could not be
achieved, then provide
dilution factor or QC
code and explanation
in the comment field.
Use consistent units for
QC
samples and field
samples
Verify that all units are provided in wet
weight units and consistently. Also provide
% moisture values to help translate from wet
weight and dry weight.
If it is not possible to
provide the results in
the same units
as most other analyses,
then assign a QC code
and describe the
reason for different
units in the
comments field of the
database.
Maintain completeness Completeness objective is 95% for all
parameters.
Contact the EPA Survey
QA Lead immediately if
issues affect
laboratory's ability to
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meet completeness
objective.
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12.0 FECAL INDICATOR: ENTEROCOCCI
Laboratory methods incorporated into EPA ORD Manuals/QAPP.
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APPENDIX A: MICROCYSTINS-ADDA SAES ELISA TEST KIT PROTOCOL
The microcystins-ADDA SAES ELISA test kit protocol is separate attachment of this document.
Follow steps in Section 4.6.1 to complete the 3x freeze-thaw process before continuing with
the protocol. Samples do not need to undergo the dilution process due to salinity as defined in
Section 4.6.2 as this test can run samples with salinity levels up to 38 ppt.
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APPENDIX B: TARGET FISH SPECIES FOR WHOLE FISH ANALYSES
Table B.l Northeast region primary and secondary marine target species - whole body fish
tissue collection (Ecofish)
NORTHEAST REGION PRIMARY ECOFISH TARGET SPECIES
FAMILY
SCIENTIFIC NAME
COMMON NAME
FISH PLUG LIST*
Ictaluridae
Ameiurus catus
White catfish
Primary
Ictalurus punctatus
Channel catfish
Primary
Moronidae
Morone americana
White perch
Primary
Paralichthyidae
Paralichthys dentatus
Summerflounder
Primary
Pleuronectidae
Pseudopleuronectes americanus
Winter flounder
Primary
Sciaenidae
Cynoscion regalis
Gray weakfish
Primary
Sciaenops ocellatus
Red drum
Primary
Sparidae
Stenotomus chrysops
Scup
Primary
NORTHEAST REGION SECONDARY ECOFISH TARGET SPECIES
FAMILY
SCIENTIFIC NAME
COMMON NAME
FISH PLUG LIST*
Achiridae
Trinectes maculatus
Hogchoaker
Anguill idae
Anguilla rostrata
American eel
Secondary
Atherinopsidae
Menidia menidia
Atlantic silverside
Batrachoididae
Opsanus tau
Oyster toadfish
Ephippidae
Chaetodip terus faber
Atlantic spadefish
Moronidae
Morone saxatilis
Rock fish
Secondary
Mugulidae
Mugil cephalus
Black mullet
Pomatomidae
Pomatomus saltatrix
Bluefish
Secondary
Sciaenidae
Bairdiella chrysoura
Silver perch
Menticirrhus saxatilis
Northern kingfish
Serranidae
Centropristis striata
Black sea bass
Triglidae
Prionotus carolinus
Northern searobin
Prionotus evolans
Striped searobin
* Indicates whether species also occurs in the primary or secondary fish plug list
Table B.2 Southeast region primary and secondary marine target species - whole body fish
tissue collection (Ecofish)
SOUTHEAST REGION PRIMARY ECOFISH TARGET SPECIES
FAMILY
SCIENTIFIC NAME
COMMON NAME
FISH PLUG LIST*
Ariidae
Ariopsisfelis
Hardhead sea catfish
Pr
imary
Bagre marinus
Gafftopsail sea catfish
Pr
imary
Paralichthyidae
Paralichthys albigutta
Gulf flounder
Pr
imary
Paralichthys dentatus
Summerflounder
Pr
imary
Paralichthys lethostigma
Southern flounder
Pr
imary
Sciaenidae
Cynoscion arenarius
Sand weakfish (or seatrout)
Pr
imary
Cynoscion nebulosus
Speckled trout
Pr
imary
Cynoscion regalis
Gray weakfish
Pr
imary
Leiostomus xanthurus
Spot croaker
Pr
imary
Sparidae
Lagodon rhomboides
Pinfish
SOUTHEAST REGION SECONDARY ECOFISH TARGET SPECIES
FAMILY
SCIENTIFIC NAME
