svEPA
United States Environmental Protection Agency
Office of Water
Washington, DC
EPA 841-B-21-010
National Lakes Assessment 2022
Laboratory Operations
Manual
Version 1.1, May 2022
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Laboratory Operations Manual
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Version History
Version
Date
Revisions or Comments
0.0
February 2022
Internal EPA version for project QAC review and comments
1.0
February 2022
Final approved document
1.1
May 2022
• Corrected the atrazine sample preservation so that samples should be
kept chilled until analysis, not frozen (Section 8)
• Corrected typos in Tables 5.3, 7.2 and 11.2
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NOTICE
The intention of the National Lakes Assessment 2022 (NLA 2022) is to provide a comprehensive
assessment for lakes, ponds, and reservoirs across the United States. The complete documentation of
overall project management, design, methods, standards, and Quality Assurance/Quality Control
measures, is contained in companion documents, including:
National Lakes Assessment 2022: Quality Assurance Project Plan (QAPP) (EPA 841-B-21-009)
National Lakes Assessment 2022: Site Evaluation Guidelines (SEG) (EPA 841-B-21-008)
National Lakes Assessment 2022: Field Operations Manual (FOM) (EPA 841-B-21-011)
This document (Laboratory Operations Manual) contains information on the methods for analyses of the
samples for nine indicators: algal toxins (cylindrospermopsin and microcystins), atrazine screen, benthic
macroinvertebrates, fecal indicator (enterococci), phytoplankton, water chemistry and chlorophyll a,
and zooplankton to be collected during the project, quality assurance objectives, sample handling, and
data reporting. Environmental DNA (eDNA) and fish fillet contaminant analysis are also included as part
of the NLA 2022, and those methods are available separately. The NLA laboratory methods are based on
guidelines developed by federal agencies and methods employed by several key states that were
involved in the planning phase of this project. Methods described in this document are to be used
specifically in work relating to the NLA 2022. All project cooperator laboratories must follow these
guidelines. Mention of trade names or commercial products in this document does not constitute
endorsement or recommendation for use. Details on specific methods for site evaluation and sampling
can be found in the appropriate companion document. Revision history information is found in the
associated QAPP document.
The suggested citation for this document is:
U.S. EPA. 2022. National Lakes Assessment 2022. Laboratory Operations Manual. V.l.l. EPA 841-B-21-
010. U.S. Environmental Protection Agency, Washington, DC.
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TABLE OF CONTENTS
TITLE PAGE COVER PAGE
NOTICE III
TABLE OF CONTENTS IV
LIST OF TABLES VIII
LIST OF FIGURES VIII
LIST OF EQUATIONS VIII
LIST OF ACRONYMS AND ABREVIATIONS X
1.0 GENERAL LABORATORY GUIDELINES 2
1.1 Responsibility and Personnel Qualifications 2
1.2 Roles and Contact Information 2
1.3 Sample Tracking 2
1.4 Reporting 3
2.0 LABORATORY QUALITY CONTROL 4
2.1 Laboratory Verification Process/Technical Assessment 5
2.2 Inter-laboratory Comparison 6
3.0 ALGAL TOXIN IMMUNOASSAY PROCEDURE: CYLINDROSPERMOPSIN 7
3.1 Summary of Method 7
3.2 Health and Safety Warnings 7
3.3 Definitions and Required Resources (Personnel Qualifications, Laboratories, and Equipment) 7
3.3.1 Definitions 7
3.4 General Requirements for Laboratories 9
3.4.1 Expertise 9
3.4.2 Quality assurance and quality control requirements 9
3.4.3 Personnel 10
3.5 Equipment/Materials 10
3.6 Sample Receipt 10
3.7 Procedure 12
3.7.1 Sample Preparation 12
3.7.2 Kit Preparation 12
3.7.3 Insertion of Contents into Wells 13
3.7.4 Dilutions (if needed) 18
3.8 Pertinent QA/QC Procedures 19
3.8.1 QC Samples 19
3.8.2 Summary of QA/QC Requirements 19
3.9 Sample and Record Retention 21
4.0 ALGAL TOXIN IMMUNOASSAY PROCEDURE: MICROCYSTIN 22 on
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4.1 Summary of Method 22 lu
4.2 Health and Safety Warnings 22 z
4.3 Definitions and Required Resources (Personnel, Laboratories, and Equipment) 23 8
4.3.1 Definitions 23 q
4.4 General Requirements for Laboratories 24 u-j
4.4.1 Expertise 24 ^
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4.4.2 Quality assurance and quality control requirements 24
4.4.3 Personnel 24
4.4.4 Equipment/Materials 24
4.5 Sample Receipt 25
4.6 Procedure 26
4.6.1 Sample Preparation 26
4.6.2 Kit Preparation 27
4.6.3 Insertion of Contents into Wells 28
4.6.4 Dilutions (if needed) 33
4.7 Quality Measures 33
4.7.1 Assist an ce Visits 33
4.7.2 QC Samples 34
4.7.3 Summary of QA/QC Requirements 34
4.8 Sample and Record Retention 36
5.0 BENTHIC MACROINVERTEBRATE METHODS 37
5.1 Responsibility and Personnel Qualifications 37
5.2 Precautions 37
5.2.1 Sorting and Subsampling Precautions 37
5.2.2 Taxonomy Precautions 37
5.3 Equipment/Materials 38
5.3.1 Sorting and Subsampling Equipment/Materials 38
5.3.2 Taxonomy Equipment/Materials 38
5.4 Sample Receipt 39
5.5 Procedure 40
5.5.1 General 40
5.5.2 Subsampling 40
5.5.3 Sorting 42
Empty snail or bivalve shells 43
Specimens of surface-dwelling or strict water column2 arthropod taxa (e.g., Collembola, Veliidae, Gerridae,
Notonectidae, Corixidae, Culicidae, Cladocera, or Copepoda) 43
Incidentally-collected terrestrial taxa 43
5.5.4 Taxonomy Procedures 44
5.5.4.1 Taxonomic Level of Effort 46
5.6 Pertinent QA/QC Procedures 47
5.6.1 Sorting and Subsampling QC 47
5.6.2 Taxonomic QC 48
5.6.2.1 Internal Taxonomic QC 48
5.6.2.2 External Taxonomic QC 48
5.6.2.3 Taxonomic QC Review & Reconciliation 49
6.0 FECAL INDICATOR: ENTEROCOCCI 50
6.1 Scope and Application 50
6.1.1 Summary of Method 50
6.1.2 Definitions of Method 50
6.2 Interferences 51
6.3 Health & Safety Warnings 51
6.4 Personnel Qualifications 52
6.5 Equipment andSupplies 52
6.6 Reagents & Standards 52
6.7 Preparations Prior to DNA Extraction & Analysis 53
6.8 Procedures for Processing & qPCR Analysis of Sample Concentrates 54
6.8.1 Sample Processing (DNA Extraction) 54
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6.8.2 Sample Analysis by Enterococcus qPCR 55
6.8.2.1 Preparation of qPCR assay mix 55
6.8.3 Sample analysis sequence for SmartCycler 57
6.9 Storage & Timing of Processing/Analysis of Filter Concentrates 57
6.10 Chain of Custody 57
6.11 Quality Assurance/Quality Control (QA/QC) Procedures 57
6.12 Method Performance 58
6.13 Record Keeping & Data Management 58
6.14 Waste Management & Pollution Prevention 58
6.15 Tables, Diagrams, Flowcharts, Checklists, and Validation Data 59
6.15.1 Enterococcus qPCR Analysis Decision Tree (ADT) 62
6.15.2 "Modified" MagNA Pure LC DNA Purification Kit III Protocol 63
7.0 PHYTOPLANKTON METHODS 65
7.1 Responsibility and Personnel Qualifications 65
7.2 Precautions 65
7.3 Equipment/Materials 65
7.4 Sample Receipt 65
7.5 Procedure 67
7.5.1 Prepare Utermohl Sedimentation Chamber 67
7.5.2 Choose Count Method 67
7.5.2.1 Determine random fields 67
7.5.2.2 Determine transects 67
7.5.3 Identify and Enumerate 400 Natural Algal Units 67
7.5.4 Identify and Enumerate Larger, Rarer Taxa 68
7.5.5 Measure Cell Biovolumes 68
7.6 Calculation and Reporting 69
7.7 Pertinent QA/QC Procedures 69
7.7.1 Internal Taxonomic QC. 69
7.7.2 External Taxonomic QC 69
7.7.2.1 Plankton Re-identification 69
7.7.3 Taxonomic QC Review & Reconciliation 70
8.0 PESTICIDE SCREEN: ATRAZINE 72
8.1 Responsibility and Personnel Qualifications 72
8.2 Precautions 72
8.2.1 Storage and Stability 72
8.3 Equipment 73
8.4 Sample Receipt 73
8.5 Procedure 74
8.5.1 Test preparation 74
8.5.2 Procedural notes and precautions 74
8.5.3 Assay procedure 75
8.5.4 Results 75
8.5.5 Data Entry 76
8.6 Pertinent QA/QC Procedures 76
8.6.1 Internal QC 76
8.6.2 External QC 77
9.0 FISH TISSUE FILLET (WHOLE FISH COMPOSITE SAMPLE) 78
10.0 WATER CHEMISTRY AND CHLOROPHYLLS 79
10.1 Analytical Parameters 79
10.2 Sample Receipt 80
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10.3 Sample Processing and Preservation 81
10.3.1 Water Chemistry Samples 81
10.3.2 Chlorophyll a Samples 83
10.4 Performance-based Methods 83
10.5 Pertinent QA/QC Procedures 85
10.5.1 Laboratory Performance Requirements 85
10.5.2 Laboratory Quality Control Samples 85
10.5.3 Data Reporting, Review, and Management 90
10.5.4 Data Entry 92
11.0 ZOOPLANKTON METHODS 93
11.1 Responsibility and Personnel Qualifications 93
11.2 Precautions 93
11.3 Equipment/Materials 93
11.4 Sample Receipt 94
11.5 Procedure 95
11.5.1 Zooplankton Stratified Splitting 95
11.5.2 Taxonomy Procedures 96
11.5.2.1 Taxonomic Level of Effort 96
11.5.2.2 Macrozooplankton Identification and Enumeration (Excluding Rotifers and Nauplii) 96
11.5.2.2.1 General Analysis and Guidelines 97
11.5.2.2.2 Large Taxa Scan 97
11.5.2.3 Microzooplankton (Rotifers, Nauplii, and Crustaceans) 97
11.5.2.3.1 Preparation and Microzooplankton Enumeration 97
11.5.2.4 Measurement of Macrozooplankton and Microzooplankton 98
11.5.2.4.1 Crustaceans 98
11.5.2.4.2 Rotifers 98
11.6 Calculating and Reporting 98
11.6.1 Volume of water filtered 98
11.6.2 Macrozooplankton Densities 99
11.6.3 Microzooplankton Densities 99
11.6.4 Zooplankton Biomass Estimates 99
11.6.5 Results of Laboratory Processing, Sample Archiving 99
11.7 Pertinent QA/QC Procedures 100
11.7.1 Taxonomic QC 100
11.7.1.1 Internal Taxonomic QC 100
11.7.1.2 External Taxonomic QC 100
11.7.1.3 Taxonomic QC Review & Reconciliation 100
12.0 RESEARCH INDICATOR: ENVIRONMENTAL DNA (EDNA) 102
13.0 LITERATURE CITED 103
APPENDIX A: LABORATORY REMOTE EVALUATION AND VERIFICATION FORMS 107
APPENDIX B: SAMPLE LABORATORY FORMS 112
Benthic Macroinvertebrate Laboratory Bench Sheet 113
Phytoplankton Measurement Datasheet 114
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Zooplankton Sample Log In Form 115 h
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Zooplankton Enumeration Datasheet 116 llj
Zooplankton Measurement Datasheet 117 z
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APPENDIX C: STATE SAMPLE TRACKING SPREADSHEET
APPENDIX D: REPORTING TEMPLATES
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LIST
Table 1.1 Contact information 2
Table 3.1 Cylindrospermopsin login: required data elements 11
Table 3.2: Cylindrospermopsin: required data elements- data submission 16
Table 3.3: Cylindrospermopsin: quality control- sample analysis 19
Table 4.1 Microcystin: required data elements - login 26
Table 4.2 Microcystin: required data elements - data submission 31
Table 4.3 Microcystin: quality control - sample analysis 34
Table 5.1 Benthic macroinvertebrate login: required data elements 39
Table 5.2 Required level of taxonomic identification for benthic macroinvertebrates 46
Table 5.3 Laboratory quality control: benthic indicator 49
Table 6.1 Enterococci: PCR assay mix composition (according to draft EPA method 1606) 59
Table 6.2 Enterococci: batch calibrator & enterococcus standards PCR run - 7 samples 59
Table 6.3 Enterococci: sub batch test sample PCR run - 26 samples & 1 method blank 60
Table 6.4 Enterococci: laboratory methods 60
Table 6.5 Enterococci: parameter measurement data quality objectives 61
Table 6.6 Enterococci: laboratory QC procedures - enterococci DNA sequences 61
Table 7.1 Phytoplankton login: required data elements 66
Table 7.2 Laboratory quality control: phytoplankton indicator 70
Table 8.1 Atrazine login: required data elements 74
Table 8.2 Test tube labeling for atrazine assay 76
Table 8.3 Atrazine: quality control requirements 76
Table 10.1 Water chemistry parameters measured for the National Lakes Assessment 2022 79
Table 10.2 Water Chemistry login: required data elements 80
Table 10.3 Acid preservatives added for various analytes 82
Table 10.4 Summary of analytical methods used by NLA 2022 (PESD Laboratory, USEPA ORD-Corvallis) 84
Table 10.5 Laboratory method performance requirements for water chemistry and chlorophyll a sample analysis.
86
Table 10.6 Laboratory quality control samples: water chemistry indicator 88
Table 10.7 Data validation quality control for water chemistry indicator 90
Table 10.8 Data reporting criteria: water chemistry indicator 91
Table 10.9 Constants for converting major ion concentration from mg/Lto neq/L 92
Table 10.10 Factors to calculate equivalent conductivities of major ions 92
Table 11.1 Zooplankton login: required data elements 95
Table 11.2 Laboratory quality control: zooplankton indicator 101
LIST OF FIGURES
Figure 3.1 Cylindrospermopsin: sample template 14
Figure 4.1 Microcystin: sample template 29
Figure 6.1 Enterococci: qPCR analysis decision tree (ADT) 62
Figure 10.1 Water chemistry sample processing procedures 82
LIST OF EQUATIONS
Equation 3.1 Standard deviation
Equation 3.2 Percent (%) coefficient of variation
Equation 5.1 Percent sorting efficiency (PSE)
Equation 5.2 Percent difference in enumeration (PDE)
Equation 5.3 Percent taxonomic disagreement (PTD).
Equation 7.1 Phytoplankton abundance
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Equation 7.2 Percent difference 70
Equation 10.1 Percent ion difference (%IBD) 91
Equation 11.1 Volume of water filtered 98
Equation 11.2 Microcrustacean densities 99
Equation 11.3 Microzooplankton densities 99
Equation 11.4 Bray-Curtis Dissimilarity (BCd) 100
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LIST OF ACRONYMS and ABBREVIATIONS
DO dissolved oxygen
DOC dissolved organic carbon
ELISA enzyme-linked immunosorbent assay
EMAP Environmental Monitoring and Assessment Program
EtOH ethyl alcohol
FOM field operations manual
HDPE high density polyethylene
H2S04 sulfuric acid
HNO3 nitric acid
HQ Headquarters
IBD ion balance difference
IM information management
IT information technology
K potassium
LCS laboratory control sample
LOM laboratory operations manual
LRL lower reporting limit
Mg magnesium
MS matrix spike
MSD matrix spike duplicate
MDL method detection limit
MQO measurement quality objective
MPCA Minnesota Pollution Control Agency
MPN most probable number
MRL Mercury Research Laboratory
N Nitrogen
NARS National Aquatic Resource Surveys
NLA National Lakes Assessment
N20 nitrous oxide
Na sodium
NH3 ammonia
NIST National Institute of Standards
N03 nitrate
A absorbance
ANOVA analysis of variance
ANC acid neutralizing capacity
AV assistance visit
Ca calcium
CH4 methane
CI chloride
C02 carbon dioxide
COB close of business
CRM certified reference manual
CV coefficient of variation
d days
Dl deionized
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N02 nitrite
PAH polycyclic aromatic hydrocarbon
PCB polychlorinated biphenyl
PD percent difference
PDE percent difference in enumeration
PESD Pacific Ecological Systems Division
PSE percent sorting efficiency
PT proficiency tests
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 solution
QMP Quality Management Plans
RL Reporting Limit
RMSE root mean square error
RO reverse osmosis
RPD relative percent difference
RSD relative standard deviation
S standard deviation
SEG site evaluation guidelines
Si02 silica
S04 sulfate
SOPs standard operating procedures
SRM standard reference material
TMB tetramethylbenzidine
TN total nitrogen
TOC total organic carbon
TOCOR Task Order Contracting Officer's Representative
TP total phosphorus
USEPA United States Environmental Protection Agency
USGS United States Geological Survey
WRS Willamette Research Station
(now known as Pacific Ecology Studies Division in EPA's Office of Research and Development)
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NATIONAL LAKES ASSESSMENT 2022
LABORATORY OPERATIONS MANUAL
The U.S. Environmental Protection Agency (USEPA), in partnership with state and tribes, has designed
the National Lakes Assessment (NLA) 2022 to assess the condition of lakes, ponds and reservoirs
(referred to collectively as lakes throughout the document) in the United States. The NLA is one in a
series of National Aquatic Resource Surveys (NARS) conducted to provide the public with a
comprehensive assessment of the condition of waters in the U.S.
This manual contains procedures for laboratory analysis of samples collected from lakes throughout the
lower 48 states of the United States. The purposes of this manual are to:
1) document the standardized sample processing and analysis procedures used in the various
laboratories for the NLA 2022; and
2) provide guidance for data quality and a performance-based method approach to obtain
comparable results across all participating laboratories.
Detailed laboratory procedures are described for the following indicators: algal toxins, benthic
macroinvertebrates, fecal indicator (enterococci), phytoplankton, atrazine pesticide screen, water
chemistry, chlorophyll a, and zooplankton. In addition to the indicators listed above, samples for two
research indicators (i.e., environmental DNA (eDNA) and fish fillet contaminants) will be collected.
These will be done in collaboration with the USEPA's Office of Research and Development (ORD) and
Office of Science and Technology (OST), respectively. Methods for the fish fillet contaminants indicator
are maintained by OST.
Specific laboratory analysis procedures for water chemistry samples are not presented here. A list of
parameters to be analyzed as well as the performance-based methods and pertinent quality
assurance/quality control (QA/QC) procedures are outlined as requirements for laboratories to follow.
Alternative analytical methods for water chemistry are acceptable if they meet all specified performance
requirements described in this document. Acceptability is determined by the NLA project management
team (USEPA Office of Water).
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1.0 GENERAL LABORATORY GUIDELINES
1.1 Responsibility and Personnel Qualifications
All laboratory personnel shall be trained in advance in the use of equipment and procedures used for
the standard operating procedure (SOP) in which they are responsible. All personnel shall be responsible
for complying with all of the QA/QC requirements that pertain to the samples to be analyzed. Each
laboratory shall follow its institutional or organizational requirements for instrument maintenance.
Specific laboratory qualification documentation required for analysis is contained in the Quality
Assurance Project Plan (QAPP).
1.2 Roles and Contact Information
Table 1.1 presents contact information for the key personnel associated with NLA 2022. The USEPA
Headquarters Project Management Team consists of the Project Leader, Alternate Project Leaders, and
Project QA Lead. The Team is responsible for overseeing all aspects of the project and ensuring technical
and quality assurance requirements are properly carried out. The Team is the final authority on all
decisions regarding laboratory analysis.
The Contractor Logistics Coordinator and the NARS Information Management (IM) Coordinator track
the location of each NLA 2022 sample that involves laboratory processing. These coordinators will be
the laboratories main point of contact in regard to sample tracking.
Table 1.1 Contact information.
Title Name Contact Information
USEPA HQ Project Lead
Lareina Guenzel, OW
guenzel. Iareina(® eoa.gov
202-566-0455
USEPA HQ Project QA
Coordinator
Sarah Lehmann, OW
lehmann.sarah(® eoa.gov
202-566-1379
USEPA HQ Logistics Lead
Brian Hasty, OW
hastv.brain(® eoa.gov
202- 564-2236
USEPA HQ Laboratory
Review Coordinator
Kendra Forde, OW
forde. kendra(® eoa.gov
202-566-0417
Information
Management (IM) Center
Coordinator
Michelle Gover, GDIT
gover. michelle(® eoa.gov
541-754-4793
Contractor Logistics
Coordinator
Chris Turner, GLEC
cturner(®glec.com
715-829-3737
1.3 Sample Tracking
Samples are collected by a large number of field crews during the index period (June through
September). The actual number of lakes sampled on a given day will vary widely during this time. Field
crews submit electronic forms when they have shipped samples and the NARS IM Center inputs each
sample into the NARS IM database. Processing laboratories can track sample shipment from field crews
by accessing the information uploaded in the NARS IM database by way of the NARS SharePoint site.
Participating laboratories and all pertinent personnel will be given access to the NARS SharePoint site,
where they can acquire site and sample status information. This will include check-in and batching
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information on samples that have been shipped to the batch laboratory by field crews (either by
overnight shipment for perishable samples or batch shipments for preserved samples). The NARS IM
Center provides laboratories with spreadsheets of samples that the batch lab or field crews (as
appropriate) have sent to them. Upon sample receipt, the analysis laboratory must immediately
complete and email the sample tracking spreadsheet (containing the sample login and sample condition
information) to the IM Center Coordinator for confirmation of sample receipt. Each laboratory will make
arrangements with the USEPA HQ Laboratory Review Coordinator for access to the NARS SharePoint site
and the NARS IM Center Coordinator, both listed above, to ensure the process of sample check-in has
been organized before samples begin to arrive.
When the samples arrive from the field crews, laboratories also receive tracking forms in the shipment
(refer to the NLA 2022 FOM). These forms 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 IM Center Coordinator and/or
Contractor Logistics Coordinator immediately. For state laboratories conducting analyses in their own
laboratories, a state sample tracking spreadsheet is available from EPA.
1.4 Reporting
All laboratories must provide data analysis information to the HQ Project Management Team and the
NARS IM Center by March 1, 2023 or earlier as stipulated in contractual agreements. These reports must
include the following information and be reported in the data templates available separately from EPA.
• Sample Type (indicator)
• Site ID (ex: NLA22_AL-10007)
• Sample ID (ex: 999000)
• Pertinent information to the indicator
• Metadata for all fields
The submitted file name must state the following:
• Indicator name (ex: microcystins)
• Date of files submission by year, month, and day (ex: 2022_11_01)
• Laboratory name (ex: MyLaboratory)
Combined, the file name would look as follows: Microcystin_2022_ll_01_MyLaboratory.xlsx
As specified in the QAPP, remaining sample material and specimens must be maintained by the USEPA's
designated laboratory or facilities as directed by the NLA 2022 Project Lead. All samples and raw data
files (including logbooks, bench sheets, and instrument tracings) are to be retained by the laboratory for
3 years (or as outlined in EPA contract documents) or until authorized for disposal, in writing, by the
USEPA Project Leader. Deliverables from contractors and cooperators, including raw data, are
permanent as per USEPA Record Schedule 258. USEPA's project records are scheduled 501 and are also
permanent.
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2.0 LABORATORY QUALITY CONTROL
As part of the NLA 2022, field samples will be collected at each assessment site unless otherwise
specified. These samples will be sent to laboratories cooperating in the assessment. To ensure quality,
each Project Cooperator laboratory analyzing samples from the NLA 2022 will participate in a laboratory
verification process. All Project Cooperator laboratories will follow these guidelines.
No national program of accreditation for laboratory processing for most of our indicators currently
exists. For this reason, a rigorous program of laboratory evaluation and verification has been developed
to support the NLA 2022.
Given the large number of laboratories participating in the NLA 2022, it is not feasible to perform an
assistance visit3 (AV) on each of these laboratories. An AV would include an on-site visit to the
laboratory lasting at least a day. As a result, the USEPA Headquarters Project Management Team will
conduct remote review of laboratory certifications and accreditations of all laboratories and an inter-
laboratory comparison will be performed between some laboratories (mainly for biological indicators).
This process is called laboratory verification and is conducted before sample processing and analysis
begins. If issues arise from the remote review or inter-laboratory comparison that cannot be resolved
remotely then an on-site visit to the laboratory will be performed. The NLA 2022 Project Management
Team believes this approach meets the needs of this assessment and can ensure quality control on data
generated by the participating laboratories. General information is provided here and more specifics are
provided in Sections 2.1 and 2.2.
Competency
To demonstrate its competency, the laboratory shall provide analyte and matrix specific information to
the USEPA; or information specific to the relevant biological indicator. The USEPA will accept one or
more of the following as a demonstration of competency:
• Memorandum that identifies the relevant services that the laboratory provided for the National
Aquatic Resource Surveys in the past five years.
• Documentation detailing the competency of the organization, including professional
certifications for water-related analyses, membership in professional societies, a curriculum vita
for taxonomists, and experience with analyses that are the same or similar to the requirements
of this method.
• Demonstration of competency with sediment and water chemistry samples in achieving the
method detection limits, accuracy, and precision targets.
Quality assurance and quality control requirements q
QC
To demonstrate its competency in quality assurance and quality control procedures, each laboratory z
shall provide the USEPA with copies of the quality-related documents relevant to the procedure. 8
Examples include Quality Management Plans (QMP), QAPPs, and applicable SOPs. h
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To demonstrate its ongoing commitment, the person in charge of quality issues for the participating
laboratory shall sign the NLA QAPP Certification Page, which will be maintained at the USEPA in a quality
assurance file.
2.1 Laboratory Verification Process/Technical Assessment
Procedural review and assistance personnel are trained to the specific implementation and data
collection methods detailed in this NLA 2022 LOM. Laboratory evaluation and verification reinforces the
specific techniques and procedures for both field and laboratory applications. A remote evaluation and
verification procedure has been developed for performing assessment of all laboratories.
Laboratory evaluation and verification process will be conducted prior to data analysis to ensure that
specific laboratories are qualified and that techniques are implemented consistently across the multiple
laboratories generating data for the program. Laboratory evaluation and verification plans have been
developed to ensure uniform interpretation and guidance in the procedural reviews.
The procedure being utilized involves requesting the laboratory to provide documentation of its policies
and procedures. For the NLA 2022 project, we have requested that each participating laboratory provide
the following documentation:
• The laboratory's Quality Manual, (QMP) or similar document.
• SOPs for each analysis to be performed.
• Long term Method Detection Limits (MDLs) for each instrument used and Demonstration of
Capability for each analysis to be performed.
• A list of the laboratory's accreditations and certifications (e.g. NELAP, ISO, etc.), if any.
• Results from Proficiency Tests (PT) for each analyte to be analyzed under the NLA project.
• Relevant curriculum vitae and documents demonstrating previous survey participation.
If a laboratory has clearly documented procedures for sample receiving, storage, preservation,
preparation, analysis, and data reporting; has successfully analyzed PT samples (if required by the
USEPA, the USEPA will provide the PT samples); has a Quality Manual that thoroughly addresses
laboratory quality including standard and sample preparation, record keeping and QA non-conformance;
participates in a nationally recognized or state certification program; and has demonstrated the ability
to perform the testing for which program/project the audit is intended, then the length of an on-site
visit will be minimum, if not waived entirely. A final decision on the need for an actual on-site visit
should be made after the review and evaluation of the documentation requested.
If a laboratory meets or exceeds all of the major requirements and is deficient in an area that can be
corrected remotely, suggestions will be offered, and the laboratory will be given an opportunity to
correct the issue. A correction of the deficiency will then be verified remotely. The on-site visit should
only be necessary if the laboratory fails to meet the major requirements and is in need of assistance or
fails to produce the requested documentation.
All laboratory personnel responsible for quality must sign the NLA 2022 QAPP signature page.
In addition, all laboratories must sign a Laboratory Signature Form (in APPENDIX A: LABORATORY
REMOTE EVALUATION AND VERIFICATION FORMS) indicating that they will abide by the following:
1. Utilize procedures identified in the NLA 2022 Laboratory Operations Manual (or equivalent). If
using equivalent procedures, please provide procedural manual to demonstrate ability to meet
the required MQOs.
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2. Read and abide by the NLA 2022 Quality Assurance Project Plan (QAPP) and related SOPs.
3. Have an organized IT system in place for recording sample tracking and data analysis.
4. Provide data to the USEPA using the template provided in the Laboratory Operations Manual.
5. Provide data results in a timely manner. This will vary with the type of analysis and the number
of samples to be processed. Sample data must be received no later than May 1, 2018 or as
otherwise negotiated with the USEPA.
6. Participate in a laboratory technical assessment or audit if requested by the USEPA NLA staff
(this may be a conference call or on-site audit).
If a laboratory is participating in biological analyses, they must, in addition, abide by the following:
1. Use taxonomic standards outlined in the NLA 2022 Laboratory Manual.
2. Participate in taxonomic reconciliation exercises during the field and data analysis season, which
include conference calls and other laboratory reviews (see more below on Inter-laboratory
comparison).
2.2 Inter-laboratory Comparison
An inter-laboratory investigation is being implemented for the laboratories performing analysis on
benthic macroinvertebrates and zooplankton data for the NLA 2022. This process is defined as an inter-
laboratory comparison since the same protocols and method will be used by all participating
laboratories as described in this manual. No on-site assistance visit is envisioned for these laboratories
unless the data submitted and reviewed by the USEPA does not meet the requirements of the inter-
laboratory comparison described.
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3.0 ALGAL TOXIN IMMUNOASSAY PROCEDURE:
CYLINDROSPERMOPSIN
This chapter describes an enzyme-linked immunosorbent assay procedure that measures
concentrations of total cylindrospermopsin in water samples. The laboratory uses Eurofins
Technologies (formerly Abraxis) Cylindrospermopsin Test Kits ("kits") to conduct the analyses.
3.1 Summary of Method
Frozen cylindrospermopsin samples will be shipped on dry ice from the field crews to the contract
batching laboratory. The contract batching laboratory will maintain frozen samples and send the
batched samples to the analysis laboratory in coolers on ice. Samples will arrive in the analysis
laboratory frozen and they can be held in a freezer for several weeks. Cylindrospermopsin analyses
laboratories will need to process the samples within the 90-day holding time and in accordance with
timeframes outlined in contractual agreements.