COMMON NAME
FISH PLUG LIST*
Cichlidae
Tilapia mariae
Spotted tilapia
Haemulidae
Haemulon aurolineatum
Tomtate
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Sciaenidae
Bairdiella chrysoura
Silver perch
Menticirrhus americanus
Southern kingfish
Serranidae
Centropristis striata
Black sea bass
* Indicates whether species also occurs in the primary or secondary fish plug list
Table B.3 Gulf region primary and secondary marine target species - whole body fish tissue
collection (Ecofish)
GULF REGION PRIMARY ECOFISH TARGET SPECIES
FAMILY
SCIENTIFIC NAME
COMMON NAME
FISH PLUG LIST*
Ariidae
Ariopsis felis
Hardhead sea catfish
Pr
mary
Bagre marinus
Gafftopsail sea catfish
Pr
mary
Paralichthys albigutta
Gulf flounder
Pr
mary
Paralichthyidae
Paralichthys dentatus
Summer flounder
Pr
mary
Paralichthys lethostigma
Southern flounder
Pr
mary
Cynoscion arenarius
Sand weakfish (or seatrout)
Pr
mary
Cynoscion nebulosus
Speckled trout
Pr
mary
Sciaenidae
Cynoscion regalis
Gray weakfish
Pr
mary
Leiostomus xanthurus
Spot croaker
Pr
mary
Micropogonias undulatus
Atlantic croaker
Pr
mary
Sciaenops ocellatus
Red drum
Pr
mary
Sparidae
Lagodon rhomboides
Pinfish
GULF REGION SECONDARY ECOFISH TARGET SPECIES
FAMILY
SCIENTIFIC NAME
COMMON NAME
FISH PLUG LIST*
Carangidae
Caranx hippos
Crevalle jack
Chloroscombrus chrysurus
Atlantic bumper
Diodontidae
Chilomycterus schoepfii
Burrfish
Gerreidae
Eucinostomus gula
Silver jenny
Haemulidae
Orthopristis chrysoptera
Pigfish
Ictaluridae
Ictalurus furcatus
Blue catfish
Lepisosteidae
Lepisosteus oculatus
Spotted gar
Lutjanidae
Lutjanus griseus
Gray snapper
Sciaenidae
Pogonias cromis
Black drum
Serranidae
Diplectrum formosum
Sand perch
Triglidae
Prionotus scitulus
Leopard searobin
Indicates whether species also occurs in the primary or secondary fish plug list
Table B.4 Western region primary and secondary marine target species - whole body fish
tissue collection (Ecofish)
WESTERN REGION PRIMARY ECOFISH TARGET SPECIES
FAMILY
SCIENTIFIC NAME
COMMON NAME
FISH PLUG LIST*
Atherinopsidae
Atherinops affinis
Topsmelt silverside
Cottidae
Leptocottus armatus
Pacific staghorn sculpin
Primary
Oligocottus rimensis
Saddleback sculpin
Cynoglossidae
Symphurus atricaudus
California tonguefish
Embiotocidae
Cymatogaster aggregata
Shiner perch
Primary
Embiotoca lateralis
Striped seaperch
Primary
Gasterosteidae
Gasterosteus aculeatus
Three-spined stickleback
Paralichthys californicus
California flounder
Primary
Paralichthyidae
Citharichthys sordidus
Pacific sanddab
Primary
Citharichthys stigmaeus
Speckled sanddab
Pleuronectidae
Isopsetta isolepis
Butter sole
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Parophrys vetulus
English sole
Primary
Psettichthys melanostictus
Pacific sand sole
Platichthys stellatus
Starry flounder
Primary
Sciaenidae
Genyonemus lineatus
White croaker
Primary
Serranidae
Paralabrax nebulifer
Barred sand bass
Primary
Paralabrax maculatofasciatus
Spotted sand bass
Primary
WESTERN REGION SECONDARY ECOFISH TARGET SPECIES
FAMILY SCIENTIFIC NAME COMMON NAME FISH PLUG LIST*
Echinodermata/
Toxopneustidae
Tripneustes gratilla
(Hawaii ONLYJ
Collector urchin
Batrachoididae
Porichthys notatus
Plainfin midshipman
Porichthys myriaster
Specklefin midshipman
Embiotocidae
Amphistichus argenteus
Barred surfperch
Secondary
Paralichthyidae
Xystreurys liolepis
Fantail sole
Pleuronectidae
Pleuronichthys guttulatus
Diamond turbot
Secondary
Microstomus pacificus
Dover sole
Secondary
Lepidopsetta bilineata
Rock sole
Lyopsetta exilis
Slender sole
Sciaenidae
Umbrina roncador
Yellowfin croaker
* Indicates whether species also occurs in the primary or secondary fish plug list.