The procedure is an adaption of the instructions provided by Eurofins Technologies (formerly Abraxis)
for determining total cylindrospermopsin concentrations using its ELISA kits. For freshwater samples,
the procedure's reporting range is 0.1 ng/Lto 2.0 ng/L, although, theoretically, the procedure can
detect, not quantify, cylindrospermopsin concentrations as low as 0.05 ng/L. For samples with
concentrations higher than 2.0 ng/L of cylindrospermopsin, the procedure includes the necessary
dilution steps.
3.2 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 p
sulfuric acid. In addition to the laboratory's usual requirements such as a Chemical Hygiene Plan, the o
laboratory must adhere to the following health and safety procedures: ^
LU
1. Laboratory facilities must properly store and dispose of solutions of weak acid. Si
0
2. Laboratory personnel must wear proper personal protection clothing and equipment (e.g., lab g
coat, protective eyewear, gloves). ?
3. When working with potential hazardous chemicals (e.g., weak acid), laboratory personnel must b
avoid inhalation, skin contact, eye contact, or ingestion. Laboratory personnel must avoid a:
contacting skin and mucous membranes with the TMB and stopping solution. If skin contact Q
occurs, remove clothing immediately. Wash and rinse the affected skin areas thoroughly with q
QC
large amounts of water. cl
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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.
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 ng/L to a maximum value of 2.0 ng/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 ng/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 ng/L and the reporting limit of 0.1 ng/L (i.e., >0.05 ng/L and <0.1 ng/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 ng/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 5 (standard deviation) as follows:
Equation 3.1 Standard deviation
1/2
1 V
S =
y ^ - Ay
n — 1 Z_i
t_1 q!
where n is the number of replicate samples, A„ is the absorbance measured for the /'th replicate. Per O
Section Error! Reference source not found., 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. o-
O
QC
Q
Then, calculate %CV as: O
Equation 3.2 Percent (%) coefficient of variation
S
%cv =
A
X 100
b
LU
QC
3
Q
LU
U
Dark or Dimly Lit: Away from sunlight, but under incandescent lighting is acceptable. §
CL
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limit of 0.1 ng/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.
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 ng/L. 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 limit equal to the
method detection limit of 0.05 ng/L. For diluted samples, the sample-specific detection limit will be the
product of the method detection limit of 0.05 ng/L and the dilution factor. Typical values for the
dilution factor will be 10 or 100.
Standard Deviation (S) shows variation from the average.
~z.
i/i
3.4 General Requirements for Laboratories o
3.4.1 Expertise
QC
LU
O.
To demonstrate its expertise, the laboratory shall provide EPA with one or more of the following: o
cc
O
• Memorandum that identifies the relevant services that the laboratory provided for the 2
1
National Aquatic Resource Surveys in the past five years. £5
• Documentation detailing the expertise of the organization, including professional certifications ^
for water-related analyses, membership in professional societies, and experience with analyses q
LU
that are the same or similar to the requirements of this method. y
O
QC
CL
3.4.2 Quality assurance and quality control requirements >
CO
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 O
Quality Management Plans (QMP), QAPPs, and applicable Standard Operating Procedures (SOPs). ^
To demonstrate its ongoing commitment, the person in charge of quality issues for the organization ^
shall sign the NLA 2022 QAPP Certification Page. ?
O
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3.4.3 Personnel
Laboratory Technician: This procedure may be used by any laboratory technician who is familiar with
the NLA 2022 QAPP, and this procedure in the NLA 2022 LOM. The laboratory technician also must be
familiar with the use of a multichannel pipette and plate readers.
External QC Coordinator is an EPA staff person who is responsible for selecting and managing the "QC
contractor." To eliminate the appearance of any inherent bias, the QC contractor must be dedicated to
QA/QC functions, and thus, must not be a primary laboratory or a field sampling contractor for NLA QC
contractor is responsible for complying with instructions from the External QC Coordinator;
coordinating and paying for shipments of the performance samples to participating laboratories;
comparing immunoassay results from the laboratories; and preparing brief summary reports.
3.5 Equipment/Materials
The procedures require the following equipment and information:
• Eurofins Technologies Cylindrospermospin ELISA (Microtiter) Test Kit, Product # 522011 (see
items in Section Error! Reference source not found.).
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).
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). ?
l/l
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. o
Project Quality Control Samples. o
Reagent Reservoirs (Costar Cat Number 4870): Plain plastic reservoir for reagents that zi
accommodate the use of a multi-channel pipette. u
Test tubes: For dilutions, if needed. ^
Timer: For measuring incubation times. o
LU
Vortex Genie: For mixing dilutions. u
Whatman Glass fiber syringe filter (25mm, GF 0.45 pim filter). ^
3.6 Sample Receipt
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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
Error! Reference source not found, 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
o If any sample is damaged or missing, contact the USEPA HQ Laboratory Review
Coordinator to discuss whether the sample can be analyzed.
3. Store samples in the freezer until sample preparation begins.
4. Maintain the chain of custody or 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
SITEJD
text
NLA site ID as used on sample label
VISIT_NO
numeric
Sequential visits to site (1 or 2)
SAMPLEJD
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.
l/l
CL
O
QC
LU
CL
CO
O
cc
o
b
QC
3
Q
LU
U
o
QC
CL
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3.7 Procedure
The following sections describe the sample, kit preparation and analysis.
3.7.1 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:
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:
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.
3. Second freeze-thaw cycle:
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.
4. 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 pim) into a new, labeled 20 mL
glass scintillation vial. Norm-ject syringes and Whatman Glass fiber syringe filters (25mm, ?
CO
GF 0.45 nm filter) or similar alternatives are acceptable. One new syringe and filter should q
be used per sample.
QC
LU
O.
3.7.2 Kit Preparation o
The technician prepares the kits using the following instructions: 2
Check the expiration date on the kit box and verify that it has not expired. If the kit has b
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 o
LU
for training activities.) ^
1. Verify that each kit contains all the required contents: cl
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o S5: 1.0 ng/L
o S6: 2.0 ng/L
• Kit Control (KC): 0.75 ng/L
• Cylindrospermospin-HRP conjugate Solution (vortex before use)
• Antibody solution (rabbit anti-Cylindrospermopsin)
• Wash Solution 5X Concentrate
• Substrate (Color) Solution
• Stop Solution
• Dilutant
• Foil bag with 12 microtiter plate strips
2. 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.
3. Adjust the microtiter plate, samples, standards, and the reagents to room temperature.
4. 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).
5. Prepare a negative control (NC) using distilled water.
6. The standards, controls, antibody solution, enzyme conjugate, color solution, and stop
solutions are ready to use and do not require any further dilutions.
7. 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 g
microtiter wells later. O
8. Handle the stop solution containing diluted H2S04 with care. 2j
CL
l/l
3.7.3 Insertion of Contents into Wells §
Q
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 b
the kits instead mechanized shaking. ^
3
Q
LU
U
9. While preparing the samples and kit, turn the plate reader on so it can warm up. The plate O
reader needs a minimum of 30 minutes to warm up.
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10. Turn on the computer so that it can control and access the plate reader. $
11. Print the template (Figure 3.1) to use as reference when loading the standards, controls, and o
samples as described in the next step. Templates contain rows, labeled with a marking pen, of 3
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 z
first use. O
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1
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12. Using the lOO-piL pipette, add 50 piL, 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
Hg/L), the kit control (0.75 piL), 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.
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
l/l
CL
o
13. Add 50 piL of the conjugate solution to each well using the multi-channel pipettor and a reagent cz
reservoir. Add 50 piL of the cylindrospermopsin antibody solution to each well using the multi- Si
O
channel pipettor and a reagent reservoir. Use dedicated reagent reservoirs for each reagent to g
avoid contamination from one reagent to another. ?
14. Place the sealing Parafilm over the wells. b
15. 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. a
16. Place the plate in an area away from light for 45 minutes. q
17. After 45 minutes, carefully remove the Parafilm. cl
18. Empty the contents of the plate into the sink, pat inverted plate dry on a stack of paper towels, <
CO
and then wash the wells of the plate four times with 250 piL 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. ^
19. Add 100 piL of substrate/ color solution to all wells using the multi-channel pipettor. ~
20. Cover the wells with Parafilm. ><
O
21. Manually mix the contents by moving the strip holder in a rapid circular motion on the [3
benchtop for 30 seconds. Be careful not to spill the contents. ej
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22. Place the strip holder in an area away from light for 30-45 minutes.
23. After 30-45 minutes, remove the Parafilm, add 100 piL of stop solution to the wells using the
multi-channel pipette and reagent reservoir in the same sequence as the substrate solution.
24. 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.
25. Dispose of solution in plates in a laboratory sink. Rinse plates and sink with water to dilute the
weak acid present.
26. 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 A, is the absorbance average for S,, then the
absorbance averages must be: A0> Ai> A2> A3> A4>A5>A6
iii. The average absorbance of the standard SO less than 0.8 (i.e., A0< 0.8).
iv. Two or more negative control samples with detectable concentrations of
Cylindrospermopsin (i.e., values > 0.1 ng/L). If this occurs, then evaluate possible
causes (e.g., cross-contamination between samples), and if appropriate, modify
laboratory processes before the next analytical run.
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. ^
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b. If either, or both, of the following failures occur for the sample, then the sample must be q
reanalyzed (maximum of two analyses, consisting of the original analysis and, if necessary, ^
one reanalysis): £
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i. The concentration value registers as HIGH (exceeds the calibration range). Dilute the §
sample for the reanalysis per Section Error! Reference source not found.. z
ii. The %CV > 15% between the duplicate absorbance values for a sample. ^
27. Record the results, even if the data failed the quality control requirements in #26b, for each ^
well in the USEPA's data template (Table 3.2). The required entries are for the following ^
columns: u
O
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
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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.b
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 0.1 ng/L (for
undiluted samples);
iii. H if the sample did not meet the holding time and was not analyzed within 90
days.
iv. HI if the concentration value registers as HIGH (exceeds the calibration range).
d. QUALITY FLAGS have codes for the following special cases:
i. QCF if there is a QC failure per step 26 above. The QCF code must be used for
all failures to facilitate data analysis.
ii. Qfor any other quality issue (describe in COMMENTS)
e. DILUTION FACTOR is only required if the sample was diluted.
f. 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
SITEJD
Text
NLA site ID code as recorded on sample label or
tracking form (blank if standard or control)
VISIT NUMBER
VISIT_NO
Numeric
Sequential visits to site (1 or 2) (blank if standard or
control)
SAMPLE ID
SAMPLEJD
Numeric
6-digit Sample ID number as recorded on sample
jar or tracking form (blank if standard or control)
DATE COLLECTED
DATE_COL
MMDDYY
Date sample was collected (blank if standard or
control)
CONDITION CODE
CONDITION_CODE
Text
Sample condition upon arrival at the laboratory
(blank if standard or control)
Flag
Definition
Blank or N
Not a sample (blank, standard,
or control)
OK
Sample is in good condition
C
Sample container is cracked
l/l
CL
O
QC
LU
CL
CO
O
cc
o
b
QC
3
Q
LU
U
o
QC
CL
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CONDITION
COMMENT
BATCH
IDENTIFICATION
TECHNICIAN
DATE ANALYZED
KIT EXPIRE DATE
KIT ID
R2
SAMPLE CODE
COND_COMMENT Text
'BATaTlF~^^ Numeric
TECHNICIAN
DATE_ANALYZED
KIT EXPIRE DATE
KIT ID
R2
SAM CODE
Text
MMDDYY
MMDDYY
Text
Numeric
Text
LOCATION
LOCATION
Text
PRIMARY OR
DUPLICATE
PRIM_DUP
Text
CONCENTRATION
CONC
Numeric
UNITS
MDL*
UNITS
MDL
Text
Numeric
RL
ABSORBANCE
DILUTION FACTOR
RL
ABSORBANCE
DILUTION_FACTOR
Numeric
Numeric
Numeric
CV ABSORBANCE
CV_ABSORB
Numeric
AVERAGE
ABSORBANCE
AVG_ABSORB
Numeric
AVERAGE
CONCENTRATION
AVG_CONC
Numeric
OA FLAG (if
appropriate)
QA_FLAG
Text
L Sample or container is leaking
ML Sample label is missing
NF Sample is not frozen
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 well
Code
KC
NC
Definition
Kit Control
Negative Control
SO, SI, S2, S3, S4,
S5, S6
QC
Standard
Quality Control
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)
Concentration or sample-specific detection limit of
contents of well in ng/L. Sample-specific detection
limit should be 0.1 ng/L if the sample hasn't been
diluted.
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/Lfor any result recorded
as <0.1 ng/L
Data qualifier codes associated with specific
identifications of voucher samples. These codes
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LU
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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. Unless the sample
was diluted, the concentration
will be 0.05 ng/L
H
HI
QCF
Q
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 ng/L
QC failure
Other quality concerns, not
identified above
Explanation for data flag(s) (if needed) or other
comments.
LABORATORY LAB_COMMENT Text
COMMENT
*ln the event for sample dilution is necessary to overcome the matrix effect, please notify EPA Laboratory Coordinator
3.7.4 Dilutions (if needed)
Dilutions if needed are prepared as follows (using clean glass tubes):
1. 1:10 dilution
a. Add 900 piL 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.)
Pipette 100 piL from the sample into the vial. (To provide more accurate dilutions and less
chance of contaminating the diluent, the diluent should be added to the vial before the
sample.)
Mix by vortexing.
Multiply final concentration and Abraxis' method detection limit of 0.05 ng/L by 10 to
obtain the sample-specific detection limit of .5 ng/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 piL from the sample and add to the
water (or diluent) in the appropriate labeled vial. Vortex the sample again.
b.
c.
d.
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3.
Multiply the final concentration and Abraxis' method detection limit of 0.05 ng/L by 100 to
obtain the sample-specific detection limit of 50 ng/L.
Other dilutions can be calculated in the same manner as #1 and #2 if needed.
3.8 Pertinent QA/QC Pro cedures
This section describes the quality assurance and quality control measures used to ensure that the data
will meet NCCA requirements.
3.8.1 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 NLA 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.
3.8.2 Summary of QA/QC Requirements
Table 3.3 provides a summary of the quality control requirements described in Sections Error!
Reference source not found, and Error! Reference source not found.. 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 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
Activity
Description and Requirements
Corrective Action
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.
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Quality Control
Description and Requirements
Corrective Action
Activity
Calibration
All of the following must be met:
o Standard curve must have a correlation
coefficient of >0.99;
o Average absorbance value, Ao, for SO must
be >0.80; and
o Standards S0-S6 must have decreasing
average absorbance values. That is, if Ai is
the average of the absorbance values for
Si, then the absorbance average values
must be: Ao > Ai > A2 > A3 > A4 >As>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 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 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.
• 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%).
ResuIts Within
Calibration Range
All samples are run in duplicate. If both of the
values are less than the upper calibration range
If a result registers as "HIGH", then
record the result with a data flag of
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Quality Control
Description and Requirements
Corrective Action
Activity
(i.e., 2.0 ng/Lfor undiluted samples), then the
requirement is met.
"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.
3.9 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
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periodically check the sample materials for degradation. O
Q
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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. ^
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After the stated time periods, the laboratory shall follow its internal protocols for disposal. q
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4.0 ALGAL TOXIN IMMUNOASSAY PROCEDURE: MICROCYSTIN
This chapter describes an immunoassay procedure that measures concentrations of total microcystins
in water samples using Eurofins Technologies (formerly Abraxis) Microcystins-ADDA Test Kits ("kits")c.