Table B.5 Great Lakes primary and secondary target species - whole body fish tissue collection
(Ecofish)
GREAT LAKES PRIMARY ECOFISH TARGET SPECIES
FAMILY
SCIENTIFIC NAME
COMMON NAME
FISH PLUG LIST*
Catostomidae
Moxostoma macrolepidotum
Shorthead redhorse
Primary
Centrarchidae
Ambloplites rupestris
Rock bass
Primary
Lepomis gibbosus
Pumpkinseed
Primary
Lepomis macrochirus
Bluegill
Primary
Micropterus dolomieu
Smallmouth bass
Primary
Pomoxis annularis
White crappie
Pomoxis nigromaculatus
Black crappie
Cottidae
Cottus bairdii
Mottled sculpin
Cottus co gnat us
Slimy sculpin
Cyprinidae
Couesius plumbeus
Lake chub
Cyprinus carpio
Common carp
Primary
Pimephales notatus
Bluntnose minnow
Esocidae
Esox lucius
Northern pike
Primary
Esox masguinongy
Muskellunge
Primary
Gasterosteidae
Gasterosteus aculeatus
Three-spined stickleback
Gobiidae
Neogobius melanostomus
Round goby
Proterorhinus marmoratus
Tubenose goby
Ictaluridae
Ameiurus nebulosus
Brown bullhead
Primary
Ictalurus punctatus
Channel catfish
Primary
Noturus flay us
Stonecat
Gadidae
Lota lota
Burbot
Primary
Moronidae
Morone americana
White perch
Primary
Morone chrysops
White bass
Primary
Osmeridae
Osmerus mordax
American/ rainbow smelt
Percidae
Gymnocephalus cernuus
Ruffe
Perca flavescens
Yellow perch
Primary
Percina caprodes
Logperch
Sander canadensis
Sauger
Sander vitreus
Walleye
Primary
Percopsidae
Percopsis omiscomaycus
Trout-perch
Salmonidae
Coregonus artedi
Cisco/ lake herring
Coregonus clupeaformis
Lake whitefish
Primary
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Oncorhynchus qorbuscha
Pink salmon
Oncorhynchus kisutch
Coho salmon
Primary
Oncorhynchus mykiss
Rainbow trout
Primary
Oncorhynchus tshawytscha
Chinook salmon
Primary
Salvelinus namaycush
Lake trout
Primary
Sciaenidae
Aplodinotus arunniens
Freshwater drum
Primary
GREAT LAKES SECONDARY ECOFISH TARGET SPECIES
FAMILY
SCIENTIFIC NAME
COMMON NAME
FISH PLUG LIST*
Catostomus catostomus
Longnose sucker
Catostomidae
Catostomus commersonii
White sucker
Secondary
Moxostoma anisurum
Silver redhorse
Centrarchidae
Micropterus salmoides
Largemouth bass
Clupeidae
Alosa pseudoharenqus
Alewife
Dorosoma cepedianum
American gizzard shad
Cyprinella spiloptera
Spotfin shiner
Cyprinidae
Luxilus cornutus
Common shiner
Notropis stramineus
Sand shiner
Esocidae
Esox niger
Chain pickerel
Fundulidae
Fundulus diaphanus
Banded killifish
Fundulus majalis
Striped killifish
Ictaluridae
Ameiurus melas
Black bullhead
Prosopium cylindraceum
Round whitefish
Salmonidae
Salmo trutta
Brown trout
Secondary
Salvelinus fontinalis
Brook trout
Salvelinus fontinalis x namaycush
Splake
* Indicates whether species also occurs in the primary or secondary fish plug list
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APPENDIX C: EXAMPLE SOPS FOR MERCURY IN FISH TISSUE PLUG ANALYSES
Example SOPs for Mercury in Fish Tissue Plug Analyses are attached at the end of this
document.
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APPENDIX D: RESEARCH INDICATOR - MICRO PLASTICS IN SEDIMENT
All lab work for this supplemental indicator is expected to be done by EPA Office of Research
and Development labs.
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APPENDIX E: RESEARCH INDICATOR- TOTAL ALKALINITY
All lab work for this supplemental indicator is expected to be done by EPA Office of Research
and Development labs.
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APPENDIX F: RESEARCH INDICATOR- A N15 ISOTOPE IN BENTHIC ORGANIC
MATTER
All lab work for this supplemental indicator is expected to be done by EPA Office of Research
and Development labs.
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APPENDIX G: LABORATORY REMOTE EVALUATION FORMS
Email the completed and signed forms to Kendra Forde (forde.kendra@epa.gov).
Questions: Contact Kendra Forde at forde,kendra@epa.gov or 202-566-0417
NCCA 2020 DOCUMENT REQUEST FORM - CHEMISTRY LABORATORIES
EPA and its state and tribal partners will conduct the 2020 National Coastal Condition
Assessment. NCCA is a survey of the nation's coastal waters and Great Lakes. It is designed to
provide statistically valid regional and national estimates of the condition of coastal waters and
the Great Lakes. Consistent sampling and analytical procedures ensure that the results can be
compared across the country.