Each kit is an enzyme-linked immunosorbent assay (ELISA) for the determination of microcystins and
nodularins in water samples. Microcystins refers to the entire group of toxins, all of the different
congeners, rather than just one congener. Algae can produce one or many different congeners at any
one time, including Microcystin-LR (used in the kit's calibration standards), Microcystin-LA, and
Microcystin-RR. The different letters on the end signify the chemical structure (each one is slightly
different) which makes each congener different.
4.1 Summary of Method
Frozen microcystin samples will be shipped on dry ice from the field crews to the contract batching
laboratory. The contract batching laboratory will send the batched frozen samples to the analysis
laboratory in coolers on ice where they can be held in a freezer until ready for analysis. Microcystin
analyses laboratories will need to process the samples within the 90-day holding time and in
accordance with timeframes outlined in contractual agreements.
The procedure is an adaption of the instructions provided by Abraxis for determining total microcystins
concentrations using its ELISA-ADDA kits.d For freshwater samples, the procedure's reporting range is
0.15 ng/Lto 5.0 ng/L, although, theoretically, the procedure can detect, not quantify, microcystins
concentrations as low as 0.10 ng/L. For samples with higher concentrations of microcystins, the
procedure includes the necessary dilution steps.
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 z
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: b
O
1. Laboratory facilities must properly store and dispose of solutions of weak acid. y
2. Laboratory personnel must wear proper personal protection clothing and equipment (e.g., lab ^
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coat, protective eyewear, gloves). ^
3. When working with potential hazardous chemicals (e.g., weak acid), laboratory personnel must £
u
avoid inhalation, skin contact, eye contact, or ingestion. Laboratory personnel must avoid O
contacting skin and mucous membranes with the TMB and stopping solution. If skin contact ^
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occurs, remove clothing immediately. Wash and rinse the affected skin areas thoroughly with
large amounts of water.
4.3 Definitions and Required Resources (Personnel, Laboratories, and
Equipment)
This section provides definitions and required resources for using the procedure.
4.3.1 Definitions
The following terms are used throughout the procedure:
Absorbance (A) is a measure of the amount of light in a sample. A standard statistical curve is used to
convert the absorbance value to the concentration value of microcystins.
Calibration Range is the assay range for which analysis results can be reported with confidence. For
undiluted samples, it ranges from the reporting limit of 0.15 ng/L to a maximum value of 5.0 ng/L.
Values outside the range are handled as follows. If the value is:
• < 0.10 ng/L, then the laboratory reports the result as being non-detected ("<0.10 ng/L").
• Between 0.10 ng/L and the reporting limit of 0.15 ng/L (i.e., >0.10 ng/L and <0.15 ng/L), the
laboratory should record the value, but assign a QC code to the value (i.e., DATA_FLAG=J).
• 5.0 ng/L, the laboratory must dilute and reanalyze the sample.
Coefficient of Variation (CV): The precision for a sample is reported in terms of the %CV of its
absorbance values. To calculate the %CV, first calculate 5 (standard deviation) as follows:
1/2
1 V
S =
^rl>< - &
x 100 O
QC
u
n
i=i
where n is the number of replicate samples, A„ is the absorbance measured for the /'th replicate.
Samples are evaluated in duplicate (i=l or 2); controls are either evaluated in duplicate or triplicate
(i=l, 2,3). A is the average absorbance of the replicates. Then, calculate %CV as:
S
o/o CV = -=
A
Dark or Dimly Lit: Away from sunlight, but under incandescent lighting is acceptable. ^
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Duplicate samples (D) are defined as the second aliquots of an individual sample within a well plate. ^
Each sample, including the standards, are urn in pairs and both results for the primary (P) and duplicate g
aliquot are reported in the result column of the lab deliverable. ^
QC
CL
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). The detection limit is less than the reporting limit of 0.15 ng/L at which the o
measured value of the analyte can be reported with confidence. Also see "Sample-Specific Detection §
Limit." |
Primary samples (P) are defined as the first aliquot of a sample within a well plate. Each sample is z
analyzed in pairs. The result of both the primary aliquot and secondary, duplicate aliquot are reported q
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 provides the CV in their outputs, the procedure presents the
quality control requirement in terms of %CV instead of RSD.
Reporting Limit: For undiluted freshwater sample, the reporting limit is 0.15 ng/L. A reporting limit is
the point at which the measured value of the analyte can be reported with confidence.
Sample-Specific Detection Limit: Most samples will have a sample-specific detection equal to the
method's detection limit of 0.1 ng/L. For diluted samples, the sample-specific detection limit will be the
product of the method's detection limit of 0.1 ng/L and the dilution factor. Typical values for the
dilution factor will be 10 or 100.
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).
To demonstrate its ongoing commitment, the person in charge of quality issues for the organization
shall sign the NLA 2022 QAPP Certification Page. j=
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4.4.3 Personnel u
QC
Laboratory Technician: This procedure may be used by any laboratory technician who is familiar with y
the NLA 2022 QAPP, and this procedure in the NLA 2022 LOM. The laboratory technician also must be ^
familiar with the use of a multichannel pipette and plate readers. ^
External QC Coordinator is an EPA staff person who is responsible for selecting and managing the "QC £
contractor." To eliminate the appearance of any inherent bias, the QC contractor must be dedicated to O
QA/QC functions, and thus, must not be a primary laboratory or a field sampling contractor for NLA. °-
The QC contractor is responsible for complying with instructions from the External QC Coordinator; <
coordinating and paying for shipments of the performance samples to participating laboratories; <
comparing immunoassay results from the laboratories; and preparing brief summary reports. §
4.4.4 Equipment/Materials ^
The procedures require the following equipment and information: 2
x
• Eurofins Technologies (formerly Abraxis ADDA Test Kit, Product #520011 O
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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 - See Error! Reference source not found.2
Distilled or Deionized Water: For diluting samples when necessary.
ELISA evaluation software
Glass scintillation, LC, vials (two vials of 2 mL each)
Glass vials with Teflon-lined caps of size:
o 20 mL
o 4 mL (for dilutions)
• Multichannel Pipette & Tips: A single-channel and an 8-channel pipette are used for this
method.
• Norm-ject syringes (or equivalent)
• Paper Towels: For blotting the microtiter plates dry after washing.
• Permanent Marker (Sharpie Fine Point): For labeling samples, bottles, plates and covers.
• Plate Reader (e.g., Metertech Model M965 AccuReader; ChroMate®; or equivalent readers with
software to read the microtiter plates and measure absorbances).
• Reagent Reservoirs (e.g., Costar Cat Number 4870): Plain plastic reservoir for reagents that
accommodate the use of a multi-channel pipette.
• Test tubes: For dilutions, if needed.
• Timer: For measuring incubation times.
• Vortex Genie: For mixing dilutions.
• Whatman Glass fiber syringe filter (25mm, GF 0.45 pim filter)
4.5 Sample Receipt
Microcystins samples are kept on ice while in the field, frozen as soon as possible and kept frozen until -z.
they are shipped on dry ice to a central facility ("batching laboratory") or the State's laboratory. fci
Periodically, the batching laboratory ships the frozen samples to the microcystins laboratory. The u
batching and microcystins laboratory may retain the frozen samples for several weeks but samples g
must be analyzed within the 90-day holding time. ^
Because EPA initiates tracking procedures designed to recover any missing shipment, the laboratory a:
personnel responsible for tracking samples must start the following login steps within 24 clock hours of q
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receiving a delivery (Table 4.1 Microcystin: required data elements - login. u
QC
1. Report receipt of samples in the NARS IM sample tracking system (within 24 clock hours). Q-
2. Inspect each sample THE SAME DAY THEY ARE RECEIVED: <
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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. g
b. Record the information in into NARS IM, including the Condition Code for each sample:
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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.
3. Store samples in the freezer until sample preparation begins.
4. Maintain the chain of custody or sample tracking forms with the samples.
Table 4.1 Microcystin: required data elements - login
FIELD
FORMAT
DESCRIPTION
LAB ID
text
Name or abbreviation for QC laboratory
DATE RECEIVED
MMDDYY
Date sample was received by lab
SITE ID
text
LA 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
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 concerns, not identified above
CONDITION
text
Comments about the condition of the sample. Required for "Q". Optional for
COMMENT
others.
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c. Shake well to homogenize the sample, then transfer 10 mL to an appropriately labeled
clean 20 mL glass vial.
7. Second freeze-thaw cycle:
a. Freeze the vial.
b. Keep the large sample bottle (from the 500 mL initial sample) frozen for future use.
c. Thaw the sample vial contents to room temperature.
8. 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 pim) into a new, labeled 20 mL
glass scintillation vial. Norm-ject syringes and Whatman Glass fiber syringe filters (25mm,
GF 0.45 nm filter) or similar alternative are acceptable. One new syringe and filter should
be used per sample.
4.6.2 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 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 ng/L
o SI: 0.15 ng/L
o S2: 0.40 ng/L
o S3: 1.0 ng/L
o S4: 2.0 ng/L
o S5: 5.0 ng/L
Kit Control (KC): 0.75 ng/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.
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5. Remove 12 microtiter plate strips (each for 8 wells) from the foil bag for each kit. The plates
contain 12 strips of 8 wells. If running less than a whole plate, remove unneeded strips from
the strip holder and store in the foil bag, ziplocked closed, and place in the refrigerator.
6. Store the remaining strips in the refrigerator (4-8° C).
7. Prepare a negative control (NC) using distilled water
8. The standards, controls, antibody solution, enzyme conjugate, color solution, and stop
solutions are ready to use and do not require any further dilutions.
9. Dilute the wash solution with deionized water. (The wash solution is a 5X concentrated
solution.) In a 1L container, dilute the 5X solution 1:5 (i.e., 100 mL of the 5X wash solution plus
400 mL of deionized water). Mix thoroughly. Set aside the diluted solution to wash the
microtiter wells later.
10. Handle the stop solution containing diluted H2S04 with care.
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 (Error! Reference source not found.) 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.
14. Using the lOO-piL pipette, add 50 piL, 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 ng/L),
the kit control (0.75 piL), and negative control, in pairs, starting in the well in the upper left-
hand corner of the kit as shown in Error! Reference source not found.. 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.
4.6.3 Insertion of Contents into Wells
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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.
15. Add 50 piL of the pink antibody solution to each well using the multi-channel pipettor and a
reagent reservoir. Use dedicated reagent reservoirs for each reagent to avoid contamination
from one reagent to another.
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 90 minutes.
19. After 90 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 three times with 250 piL of washing solution using the
l/l
multi-channel pipette. After adding the washing solution each time, empty the solution into the fa
O
QC
u
sink and use the paper towels as before. §
21. Add 100 piL of enzyme conjugate solution to all wells using the multi-channel pipettor.
22. Cover the wells with Parafilm. ^
23. Manually mix the contents by moving the strip holder in a rapid circular motion on the q
LU
benchtop for 30 seconds. Be careful not to spill the contents. u
24. Place the strip holder in an area away from light for 30 minutes. ^
25. After 30 minutes, remove the Parafilm, decant, and rinse the wells three times again with 250 <
benchtop for 30 seconds. Be careful not to spill the contents. h
29. Place the plate in an area away from light for 20 minutes. ^
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30. After 20 minutes, remove the Parafilm and add 50 piL of stopping solution to the wells in the
same sequence as for the color solution. This will turn the contents a bright yellow color. After
adding the stopping solution, read the plate within 15 minutes.
31. 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.
32. Dispose of solution in plates in a lab sink. Rinse plates and sink with water to dilute the weak
acid present.
33. Perform QC evaluations of the data as follows:
a. If the following failures occur, then the laboratory must reanalyze all samples in the
analytical run:
i. Standard curve with a correlation coefficient of less than 0.99 (i.e., R<0.99)
ii. Standards S0-S5 must have decreasing absorbance values. First, calculate the average
values for each standard. That is, if A, is the absorbance average for S,, then the
absorbance averages must be:
Ao > Ai > A2 > A3 > A4 >As
iii. The average absorbance of the standard SO less than 0.8 (i.e., A0< 0.8).
iv. Two or more negative control samples with detectable concentrations of microcystins
(i.e., values > 0.1 ng/L). If this occurs, then evaluate possible causes (e.g., cross-
contamination between samples), and if appropriate, modify laboratory processes
before the next analytical run.
v. Results for control samples of outside the acceptable range of 0.75 +/- 0.185 ppb. That
is, results must be between 0.565 and 0.935.
b. If either, or both, of the following failures occur, then the sample must be reanalyzed
(maximum of two analyses, consisting of the original analysis and, if necessary, one
reanalysis): O
i. The concentration value registers as HIGH (exceeds the calibration range). Dilute the ^
sample for the reanalysis per Section Error! Reference source not found.. ^
ii. The %CV > 15% between the duplicate absorbance values for a sample. 3
Q
34. Record the results, even if the data failed the quality control requirements in #23b, for each
well in EPA's data template (see Table 4.2Error! Reference source not found, for required §
CL
elements). The required entries are for the following columns: >
<
a. TYPE should be one of the following codes: S0-S5 for standards; KC, NC, or SC for controls; $
P (primary) or D (for duplicate) of unknown sample. O
b. CONC contains the numeric concentration value. Two special cases: 3
i. Non-detected concentrations: If the sample is non-detected, then provide the sample- ^
specific detection limit which is 0.1 ng/L if the sample is undiluted with a salinity <3.5 2
ppt. See Section Error! Reference source not found, for calculating the sample-specific q
detection limit for a diluted sample.
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ii. If the result shows that it is HI'/' this indicates that the sample value is outside of the
calibration range and must be diluted and re-run using another analytical run. Leave
the CONC column blank and record 'HI' in the DATA FLAG column.
c. DATA FLAGS have codes for the following special cases:
i. ND if the sample was non-detected;
ii. J if the value is detected but at a level below the reporting limit of 0.15 ng/L (for
undiluted samples);
iii. H if sample did not meet the holding time and was not analyzed within 90 days.
iv. HI if the concentration value registers as HIGH (exceeds the calibration range).
d. QUALITY FLAGS have codes for the following special cases:
i. QCF if there is a QC failure per step 23 above. The QCF code must be used for all
failures to facilitate data analysis.
ii. Qfor any other quality issue (describe in COMMENTS)
e. DILUTION FACTOR is only required if the sample was diluted.
f. DUP AVG and DUP CV are required for duplicate samples and control samples (use all three
values if the controls are used in triplicate).
g-
Table 4.2 Microcystin: required data elements - data submission
STAGE
FIELD
FORMAT
DESCRIPTION
LOGIN
LAB ID
text
Name or abbreviation for QC laboratory
DATE RECEIVED
text
Date sample was received by lab
SITE ID
text
NLA site ID code as recorded on sample label or tracking form
(blank if standard or control)
VISIT NUMBER
numeric
Sequential visits to site (1 or 2) (blank if standard or control)
SAMPLE ID
numeric
6-digit Sample ID number as recorded on sample jar or tracking
form (blank if standard or control)
DATE COLLECTED
MMDDYY
Date sample was collected (blank if standard or control)
CONDITION CODE
text
Sample condition upon arrival at the laboratory (blank if standard
or control)
Flag Definition
Blank or N Not a sample (blank, standard, or control)
OK Sample is in good condition
C Sample container is cracked
L Sample or container is leaking
ML Sample label is missing
NF Sample is not frozen
Q Other quality concerns, not identified above
CONDITION
text
Comments about the condition of the sample.
COMMENT
O
QC
u
QC
3
Q
LU
U
o
QC
CL
>
<
l/l
l/l
<
o
X
o
<
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STAGE
FIELD
FORMAT
DESCRIPTION
ANALYSIS
BATCH ID
Numeric
Batch identification code, assigned by lab
TECHNICIAN
text
Name or initials of technician performing the procedure
KIT EXPIRE DATE
MMDDYY
Expiration date on kit box
KIT ID
text
Kit identification code. If one does not exist, assign a unique code
to each kit.
R2
numeric
R2 from curve fit to the average absorbance values for the
standards. Value is between 0 and 1.
TYPE
text
Type of solution being tested in the well
Code
Definition
KC
Kit Control
NC
Negative Control
SO, SI, S2, S3, S4,
S5
Standard
QC
Quality control sample
U
Sample of unknown concentration
LOCATION
text
Location of well in the kit (e.g., B5 would be the fifth well from the
left in the second row B)
PRIM_DUP
text
Primary or duplicate run of a sample of unknown concentration
(see Figure 4.1)
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.
UNITS
text
The units of the concentration of the CONC column
MDL
numeric
Minimum detection limit in the same units as the CONC column
RL
numeric
Reporting Limit in same units as the CONC column
ABSORBANCE
numeric
Absorbance value
DILUTION FACTOR
numeric
10, 100, etc for number of times the sample was diluted. If not
diluted, leave blank or record 1
CV_ABSORB
numeric
Calculated %CV of duplicate values of absorbance for a sample.
Only calculated for TYPE=U, KC, or NC. Enter %CV. Value is
between 0 and 100%.
AVG_ABSORB
numeric
Calculated average of absorbance values for a sample. Only
provided for TYPE=U, KC, NC, or SC. Average value of the original
sample and its duplicate (or replicates for KC and NC).
AVG_CONC
numeric
Calculated average of concentration values for a sample.
QA FLAG (if
appropriate)
text
Data qualifier codes associated with specific identifications of
voucher samples. These codes provide more information that
those used when reporting receipt of samples. A technician may
use alternative or additional qualifiers if definitions are provided
as part of the submitted data package (e.g., as a separate
worksheet page of the data submission file).
Flag
Definition
ND
Concentration below detection. Unless the sample was
diluted, the concentration will be 0.1 ng/L
H
Sample did not meet the holding time and was not
analyzed within 90 days.
l/l
b
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QC
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QC
3
Q
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QC
CL
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STAGE FIELD FORMAT DESCRIPTION
HI
Result indicated that a high concentration (i.e., outside
calibration range)
J
Concentration above detection but below reporting limit.
QCF
QC failure
Q
Other quality concerns, not identified above
LAB COMMENTS
text
Explanation for data flag(s) (if needed) or other comments.
4.6.4 Dilutions (if needed)
Dilutions if needed are prepared as follows (using clean glass tubes):
1:10 dilution
a. Add 900 piL of distilled water to a clean vial. (Note: Dilutions may also be made using the kit's
diluent rather than distilled water.)
b. Pipette 100 piL from the sample into the vial. (To provide more accurate dilutions and less
chance of contaminating the diluent, the diluent should be added to the vial before the
sample.)
c. Mix by vortexing.
d. Multiply final concentration and Abraxis' detection limit of 0.1 ng/L by 10 to obtain the sample-
specific detection limit of 1.0 ng/L.
• 1:100 dilution
a. Add 3.96 mL of distilled water to a clean, appropriately labeled glass vial. (Note: Dilutions may
also be made using the kit's diluent rather than distilled water.)
b. Vortex the sample to mix thoroughly, then pipette 40 piL from the sample and add to the water
(or diluent) in the appropriate labeled vial. Vortex.
Other dilutions can be calculated in the same manner as #1 and #2 if needed.
l/l
c. Multiply the final concentration and Abraxis' detection limit of 0.1 ng/L by 100 to obtain the (j>
sample-specific detection limit of 10 ng/L. oe
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4.7 Quality Measures 2
This section describes the quality assurance and quality control measures used to ensure that the data §
Q_
will meet NLA's requirements. >
<
4.7.1 Assistance Visits <2
<
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Assistance visits are intended to familiarize EPA with actual procedures being implemented by different z
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. 5
O
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4.7.2 QC Samples
Once or twice during the survey, sets of five identical Performance Evaluation (PE) samples will be
provided to all participating laboratories by an External QC Coordinator. As determine by the External
QC Coordinator, the PE samples may be synthetic; aliquots of additional samples collected at NLA sites;
or reference samples obtained from an organization such as the National Institute of Standards and
Technology or Eurofins Technologies (formerly Abraxis). Each laboratory shall analyze the PE samples
following the same procedures used for the other samples analyzed. The External QC Coordinator will
compare the results to the expected value. The results of the comparisons shall be made available to
the EPA QC Coordinator for review. Based upon the evaluation, the External QC Coordinator may
request additional information from one or more laboratories about any unique laboratory practices
that might account for differences and may convene a conference call with all participating labs to
identify causes of and if possible, reconcile those differences. With this additional information, the
External QC Coordinator shall determine an appropriate course of action, including no action, re-
running samples, flagging the data, or excluding some or all the laboratory's data.
4.7.3 Summary of QA/QC Requirements
Table 4.3Error! Reference source not found, provides a summary of the quality control requirements
described in Sections 4.5 and 4.6.
Table 4.3 Microcystin: quality control - sample analysis
Quality Control
Description and Requirements
Corrective Action
Activity
Kit - Shelf Life
Is within its expiration date listed on kit box.
If kit has expired, then discard or set
aside for training activities.
Kit - Contents
All required contents must be present and in
acceptable condition. This is important because
Abraxis has calibrated the standards and reagents
separately for each kit.
If any bottles are missing or damaged,
discard the kit.
Calibration
All of the following must be met:
o Standard curve must have a correlation
coefficient of >0.99;
o Average absorbance value, Ao, for SO must
be >0.80; and
Standards S0-S5 must have decreasing average
absorbance values. That is, if Ai is the average of
the absorbance values for Si, then the absorbance
average values must be: Ao> Ai> A2> A3> A4>As
If any requirement fails:
• Results from the analytical run
are flagged QCF
All samples in the analytical run are
reanalyzed until calibration provides
acceptable results.
Kit Control
The average concentration value of the duplicates
(or triplicate) must be within the range of 0.75 +/-
0.185 ng/L. That is, results must be between 0.565
and 0.935.
If either requirement fails:
• Results from the analytical run
are not reported
• The lab evaluates its processes,
and if appropriate, modifies its
processes to correct possible
contamination or other problems.
Negative Control
The values for the negative control replicates must
meet the following requirements:
o All concentration values must be < 0.15
Hg/L (i.e., the reporting limit); and
l/l
b
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QC
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QC
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QC
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Quality Control
Description and Requirements
Corrective Action
Activity
One or more concentration results must be
nondetectable (i.e., <0.10 ng/L)
The lab reanalyzes all samples in the
analytical run until the controls meet
the requirements.
Sample
Evaluations
All samples are run in duplicate. Each duplicate pair
must have %CV<15% between its absorbance
values.
If %CV of the absorbances for the
sample>15%, then:
• Record the results for both
duplicates.
• Report the data for both duplicate
results as Quality Control Failure
"QCF"; and
• Re-analyze the sample in a new
analytical run. No samples are to
be run more than twice.
If the second run passes, then the
data analyst will exclude the data
from the first run. If both runs fail, the
data analyst will determine if either
value should be used in the analysis
(e.g., it might be acceptable to use
data if the CV is just slightly over
15%).
ResuIts Within
Calibration Range
All samples are run in duplicate. If both of the
values are less than the upper calibration range
(i.e., 5.0 ng/Lfor undiluted samples), then the
requirement is met.
If 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.
l/l
b
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QC
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QC
3
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QC
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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.
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5.0 BENTHIC MACROINVERTEBRATE METHODS
This procedure is adapted from the Wadeable Streams Assessment: Benthic Laboratory Methods
(USEPA. 2004),and is modified to facilitate processing and identification of benthic organisms collected
in the littoral zone of lakes and reservoirs.
Benthic macroinvertebrate samples will be preserved in the field with ethyl alcohol (EtOH) and shipped
from field crews to a contract batching laboratory. The contract batching laboratory will send the
batched samples to the analysis laboratory. Preserved samples will arrive in the analysis laboratory and
can be held for several months. If samples are not processed soon after receipt, then periodic evaluation
of samples should occur to ensure that sufficient EtOH levels are maintained. Benthic invertebrate
analysis laboratories will need to process samples in accordance with the time frame outlined in
contractual agreements. Contractual agreements for delivery of data do not supersede indicator holding
times.
5.1 Responsibility and Personnel Qualifications
This procedure may be used by any person who has received training in identification of freshwater
benthic macroinvertebrates, i.e., taxonomy. 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. A second
taxonomist will re-identify a randomly-selected 10% of the samples for QC, as noted below, 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. Samples are sent to the laboratory from the field on a regular
basis to avoid delays in processing and sample identification.
5.2 Precautions
5.2.1 Sorting and Subsampling Precautions
Because it can be difficult to detect the organisms in lake samples (due to inexperience, detritus, etc.), a
person who has received instruction from senior biology staff familiar with processing benthic samples
must have a QC check performed by qualified personnel (laboratory QC Officers) only. These QC checks
will be performed in the pertinent QA and QC Procedures section. The laboratory QC Officers must
perform these QC checks immediately following sorting of each grid.
Thoroughly clean all sorting equipment and make sure all equipment is free of organisms prior to sorting
the next sample.
The USEPA will supply a list of taxa that have been collected from previous iterations of the National
Lakes Assessment (provided during laboratory initiation call). The laboratories will use this list as the
primary source for taxonomic names to be used in the current NLA sample processing. During the
processing of samples, if new taxa are encountered that are not part of the existing NLA taxa list then
analysts must provide either a literature citation for this new taxa or its Integrated Taxonomic
Information System (ITIS; Web at http://itis.gov) number, if available. New taxa will not be excepted
unless either of these items are provided.
5.2.2 Taxonomy Precautions
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The analyst must prepare a list of primary and secondary technical literature used in completing the
identifications and submit this list to the Project Quality Assurance Manager when samples are returned
(see below).
5.3 Equipment/Materials
5.3.1 Sorting and Subsampling Equipment/Materials
• U.S. 35 sieve (500 pim)
• Round buckets
• Standardized gridded screen (370-nm)
• Mesh screen, 30 squares (6 cm2 each) with white plastic holding tray1
• 6-cm scoop
• 6-cm2 metal dividing frame ("cookie cutter")
• White plastic or enamel pan (6" x 9") for sorting
• Scissors
• Teaspoon
• India ink pens
• Dropper
• Fine-tipped forceps (watchmaker type, straight and curved)
• Specimen vials with caps or stoppers
• Sample labels for specimen vials
• 70-80% denatured ethanol (EtOH)
• Benthic Sample Log-In Form
• Benthic Macroinvertebrate Laboratory Bench Sheet (APPENDIX B: SAMPLE
LABORATORY FORMS)
• Stereo zoom microscope (6-10X magnification)
5.3.2 Taxonomy Equipment/Materials
• Stereo dissecting microscope with fiberoptics light source (50-60X magnification)
• Compound microscope (10, 40, and 100X objectives, with phase-contrast capability)
• Petri dishes
• Microscope slides (1" x 3" flat, precleaned)
• Cover slips (appropriately sized)
• CMCP-10 (or other appropriate mounting medium)
• India ink pens
• Dropper
• Fine-tipped forceps (watchmaker type, straight and curved)
'Some laboratories may choose not to use the gridded screen in a plastic holding tray.
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• Specimen vials with caps or stoppers
• Sample labels for specimen vials
• 70 - 80% denatured ethanol in plastic wash bottle
• Benthic Macroinvertebrate Taxonomic Bench Sheet
• Hand tally counter
5.4 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 1.2 of the
manual 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 sample tracking
form.
b. Record the information in Table 5.1 for the NARS IM Team, 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
c. 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 Chapter 2 of the
Manual).
3. Store samples until sample preparation begins.
4. Maintain the sample tracking forms with the samples.
Table 5.1 Benthic macroinvertebrate login: required data elements
FIELD
FORMAT
DESCRIPTION
LAB
text
Name or abbreviation for laboratory
DATE RECEIVED
MMDDYY
Date sample was received by laboratory
SITE ID
text
NLA 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
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FIELD
FORMAT
DESCRIPTION
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
Q
Other quality concerns, not identified
above
CONDITION
COMMENT
text
Comments about the condition of the sample.
5.5 Procedure
5.5.1 General
1. Record receipt of samples in the laboratory on the Benthic Sample Log-In form (APPENDIX B:
SAMPLE LABORATORY FORMS). Assign the appropriate chronological bench number to each
sample. Store samples at room temperature until ready for processing.
2. Sample container(s) may arrive with very little alcohol to expedite shipping times and to account
for hazardous material handling requirements. Inspect each jar THE SAME DAY THEY ARE
RECEIVED and refill them with 70-80% EtOH if necessary. After refilling the sample containers,
store them until sorting begins. Check samples periodically to ensure EtOH levels are sufficiently
maintained.
3. Use a gridded screen to sort a randomized 500-organism subsample separately from the rest of
the sample. Preserve the sorted organisms in one or more specimen vials with 70-80% EtOH.
4. For each sample, document the level of effort, or proportion of sample processed (e.g., number
of grids processed), on the Benthic Macroinvertebrate Laboratory Bench Sheet (APPENDIX B:
SAMPLE LABORATORY FORMS).
5. Record the following information on internal sample labels used for vials of sorted material with ^
India ink pen on cotton rag paper or an acceptable substitute. °
a. Station Name tj
b. Station Location ^
LU
c. Station Number ^
d. Date Sorted §5
e. Sorter's Initials
f. "1 of x" or "2 of x", etc. if the sample is sorted into >1 vial (where x is the total number of
vials for the sorted sample) ?
O
5.5.2 Subsampling u
1. Remove the lid from the sample container and remove the internal sample label (save the ^
label—it will need to be returned to the sample container with the archived portion of the E
sample that does not get processed). Record the sample collection information on a Benthic z
CO
40
QC
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Macroinvertebrate Laboratory Bench Sheet. Header information required includes both project
name and date the sample was collected. Set the bench sheet aside.
2. Carefully decant the alcohol from the sample container by pouring the fluid through a sieve (U.S.
35) into a separate container (the alcohol is saved to preserve the archived portion of the
sample that does not get processed). Inspect the mesh of the sieve for any organisms and return
any organisms found back to the sample.
3. Transfer the homogenized sample material to the gridded screen portion of the grid (use more
than one subsampling device if necessary). Wash the sample thoroughly by running tap water
over it to remove any fine material. If there is more than one jar for any particular sample,
empty and wash each jar onto the Caton-type grid one at a time, making sure to spread each
jar's contents evenly across the tray. Multiple jars from the same sample should all be emptied
onto the same Caton grid (or suitable alternative subsampling tray). If the amount of leaf litter
or other detrital material exceeds that which fills the tray to the level of the wall panels (it
should be spread as evenly as possible), divide it among two or more trays.