As part of the 2020 NCCA, 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 2020
NCCA.
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 a 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 2020 NCCA:
~ Water Chemistry and chlorophyll a (all of the analytes identified in the LOM and QAPP)
~ Microcystin
~ Cylindrospermopsin
~ Mercury in Fish Tissue Plugs
~ Sediment Chemistry
~ Grain Size
~ Total Organic Carbon (TOC)
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 2020 NCCA.
~ 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.
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If you have not been approved within the last 5 years through the laboratory verification
process for the water chemistry indicator, for us to determine your ability to participate as a
laboratory in the NCCA, 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.
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 2020 NCCA LOM).
~ Documentation attesting to experience running all analytes for the 2020 NCCA,
including chlorophyll a.
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Laboratory Signature Form - Chemistry Laboratories
standards in performing the following data analysis and reporting for the 2020
National Coastal Condition Assessment (NCCA).
1.) Use procedures identified in the 2020 NCCA Laboratory Operations
Manual (or equivalent). If using equivalent procedures, please provide
the procedures and obtain approval from EPA.
2.) Read and abide by the 2020 NCCA 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 SharePoint.
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 March 1, 2021 or as otherwise negotiated with
EPA.
7.) Participate in a laboratory technical assessment or audit if requested by
EPA NCCA 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-a, for
water chemistry.
located in
certify that the laboratory,
will abide by the following
This applies to the
chemistry indicator.
Signature
Date
OGRG-2400.1
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NCCA 2020 DOCUMENT REQUEST FORM - BIOLOGY LABORATORIES
EPA and its state and tribal partners will conduct the 2020 National Coastal Condition
Assessment. NCCA is a survey of the nation's coastal waters and Great Lakes. It is designed to
provide statistically valid regional and national estimates of the condition of coastal waters and
the Great Lakes. Consistent sampling and analytical procedures ensure that the results can be
compared across the country.
As part of the 2020 NCCCA, 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 biology 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 2020
NCCA.
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 a site visit.
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 2020 NCCA:
~ Mercury in Fish Plugs
~ Benthic Macroinvertabrates
~ Sediment Toxicity
If your laboratory has been previously approved within the last 5 years for the specific
parameters:
~ A signature on the attached Laboratory Signature Form indicates that your laboratory
will follow the quality assurance protocols required for biology laboratories conducting
analyses for the 2020 NCCA.
~ A signature on the Quality Assurance Project Plan (QAPP) and the Laboratory Operations
Manual (LOM) Signature Form indicates 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 specific parameters, in order for us to determine your ability to participate as
a lab in the NCCA, 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 previous NARS for this indicator.
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.).
OGRG-2400.1
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~ Documentation of NABS (or other) certification for the taxonomists performing analyses
(if applicable).
~ An updated copy of your Laboratory's QAPP and Laboratory Quality Assurance Manuals.
~ Standard Operating Procedures (SOPs) for your lab for each analysis to be performed (if
not covered in 2020 NCCA LOM).
OGRG-2400.1
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National Coastal Condition Assessment 2020 Laboratory Operations Manual
Version 1.2 March 2021 Page 169 of 169
Laboratory Signature Form - Biology Laboratories
I certify that the laboratory
located in t will abide by the following standards in
performing biology data analysis and reporting for the 2020 National Coastal Condition
Assessment (NCAA).
This applies to the biological indicator.
Use procedures identified in the 2020 NCCA Lab Operations Manual (or equivalent). If using
equivalent procedures, please provide the procedures and obtain approval from EPA.
Read and abide by the 2020 NCCA Quality Assurance Project Plan (QAPP) and related Standard
Operating Procedures (SOPs).
Have an organized IT tracking system in place for recording sample tracking and analysis data.
Use taxonomic standards outlined in the 2020 NCCA Laboratory Operations Manual.
Participate in taxonomic reconciliation exercises during the field and data analysis season,
which include conference calls and other laboratory reviews.
Provide Quality Control (QC) data for internal QC checks, including for sorting, on a monthly
basis.
Provide data using the template provided on the NARS SharePoint.
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 March 1, 2021 or as
otherwise negotiated with EPA. Samples results for independent taxonomic QC described in the
LOM and QAPP must be provided to EPA prior to final datasets to allow for reconciliation to
take place.
Participate in a Laboratory technical assessment or audit if requested by EPA NCCA staff (this
may be a conference call or on-site audit).
Agree to utilize taxonomic nomenclature and hierarchical established for NCCA 2020.
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