4. NOTE: Elutriation of a sample is acceptable for samples with heavy amounts of inorganic
substrate (e.g., sample that has 4 or 5 jars total and 2 or 3 with gravel or sand) once it has been
delivered to the laboratory, before subsampling has begun on that particular sample. Magdych
1981 describes an inexpensive, easily constructed elutriator. An example of an acceptable
elutriation method is as follows:
a. Pour alcohol off of sample containers through sieve (at least 500 pim). Also deposit leaf litter
and any other organic material (leaves, sticks, algae) onto sieve.
b. Depending on amount of inorganic material (gravel, sand, silt), pour all or a portion of this
material into a rectangular Tupperware/Rubbermaid container and cover with water.
c. Circulate (elutriate) sample with water and allow any organisms that might be in the
gravel/sand to float to the top of the water and pour the water through the sieve.
d. Repeat this until the water runs clear.
e. Fill the plastic container (that still has the inorganic material in it) with water one more time
and take it to a well-lit, flat surface. Inspect it here under a ring light w/ 3x magnification for
any remaining organisms. Have another sorter double check for organisms.
f. Once you are certain there are no organisms remaining in the plastic container, wash the
water through the sieve and dump the inorganic material into a waste bucket.
g. Repeat this process until all of the inorganic material has been elutriated and checked for
heavier organisms, such as clams, mussels, or worms.
5. Spread the sample now in the circular sieve over the 30-grid Caton tray.
6. Place the gridded screen into the larger white tray. (Note: Some laboratories may not use the
gridded screen and holding tray). Add enough water to spread the sample evenly throughout
the grid (the water level should be relatively close to the top of the white tray). Spread the
sample material over the bottom of the pan as evenly as possible. Move the sample into the
corners of the pan using forceps, spoon, or by hand. Vibrate or shake the pan gently to help
spread the sample.
7. Lift the screen out of the white tray to drain. Pour off or siphon excess water from the white tray
and set the screen back into the tray. Leave just enough water in the bottom of the tray so that
it barely covers the screen once it is returned to the tray to allow the sample to remain moist.
8. Use a random number generator to select at least 10% of the grids (usually 3 grids in a 30-grid
tray) to process (select one letter and one number, e.g., A-5, F-2). A minimum of three grids
(Canton tray or larger grid size), or 10% of the grids (if a grid of more than 30 squares [<6 cm2
each] is used) are sorted from the sample to ensure that the subsample material is
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representative of the overall sample. Remove all the material from the first grid. If two trays are
being used to hold a large sample, remove the material from the same grid on the second pan.
Remove the material as follows:
a. Place the metal dividing frame or "cookie cutter" over the sample at the approximate
location of the grid selected for processing (based on the letters and numbers marked on
the sides of the gridded tray). Use a pair of rulers or other straight edges to facilitate lining
up the cookie cutter at the intersection if necessary.
b. Remove the material within the "cookie cutter" using the 6-cm scoop, a teaspoon, forceps,
or dropper. Depending on the consistency of what is in the sample, it might be necessary to
cut the material along the outside of the "cookie cutter" with scissors or separate it with
forceps so that only one grid's worth of sample material is used. Inspect the screen for any
remaining organisms. Use the following rules when dealing with organisms that lie on the
line between two grids:
i. An organism belongs to the grid containing its head.
ii. If it is not possible to determine the location of the head (i.e., for worms), the organism
is considered to be in the grid containing most of its body.
iii. If the head of an organism lies on the line between two grids, all organisms on the top of
a grid and those on the right side of a grid belong in that grid, and are picked with that
grid.
c. Quarter the grid (if necessary, see Section 5.5.3, #2). Place the material from the selected
grid(s) into a separate white plastic or enamel pan. Add the necessary amount of water to
the pan to facilitate sorting.
9. Set the subsampling device aside in case more grids need to be retrieved later. Cover the sample
with aluminum foil to prevent desiccation of the sample and damage to specimens (periodically
moisten the sample with water from a spray bottle if the top layer begins to dry). Between each
subsampling operation, be careful not to disturb the subsampling device to prevent
redistribution of specimens, which could possibly change the probability of selection.
5.5.3 Sorting
1. Randomly select at least 10% of the tray or three grids in the case of a Caton tray (assuming 30
grids).
2. If the number of organisms appears to exceed the target number (500 organisms) in the
collective three grids, quarter each grid, and randomly select a quarter for initial sorting. Sort
the quarter volume of the first grid. Sort the remaining two grids (quartered) in successive order
(compositing of the first three grids is not done).
3. If the number of organisms is below the target number, then process another fraction of each
grid until the target number of 500 and a maximum of 600 (500+20%) is reached. All organisms
from the selected fraction, or grid, must be sorted to minimize bias.
4. If the target is not reached when the three grids are fully processed (including organisms
recovered during QC checks), randomly select subsequent grids and pick each to completion
until 500+20% organisms is reached. If the target number of organisms is reached within the
fraction of the first or second grids, stop sorting for that sample on completion of the sorting of
the corresponding fraction (i.e., the third grid quarter would not be processed).
5. If the target level of 500 organisms is not reach within 20 hours of sorting, stop sorting and
preserve the remaining unsorted material in 70-80% denatured EtOH, and store for future
sorting, if needed.
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6. Remove the macroinvertebrates from the detritus with forceps. Sort all samples under a
minimum of 6x (maximum of lOx) dissecting microscope. Perform QC checks using the same
power microscope. Place picked organisms in an internally-labeled vial (or larger container, if
necessary) containing 70-80% denatured EtOH.
7. Keep a rough count of the number of organisms removed and enter the number of organisms
found in each grid under the appropriate column on the Benthic Macroinvertebrate Laboratory
Bench Sheet. Enter the sorter's initials in the appropriate column on the bench sheet for each
grid sorted.
8. Do not remove or count:
• Empty snail or bivalve shells
• Specimens of surface-dwelling or strict water column2 arthropod taxa (e.g., Collembola,
Veliidae, Gerridae, Notonectidae, Corixidae, Culicidae, Cladocera, orCopepoda)
• Incidentally-collected terrestrial taxa.
9. Also, do not count fragments such as legs, antennae, gills, or wings.
10. For Oligochaeta, attempt to remove and count only whole organisms and fragments that include
the head; also, do not count fragments that do not include the head. If a sorter is unsure as to
whether a specimen should be counted or not, he or she should place the organism in the sort
vial without counting it (the final count is made by the taxonomist).
11. Once it is picked by the initial sorter, an experienced, certified, laboratory QC Officer must check
each sample for missed organisms before another sample is processed. The laboratory QC
Officer will count any missed organisms found and place them into the sample vial, or other
suitable sample vial. The laboratory QC Officer will note the number of organisms missed on the
Benthic Macroinvertebrate Laboratory Bench Sheet and add that number to the final count of
the sample.
12. If the last grid (or quarter) being processed results in more than 600 organisms (i.e., > 20%
above target number), evenly redistribute all of the organisms (without detritus) in a Petri dish
(or other small container, i.e., finger bowl, etc.) divided into pie slices (1-8) containing just
enough water to cover the sample. Randomly choose slices and count organisms that are wholly
contained within the slices. If an organism is lying between two slices, use the criteria in Section
5.5.2 #8 (B) to determine which slice it belongs in. Choose slices until you reach the target
number (500 +20%). As with picking grids and quarters, you must pick an entire pie slice, even if
the sample goes over 500 organisms as long as it remains under 600 total organisms.
13. Once the QC check of the material in the pan has been completed, remove the material from
the pan and place it in a separate container with preservative (70-80% EtOH). Label the
container "Sorted Residue," on both internal and external labels ("Sorted Residue" will include
material from all grids processed for each sample). Internal sample labels should be made of
cotton rag paper or an acceptable substitute, recording the same information as before.
14. After the laboratory QC Officer completes the QC check, and the target number has been
reached, search the entire tray for 5-10 minutes, looking for large/rare organisms (Vinson and
Hawkins, 1996). Large/rare is defined as any organism larger than 0.5" long and found in less
than one eighth of the tray holding the entire sample. Place any organisms found into a vial
labeled "L/R" for "Large/Rare."
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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15. Return all material not subsampled (remaining on the grid) to the original container with the
preservative. This container will include the original sample labels. Prepare two additional labels
"Unsorted Sample Remains" and place one inside the container and attach the other to the
outside of the container. Replace the lid and tighten securely. Archive the container until all
appropriate QC checks are completed (subsampling and taxonomy). The decision to discard any
sample portion should be done only following joint approval of the laboratory QC Officer and
the Project Manager.
16. Record the sorting date each sample was completed near the top right corner of the bench
sheet.
1. The taxonomic target for benthic invertebrates is identified in Section 5.5.1 #3
2. Upon receipt of a set of sample vials from the project cooperator or the contractor batch
laboratory, remove the sample tracking form from the shipping container, and sign and date it in
the "received by" space to verify that the samples were received. Compare all sample numbers
on the form with those entered on the labels of samples that actually were in the shipment. If
any vials were broken, notify the project facilitator immediately. Maintain the sample tracking
form with the samples; it will be needed to return the samples.
3. Empty one sample vial at a time into a small Petri dish. Add 80% denatured EtOH to keep the
organisms covered. Remove the internal sample label and complete the top portion of a Benthic
Macroinvertebrate Taxonomic Bench Sheet, using the information from the label or that
provided by the project facilitator.
4. View the sample under the stereo dissecting microscope and remove similar organisms to other
dishes (keep these covered with 80% EtOH). Identify organisms to the correct taxonomic level
for the project (usually genus, Table 5.2). However, according to the laboratory manager's
discretion, a taxonomist can identify any organism finer than the target level if he or she is
confident in the identification. Record the identifications on the Benthic Macroinvertebrate
Taxonomic Bench Sheet (under taxon). Enter the number of larvae, pupae, and adults of each
taxon under those columns on the bench sheet. Use the following steps to compare the final
taxa list for each site to that of the provided USEPA NLA taxa list.
a. Merge the USEPA provided NLA taxa list with the laboratory electronic bench sheet data by
merging the TARGET_NAME from the NLA taxa list to the TAXON_NAME from the individual
sample data.
b. Any taxa in the individual sample data that do not match a name from the NLA taxa list
should be checked for the following potential issues. If after this is completed and it is
determined that the non-matched taxa is unique then this taxa name can be included, but
only after either a literature citation or an ITIS number are provided.
i. Abbreviations
ii. Extra information identifiers (e.g., sp., spp.,, nr., cf., genus 1, w/ hair chaete)
iii. Extra character (e.g., "?", "Acentrella Pturbida", blank space)
iv. The word "probably" or "prob" (e.g., "Microcylloepus prob. similis")
v. Identifying to a lower level than in ITIS (e.g., to species rather than genus)
vi. Double names (e.g., Callibaetis callibaetis)
vii. Common misspellings
viii. Tribes/subfamilies/subgenus sometimes do not appear in ITIS
5.5.4 Taxonomy Procedures
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ix. Species with incorrect genus (Hydatopsyche betteni)
x. Split level taxonomy (e.g., Cricotopus/Orthocladius)
xi. Invalid name (e.g., taxonomic change, synonym; Sphaeriidae vs. Pisiidae)
xii. Valid name, in scientific literature, but not in ITIS (e.g., appears in Merritt & Cummins
(1996) or Epler (2001), but not listed in ITIS - will not have a TSN)
5. Prepare slide mounts of Chironomidae and Oligochaeta as needed using CMCP-10 (or CMC-9,
CMC-10, or other media) and applying a coverslip. View these organisms under the compound
microscope to ensure that all necessary diagnostic characters have been observed, according to
the taxonomic key or other literature. Record the identifications on the bench sheet as above.
Label the slides with the same sample number or log-in number as the alcohol specimens.
6. 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 will be kept on file with the project QC officer.
7. If damaged organisms can be identified, they are counted ONLY if:
a. the fragment includes the head, and, in the case of arthropods, the thorax; the analysts
should match the detached head and thorax parts to ensure that double counting of
individuals does not occur.
b. oligochaetes, heads with a sufficient number of segments;
c. the mollusk shell (bivalve or gastropod) is occupied by a specimen;
d. the specimen is the sole representative of a taxon in the sample.
8. If early instar or juvenile specimens can be identified, they are counted as separate taxon.
9. Add the number of organisms from each developmental stage and enter the total on the bench
sheet.
10. Complete the bench sheet 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. Make a copy of the bench sheet for the project file.
11. Create a reference collection with at least one specimen from each genus (or lowest taxonomic
level identified). The taxonomist must choose an appropriate specimen(s) to represent each
taxon in the master taxa list. The specimen(s) must be removed from the sample and placed in
the reference collection. Circle slide-mounted specimens with a grease pencil (or other
appropriate mark) to indicate those which belong to the reference collection. For all slides
containing reference and non-reference specimens, be sure to place a label in the sample
container that does not contain the reference collection. Each laboratory must maintain a
master list of taxa recorded. The contract lead will coordinate any necessary inter-laboratory
communication and produce and integrated master taxa list for the project.
12. Carefully return the rest of the organisms to the original sample vial, fill with 70-80% denatured
EtOH, and cap tightly.
13. Re-package the samples and slide-mounted specimens carefully, and sign and date the sample
tracking form in the next "relinquished by" space. The samples must be shipped, properly
packed in a box, by overnight carrier to the Project Facilitator, and receipt must be confirmed by
the person doing the shipping. Each taxonomist must retain a full set of bench sheet copies and
ship the original bench sheets to the contract lead. Ship samples and bench sheets separately.
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5.5.4.1 Taxonomic Level of Effort
This is the Standard Taxonomic Effort list for benthic macroinvertebrates (Table 5.2). It represents the
minimum level needed for mature and well preserved specimens. The lowest targeted taxonomic level
will be genus. Due to taxonomic limitations, some groups cannot be identified to the genus level and
therefore should be taken to the level specified below. For all taxonomic groups, if the level can easily
go lower, for example monotypic genera, or if only one genus or species is known to occur in a certain
geographic area, then these specimens should be identified at the lowest possible taxonomic level (e.g.,
Ephemerellidae Drunella doddsl). If the minimum taxonomic level cannot be achieved due to immature,
damaged, or pupal specimens this should be noted in the data file with a "flag" variable (e.g., IM = y, DD
= y, PP = y).
Table 5.2 Required level of taxonomic identification for benthic macroinvertebrates.
Phylum Class
Required
Taxonomic
Identification Notes
ANNELIDA
Branchiobdellida
Family
Hirudinea
Genus
Oligochaeta
Genus
Polychaeta
Family
ARTHROPODA
Arachnoidea
Acari
Genus
Insecta
Coleoptera
Genus
Diptera
Except in the
following
cases:
Genus
Chironomidae
Genus
this may not be possible
for some groups, which
should be identified to at
least tribe or subfamily
Dolichopodidae
Family
Phoridae
Family
Scathophagidae
Family
Syrphidae
Family
Ephemeroptera
Genus
Hemiptera
Genus
Lepidoptera
Genus
Megaloptera
Genus
Odonata
Genus
Plecoptera
Genus
Trichoptera
Genus
Malacostraca
Genus
Amphipoda
Genus
Decapoda
Genus
Isopoda
Genus
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Required
Taxonomic
Phylum
Class
Identification
Notes
Mysidacea
Genus
COELENTERATA
MOLLUSCA
Bivalvia
Genus
Gastropoda
Except in the
following case:
Genus
Hydrobiidae
Family
NEMERTEA
Genus
5.6 Pertinent QA/QC Pro cedures
5.6.1 Sorting and Subsampling QC
1. A QC Analyst will use 6-10X microscopes to check all sorted grids from the first five samples
processed by a sorter to ensure that each meets the acceptable criteria for percent sorting
efficiency (PSE), which is 90%. This will not only apply to inexperienced sorters, but also to those
initially deemed as "experienced." Qualification will only occur when sorters achieve PSE > 90%
for five samples consecutively.
2. The laboratory QC Officer will calculate PSE for each sample as follows:
Equation 5.1 Percent sorting efficiency (PSE).
PSE = ——— x 100
A + B
where A = number of organisms found by the primary sorter, and B = number of recoveries
(organisms missed by the primary sort and found during the QC check).
3. If the sorting efficiency for each of these five consecutive samples is > 90% for a particular
individual, this individual is considered "experienced" and can serve as a laboratory QC Officer.
In the event that an individual fails to achieve > 90% sorting efficiency, he or she will be required
to sort an additional five samples and continue to have their sorting efficiency monitored.
However, if he or she shows marked improvement in sorting efficiency prior to completion of
the next five samples, achieving > 90% sorting efficiency, the laboratory QA Officer may, at
his/her discretion, consider this individual to be "experienced". Do not calculate PSE for samples
processed by more than one individual.
4. After individuals qualify, 10% (1 out of 10, randomly selected) of their samples will be checked.
5. If an "experienced" individual fails to maintain a > 90% PSE as determined by QC checks, the
laboratory QC Officer will perform QC checks on every grid of five consecutive samples until a >
90% sorting efficiency is achieved on all five. During this time, that individual will not be able to
perform QC checks.
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5.6.2 TaxonomicQC
5.6.2.1 Internal TaxonomicQC
As directed by the Indicator QC Coordinator, an in-house QC Analyst will conduct an internal 10% re-
identification of all samples identified by that laboratory to ensure that each meets the acceptable
criteria for percent identification efficiency which is 90%.
If the individual fails to maintain a > 90% identification as determined by QC checks, previous samples
will be re-counted and identified.
5.6.2.2 External Taxonomic QC
1. Upon receipt of the data, the Indicator QC Coordinator for macroinvertebrates will randomly
select 10% of the samples. The Indicator QC Coordinator will then have the original laboratory
send those samples to a QC taxonomist (another experienced taxonomist who did not
participate in the original identifications). The original laboratory will complete a sample
tracking form and send with the samples.
2. The QC taxonomist will perform whole-sample re-identifications, taking care to ensure inclusion
of all slide-mounted specimens and completing another copy of the Benthic Macroinvertebrate
Taxonomic Bench Sheet for each sample. Label each bench sheet with the term "QC Re-ID." As
each bench sheet is completed, fax it to the Project Facilitator.
3. The Indicator QC Coordinator will compare the taxonomic results (counts AND identifications)
generated by the primary and QC taxonomists for each sample and calculate percent difference
in enumeration (PDE) and percent taxonomic disagreement (PTD) as measures of taxonomic
precision (Stribling et al. 2003) as follows:
Equation 5.2 Percent difference in enumeration (PDE).
W -nA
PDE = \— — xlOO
nx +n2
where nl is the number of specimens counted in a sample by the first taxonomist and n2 is the
number of specimens counted by the QC taxonomist.
Equation 5.3 Percent taxonomic disagreement (PTD).
PTD =
1-
compr
r
N~
x 100
where comppos is the number of agreements (positive comparisons) and N is the total number of
specimens in the larger of the two counts.
4. The recommendation for PDE is 5% or less.
5. A PTD of 15% or less is recommended for taxonomic difference (overall mean < 15% is
acceptable). Individual samples exceeding 15% are examined for taxonomic areas of substantial
disagreement, and the reasons for disagreement investigated. A reconciliation call between the
primary and secondary taxonomist will facilitate this discussion. Results greater than this value
is investigated and logged for indication of error patterns or trends.
6. Corrective actions include determining problem areas (taxa) and consistent disagreements and
addressing problems through taxonomist interactions. These actions help to rectify
disagreements resulting from identification to a specific taxonomic level.
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5.6.2.3 Taxonomic QC Review & Reconciliation
The Indicator QC Coordinator prepares a report or technical memorandum to quantify aspects of
taxonomic precision, assess data acceptability, highlight taxonomic problem areas, and provide
recommendations for improving precision. This report is submitted to the HQ Project Management
Team, with copies sent to the primary and QC taxonomists. Another copy is maintained in the project
file. Significant differences may result in the re-identification of samples by the primary taxonomist and
a second QC check by the secondary taxonomist.
All samples are stored at the laboratory until the Project Lead notifies the laboratory regarding
disposition.
Table 5.3 Laboratory quality control: benthic indicator.
Check or Sample Frequency Acceptance Criteria Corrective Action
Description
SAMPLE PROCESSING (PICK AND SORT)
Sample residuals
examined by
different analyst
within laboratory
10% of all samples
completed per
analyst
Efficiency of picking > 90%
If < 90%, examine all residuals of
samples by that analyst and retrain
analyst
IDENTIFICATION
Sorted samples re-
identified by
different analyst
within laboratory
10% of all samples
Accuracy of contractor
laboratory picking and
identification > 90%
If picking accuracy < 90%, all
samples in batch will be reanalyzed
by contractor
Independent
identification by
outside taxonomist
All uncertain taxa
Uncertain identifications to
be confirmed by expert in
particular taxa
Record both tentative and
independent IDs
Use standard
taxonomic
references
For all
identifications
All keys and references used
must be on bibliography
prepared by another
laboratory
If other references desired, obtain
permission to use from Project
Facilitator
Prepare reference
collection
Each new taxon
per laboratory
Complete reference
collection to be maintained
by each individual
laboratory
Benthic Laboratory Manager
periodically reviews data and
reference collection to ensure
reference collection is complete
and identifications are accurate
External QC
10% of all samples
completed per
laboratory
PDE< 5%
PTD<15 %
If PDE > 5%, implement
recommended corrective actions.
If PTD > 15%, implement
recommended corrective actions.
DATA VALIDATION
Taxonomic
"reasonable-ness"
checks
All data sheets
Genera known to occur in
given lakes or geographic
area
Second or third identification by
expert in that taxon
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6.0 FECAL INDICATOR: ENTEROCOCCI
6.1 Scope and Application
This document describes the application of Draft EPA Method 1606 for the processing and qPCR analysis
of water sample concentrates from rivers and streams for the purpose of determining water quality by
Real-Time Quantitative Polymerase Chain Reaction (qPCR) assays that determine the concentration of
bacteria such as the fecal indicator, Enterococcus, by measuring the concentration of their DNA in the
water sample.
This method facilitates the microbiological determination of water quality of water bodies at remote
locations from which collected water samples cannot feasibly be analyzed for the enumeration of viable
(culturable) indicator bacteria because they cannot be transported to an analytical laboratory within 6
hours of collection time for analysis by membrane filtration and / or selective media inoculation and
incubation (e.g., MPN broth analysis) methods (EPA method 1600). Prior to qPCR analysis of the water
samples, the bacterial cells present in a water sample will have been concentrated by "field" filtration
within 6 hours after collection of the samples. The filter retentate preserved by freezing of the sample
filters on dry ice and in < -20°C freezers will be subjected to DNA extraction (e.g., bead-beating) and
purification processes leading up to qPCR analysis. This processing can be completed up to 1 year after
cell concentration if the sample filter retentates are maintained frozen at -20 to -80°C
6.1.1 Summary of Method
Each sub-sample has previously been filtered aseptically and folded inward in half three times to form
an umbrella or in half and rolled up and then inserted into sterile sample extraction tubes containing
sterile glass beads or Roche MagNA Lyser Green BeadsTM (actually siliconized white ceramic beads in a
green capped tube). Extraction tubes containing filter concentrates (retentates) have been stored on dry
ice until transport to the analytical laboratory by air courier. Filter concentrates will be shipped by air
courier on dry ice from the field to the analytical team at EPA New England Regional Laboratory. Filter
concentrates received by NERL staff will be subjected to DNA extraction procedures and subsequently
analyzed by Draft EPA Method 1606 or 1607 for Total Enterococcus along with modifications to the
QA/QC procedures described below. The laboratory methods are summarized in Table 6.4 of Section
6.15.
6.1.2 Definitions of Method
Batch Size: The number of samples that will be processed by filter extraction with the same batch
(volume) of SAE buffer and analyzed by the same qPCR assay(s) using the same batch of qPCR master
mix. A batch is covered for quantitation purposes by the same "batch" calibrator samples, a minimum of
three, analyzed during the same week.
Bottle Blank: Analyte-free water is collected into a sample container, of the same lot number as the
containers used for collection of the environmental samples. Analysis of this sample is performed to
evaluate the level of contamination, if any, introduced into the environmental and control samples from
the sample container(s) from a common vendor's lot.
DNA: Deoxyribo-Nucleic Acid, double-stranded genetic molecules containing sequences of the four
nucleotide bases, adenine, thymine, guanidine, and cytosine that encode rRNA, mRNA, and tRNA
involved in protein synthesis.
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Field Filter Blank: A volume of sterile PBS, free of target organisms (i.e. Enterococcus) filtered through a
sterile filter and processed in parallel with all other samples to serve as a sentinel for detection of
reagent contamination or contamination transferred between samples by processing and analysis.
Field Replicates: Samples collected from rivers and streams that are collected at the same sampling site
one right after the other with only slight temporal variation. They are not "splits" of the same sample
volume.
Filtrate: Sample liquid or buffer rinsate passing through the filter into the vacuum flask.
Laboratory Quality Samples: Mock samples created in the lab such as lab blanks, lab-fortified blanks
(LFBs), and Lab-Fortified Matrices (LFMs) used to assure lack of sample contamination and to measure
analytical recovery during performance of sample processing and analysis methods.
Performance Testing (PT) / Performance Evaluation Sample (PES): Calibrator samples (filters spiked
with E. faecalis grown in Brain Heart Infusion Broth) and Laboratory Fortified Blanks (Phosphate
Buffered Saline; PBS) spiked with Enterococcus faecalis cells from BHI Broth suspension) will be assayed
by EPA Method 1600 and Draft EPA Method 1606 to ascertain method performance. Ball-T Bioballs®
which contain a specified number of E. faecalis cells may also be acquired to determine the performance
of the Relative Quantitation Method. Purified E. faecalis DNA acquired from the American Type Culture
Collection and TIB Mol Biol Inc. is used to test the performance of the Absolute Quantitation Method.
Retentate: The sample residue retained by the filter after the sample is vacuum-filtered. The retentate
contains particulates, microbiota, and macrobiota from which the DNA is extracted into buffer by bead-
beating for subsequent qPCR analysis.
Rinsate: The volume of phosphate buffered saline (PBS) applied to a sample's filter retentate in order to
"wash" any residual fine particles, smaller than the filter's nominal pore size, through the retentate and
the filter.
Sample Processing Control (SPC): A surrogate homologue analyte (e.g. Salmon DNA) spiked into each
sample to determine the recovery of target analyte and/or detect assay inhibition caused by matrix
effects.
Standards: Known amounts or numbers of copies of Enterococcus genomic DNA analyzed by the
Enterococcus qPCR assay to generate a Standard Curve (Log Copy Number vs. Crossing Point Value) in
order to determine Enterococcus genomic copy numbers in "Unknown" test sample extracts by Absolute
Quantitation Method.
6.2 Interferences
• Low pH (acidic) water
• Humic and fulvic acid content
• Suspended solids (e.g. fecal matter) and particulates (sand, dirt)
• Excessive algal growth
6.3 Health & Safety Warnings
All proper personal protection clothing and equipment (e.g. lab coat, protective eyewear/goggles) must
be worn or applied.
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When working with potential hazardous chemicals (e.g. 95% ethanol) or biological agents (fecally-
contaminated water) avoid inhalation, 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. If available consult the MSDS for prompt action, and in all cases seek
medical attention immediately. If inhalation, eye contact or ingestion occurs, consult the MSDS for
prompt action, and in all cases seek medical attention immediately.
6.4 Personnel Qualifications
All laboratory personnel shall be trained in advance in the use of equipment and procedures used during
the sample extraction and qPCR analysis steps of this SOP. All personnel shall be responsible for
complying with all of the quality assurance/quality control requirements that pertain to their
organizational/technical function. All personnel shall be responsible for being aware of proper health
and safety precautions and emergency procedures.
6.5 Equipment and Supplies
• Clean powderless latex or vinyl gloves
• Goggles or Face Shield
• Roche MagNA Lyser
• Roche MagNA Pure LC (automated nucleic acid isolation and purification platform)
• High Speed Microfuge
• Micropipettors
• Semi-conical, screw cap microcentrifuge tubes (PGC, #506-636 or equivalent) pre-filled with 0.3
j^0.02 g Acid-washed glass beads (Sigma, # G-1277 or equivalent). Filled tubes are autoclaved
15-min. Liquid Cycle (Slow Exhaust) OR
• Roche MagNA Lyser Green Bead tubes (Roche Applied Science, #03-358-941-001) sterile,
siliconized 3-mm diameter ceramic beads in a siliconized 2-mL microfuge tube.
• Roche MagNA Lyser Rotor Cooling Block
• 2-mL tube racks
• Permanent marking pens (fine point and regular point) for labeling tubes
• Bench Sheets & Printouts of Computer Software Sampling Loading Screen
6.6 Reagents & Standards
Qiagen AE buffer (Qiagen 19077) u
u
Salmon DNA (Sigma D1626) O
Frozen tubes of Enterococcus faecalis (ATCC #29212) calibrator cell stock §
LU
Purified Enterococcus faecalis (ATCC #29212d) genomic DNA ^
ABI TaqMan® Universal PCR Master Mix (ABI #4304437) ^
Enterococcus PCR primers and TaqMan® probe h
<
Sketa PCR primers and TaqMan® probe ^
Bovine Serum Albumen (BSA) Sigma Cat. #B-4287) ?
I
Roche MagNA Pure LC DNA Isolation Kit III for Fungi & Bacteria ^
LU
U_
52
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6.7 Preparations Prior to DNA Extraction & Analysis
Determine/estimate the sample batch size (number of samples) for one-week of sample processing and
qPCR analysis. The batch size is the number of samples that will be processed by filter extraction with
the same batch (volume) of SAE buffer and analyzed by the same qPCR assay(s) using the same batch of
qPCR master mix. A batch is covered for quantitation purposes by the batch calibrator samples, (a
minimum of three) whose 5-fold and 25-fold diluted extracts are analyzed at the outset of the week
along with a reagent blank. The lab will fill out a batch sample analysis bench sheet.
3. Micropipettors are calibrated annually and tested for accuracy on a weekly basis. Follow
manufacturer instructions for calibration check. Measure three replicate volumes per
pipettor and keep log book of their weights on a calibrated balance scale.
4. Preparation of stock Salmon Sperm (SS) DNA: Dissolve Salmon DNA in PCR grade water
at a concentration of ~10 |ag/mL. Determine concentration of Salmon testes DNA stock
by OD260 reading in a spectrophotometer. A DNA solution with an OD26oof 1.0 has a
concentration equal to approximately 50 |ag/mL depending on the GC content of the
DNA's sequence(s).
5. Dilute Salmon testes DNA stock with AE buffer to make 0.2 |ag/mL Salmon DNA
Extraction Buffer (SAE). Extraction buffer may be prepared in advance and stored at 4 9C
for a maximum of 1 week.
Note: Determine the total volume of Salmon DNA Extraction Buffer required for each day or
week by multiplying the volume (600 /uL) times the total number of samples to be analyzed
including controls, water samples, and calibrator samples. For example, for 18 samples, prepare
enough Salmon/DNA extraction buffer for 24 extraction tubes (18 ) / 6 = 3, therefore, 3 extra
tubes for water sample filtration blanks (method blanks) and 3 extra tubes for calibrator
samples). Note that the number of samples is divided by 6 because you should conduct one
method blank for every 6 samples analyzed. Additionally, prepare excess volume to allow for
accurate dispensing of 600 /iL per tube, generally 1 extra tube. Thus, in this example, prepare
sufficient Salmon DNA Extraction Buffer for 24 tubes plus one extra. The total volume SAE
needed per sample is 60011L. Hence for the SAE volume for 25 sample tubes is equal to 15,000
jiL. Dilute the Salmon DNA working stock 1:50, for a total volume needed (15,000 juL) 50 = 300 jiL
of 10 /ug/mL Salmon DNA working stock. TheAE buffer needed is the difference between the
total volume and the Salmon testes DNA working stock. For this example, 15,00011L - 30011L =
14,700 jiL AE buffer needed.
6. Make Dilution Series of Enterococcus faecalis purified genomic DNA for use as internal standards
in individual qPCR runs and to generate the weekly Enterococcus qPCR Standard Curve for
quantitation purposes.
7. Enterococcus faecalis DNA for Standards.
8. Frozen Reference Stock (20-piL) at 2.89 x 10s GEQs per piL.
9. Dilute 10-piL of the Frozen Reference stock 363-fold to a final volume of 3,630 piL AE buffer.
Aliquot 20-piL volumes into many 200-nL microfuge tubes and store frozen at -20 2C. The net
concentration of Enterococcus GEQs is 8,000 / piL. Each week perform a series of 10-fold and 4-
fold dilutions from one thawed tube of the 8,000 GEQ/^L standard solution to create 800
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GEQ/nL, 80 GQ/nL and 20 GEQ/^L standard solutions. The analyst performs Enterococcus qPCR
upon duplicate 5-piL volumes of each of the four standards yielding a Standard Curve of Log
GEQs ENT versus Ct value from which the assays "efficiency" is subsequently calculated in the
Relative Quantitation EXCEL Spreadsheet,
a. Make Enterococcus faecalis calibrator filter samples:
i. Assemble calibrator positive control samples by thawing tubes of E. faecalis cell stocks,
diluting their contents (lO-piL) up to 1-mL AE buffer and spotting 10-piL on sterile PC
filter previously folded and inserted into a pre-chilled Green Bead tube.
ii. Spot a sufficient number of calibrator filter samples for the entire study to insure
uniform, consistent relative quantitation of study samples. Store the calibrator filter
samples in -202C freezer and thaw individual calibrators (three per week) for extraction
with each week's batch of samples.
10. The calibrator sample filters are spotted with 104 or 10s Enterococcus faecalis cells and this
number is incorporated into the Relative Quantitation EXCEL spreadsheet.
11. Prior to and after conducting work with cells and / or genomic DNA standards, disinfect and
inactivate (render non-amplifiable) DNA in the Sample Extraction Hood, the qPCR Cabinet, and
the qPCR Sample Loading Hood with 10% bleach and >_15-min. exposure to high intensity
germicidal (254 nm) ultraviolet light.
6.8 Procedures for Processing & qPCR Analysis of Sample Concentrates.
Typically, 100-mL volumes of surface water are filtered according to EPA Method 1606 for processing
and analysis by PCR assays. Due to the limitations of field crew sampling time and the performance
limitations of the manually-operated vacuum pumps used in the field sampling operations, only 50-mL
surface water samples were filtered. Lower volumes (< 50-mL) are acceptable if suspended particulates
hinder the filtering of the standard 50-mL volume but equivalent volumes for each filter replicate were
requested. Filtration of lower sample volumes necessitated modifications to Method 1606 which are
directed by the Analysis Decision Tree (ADT; Section 6.15.1).
In accordance with the ADT, if < 40-mL of a water sample is filtered per filter replicate, then the
laboratory analyst extracts two replicate filters in parallel and combines equivalent volumes of the filter
extracts to form one composite filter extract. Each individual filter is extracted with only 300-nL of SAE
Extraction Buffer instead of the usual prescribed 600-nL volume of SAE buffer. Halving the SAE buffer
volume enables the analyst to maintain an equivalent Method Detection Limit and maintain a similar
Sample Equivalence Volume (SEQ; i.e. water sample volume per extract volume) in the extract volumes
(e.g. 5-^L) of each sample filter concentrate added to the PCR reactions.
12. Pre-chill MagNA Lyser Rotor Cooling Block in -20°C freezer. Label 1.7-mL sterile microfuge tubes
with sample ID number to match them with Green Bead Tubes. Two supernatant recovery tubes
and one "5-fold" dilution tube is needed per sample and should be labeled accordingly. The
dilution tube shall be filled with 80-nL AE buffer using a micropipettor.
13. To extract sample filters, uncap green bead tube (cold) and add 0.6-mL (600-nL) SAE Buffer
(Qiagen AE Buffer spiked with Salmon DNA). Re-cap tubes tightly.
6.8.1 Sample Processing (DNA Extraction)
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14. Insert Green Bead tubes of samples into MagNA Lyser and bead-beat for 60-sec (1-min) at 5,000
rpm at Room Temperature. Transfer sample tubes to microfuge. Spin tubes at 12,000 rpm for 2-
min. Being careful to move filter aside, recover and transfer up to 400-nL of supernatant (sans
debris) to new tube with a P-200 or P-1000 micropipettor.
15. Spin the supernatant tubes for 5-min at 14,000 rpm at Room Temperature. Recover >350-nL
supernatant and transfer to new 1.7-mLtube. When all samples in a batch have been extracted
transfer dilute 20-^L of DNA extract (2nd supernatant) five-fold (5X) in 80-nL AE buffer (sans SS-
DNA) and store at 4°C for qPCR assays. (If supernatant, 5X and even 25X sample dilutions
possess dark pigment and exhibit severe qPCR inhibition in Sketa assays, consider extracting
replicate filters of samples using the Modified MagNA Pure LC DNA Isolation Protocol (see
Section 6.15.2).
6.8.2 Sample Analysis by Enterococcus qPCR
6.8.2.1 Preparation ofqPCR assay mix
16. To minimize environmental DNA contamination, routinely treat all work surfaces with a 10%
bleach solution, allowing the bleach to contact the work surface for a minimum of 15 minutes
prior to rinsing with sterile water. If available, turn on UV light for 15 minutes.
17. Using a micropipettor with aerosol barrier tips, add PCR grade water to the lyophilized primers
and probe from the vendor to create stock solutions of 500 piM primer and 100 piM probe and
dissolve by extensive vortexing. Pulse centrifuge to coalesce droplets. Store stock solutions at -
20?C.
18. Prepare working stocks of Enterococcus, and Salmon DNA primer/probe mixes by adding 10 piL
of each Enterococcus or Salmon DNA primer stock and 4 piL of respective probe stock to 676 piL
of PCR grade water, and vortex. Pulse centrifuge to create pellet. Use a micropipettor with
aerosol barrier tips for all liquid transfers. Transfer aliquots of working stocks for single day use
to separate tubes and store at 4^C.
19. Using a micropipettor, prepare assay mix of the Enterococcus, and Salmon DNA reactions in
separate, sterile, labeled 1.7 mL microcentrifuge tubes as described in Table 6.1.
20. Finger vortex the assay mix working stocks; then pulse microcentrifuge to coalesce droplets.
Return the primer/probe working stocks and other reagents to the refrigerator.
21. Thaw and finger vortex sample extract (dilution) tubes that will be assayed in PCR run.
Microfuge a few seconds to coalesce droplets. Finger mix and spin the standards and calibrator
samples (dilutions). Temporarily store all samples in 4°C refrigerators until use in assay or return
to long term storage at -20°C. Discard disposable gloves and put on a new pair.
22. Set 32 Smart tubes in Cepheid Racks in PCR cabinet along with micro-pippetors and expose to
germicidal UV lamp for 15-min.
23. Pipette 20-uL of respective Master Mix into each labeled Smart tube. Transfer Smart tubes
(racks) from PCR cabinet to disinfected Sample Loading Fume Hood.
24. Using P-10 or P-20 micro-pipettor load each Smart tube with 5-uL volume of respectively
designated sample extract (dilution), standard, or buffer blank (SAE). Cap each sample's Smart
tube after loading.
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25. Check to make sure each Smart tube is properly labeled and identifiable by sample number or I-
core position (e.g. A4). Insert loaded Smart tubes into Smart Tube microfuge. Close lid and spin
5-sec. Pop lid to stop. Remove Smart Tubes from microfuge and insert into proper position in
SmartCycler.
Enterococcus (Ludwig) and Salmon (Sketa) qPCR assays (EPA Method 1606) will be performed upon 5-uL
aliquots of un-diluted & 5X diluted extracts of sample unknowns, calibrator, field blank, and lab blank. A
"No Template Controls" (NTC) shall be analyzed on an ongoing basis to ensure that the Master Mix PCR
reagents are not contaminated. To minimize the number of Enterococcus qPCR reactions needed to be
performed upon samples, Sketa qPCR assays will be performed upon the 5-fold diluted DNA extracts of
samples before any Enterococcus qPCR assays are run in order to screen samples for the presence and
dilution of PCR inhibitors by comparison with the undiluted and 5-fold dilution DNA extract of the
calibrator samples and unused portions of SAE buffer. Each sample's lowest dilution DNA extract not
exhibiting PCR inhibition in the Sketa qPCR assay will be re-assayed by the Enterococcus qPCR assay and
it's results will be used for quantitation of Enterococcus DNA sequences and CCEs.
Detection of reduced levels of Salmon DNA (higher instrument Ct values) is indicative of technical error
during extract dilution or excessive levels of PCR inhibitors or nuclease activity which could impact
detection of the Enterococcus DNA target sequences in the Enterococcus PCR assay. Alternatively, the
high Sketa Ct value may be indicative of the occurrence of a technical error during extract dilution. If a
test sample's Ct value is less than 3 cycles different than the blank negative control and calibrator
samples, indicating only negligible or marginal inhibition (the Sketa Assay is more sensitive to inhibitors
than the ENT Assay), an aliquot of its five-fold diluted extract is analyzed in the Enterococcus Assay. If an
abundance of PCR inhibitors or DNA nucleases are present in a sample extract which are causing a
greater increase in an extract's Ct value (> 3 cycles increase), then the extract is diluted an additional
five-fold (net 25-fold dilution) and re-assayed by both the Sketa and ENT assays. If the inhibition is not
ameliorated by the additional dilution, which should restore the Sketa Ct value to that of the 25-fold
diluted calibrator samples' extracts, the following actions are taken by the analyst. First, the analyst re-
dilutes the sample's undiluted DNA extract five-fold and re-analyzes the dilution with the Sketa PCR
assay to confirm that Ct variance is not due to a dilution error. If the Ct difference is not attributed to a
dilution error, replicate sample filters of the "inhibited" samples are subjected to DNA extraction and
purification by the MagNA Pure LC automated platform loaded with the Roche DNA Kit III (Bacteria;
Fungi) reagents (see Section 6.15.2).
The EPA Modified MagNA Pure LC extraction process which includes the spiking of the Lysis Binding
Buffer with the Salmon (IPC) DNA is more effective, but more costly, than EPA Method 1606 in
neutralizing severe levels of PCR inhibitors and DNA nucleases present in some environmental samples,
especially those containing high levels of algae or phytoplankton. The purified DNA extract yielded by
MagNA Pure extraction of the few (<5%) "severely inhibited" samples is subsequently analyzed by the
Sketa and Enterococcus qPCR assays and the number of Enterococcus CCEs per 100-mL determined by
the delta Ct and delta delta Ct Relative Quantitation Methods. While the MagNA Pure LC extraction
method is not 100% conservative (no partitioning or recovery issues) like EPA Method 1606, it typically
exhibits DNA recoveries in the range of 25-50%. DNA recoveries and Enterococcus CCE concentrations
are calculated using only the Delta-Delta Ct Relative Quantitation Method. The relative DNA recoveries
are determined by comparison of the Sketa results from purified DNA eluates of each test sample with
those of the extracted lab blank and calibrator samples. The absolute DNA recovery is calculated by
comparison of the former Sketa results with those of elution buffer spiked with an amount of Salmon
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DNA equivalent to the amount in the Salmon-spiked Lysis Binding Buffer added to each sample filter
lysate during the MagNA Pure LC DNA extraction process.
The "Unknown" and "Control" sample extracts whether processed using the SAE buffer or MagNA Pure
LC Kit III reagents are analyzed according to the Cepheid SmartCycler Enterococcus and Sketa qPCR
protocols described in Appendix A of the Draft EPA Method 1606 with Ct determination made by the
software using Manual Determination (equivalent of Fit Points Method of Roche LightCycler) with the
fluorescence threshold set at 8.0 units which enables uniform analysis and comparability of all samples'
qPCR results.
Example: For analyses on a single 16-position SmartCycler, calibrator samples and water samples are
analyzed in separate runs and a maximum of 6 water samples (or 2 replicates of 3 samples) are analyzed
per run, as described in Table 6.2 and Table 6.3 of Section 6.15.
Enterococcus and Sketa (Salmon DNA = SPC) qPCR results are exported to an EXCEL spreadsheet in which
relative quantitation calculations are performed by analysts. The Method 1606 results are reported in
terms (units of measure) of Number of Enterococcus Sequences and Number of Enterococcus Calibrator
Cell Equivalents (CCEs) per 100-mL sample volume. The qPCR results are converted to this standardized
unit of measure based on the volume of water sample actually filtered (e.g., 10-mL, 25-mL, or 50-mL).
Non-detects are reported as below the reporting limit (RL) which varies proportionally to the volume of
sample filtered by each sample crew at a specific site. Reporting limits and Method Detection Limits
(MDLs) will be higher among samples for which a volume of water <50-mL was filtered.
Enterococcus qPCR results are flagged if some part of the sample collection, hold-time, processing,
shipment, storage, sample extraction, or qPCR analysis are compromised and did not meet the
requirements of the Sampling and Analysis SOPs.
6.9 Storage & Timing of Processing/Analysis of Filter Concentrates
When a sufficient number of water sample filter concentrates (filters and retentates) have been
received by NERL and qPCR analytical reagents have been obtained the samples will be logged into LIMS.
Sample processing and qPCR will commence and results will be entered into the LIMS upon completion
of analysis.
6.10 Chain of Custody
Follow the Sample Control Procedures, Field Sampling Form/Enterococci Filtration/Sample Processing
Standard Operating Procedures.
Field Sampling forms and NRSA 2018-2019 Sample Tracking EXCEL Spreadsheet shall be consulted to
determine if a sample has been properly preserved during collection and transport prior to analysis and
that it has passed all criteria permitting its analysis. The qPCR results of samples exceeding established
criteria or whose associated field/lab blanks had positive Enterococcus qPCR detections of DNA shall be
flagged.
6.11 Quality Assurance/Quality Control (QA/QC) Procedures
The Data Quality Objectives and the Laboratory QC Procedures are listed and summarized in Table 6.5
and Table 6.6 of Section 6.15.
6.8.3 Sample analysis sequence for SmartCycler
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The number of field blanks (dilution buffer only) shipped by field crews performing the resampling of 91
re-visited rivers and streams represents a frequency of 5-10% of the total number of samples extracted
and analyzed by qPCR. All field blanks (negative controls) will be extracted and analyzed by qPCR for the
detection of Enterococcus. The blanks will be analyzed in these cases to insure that positive detections in
field samples are not due to contamination by sampling crews.
One Lab / Method Blank (LB; sterile filters) will be run per batch week in order to insure the sterility
(lack of DNA contamination) in the SAE buffer and pipette tips used to process all of the samples. The LB
sample will be processed and diluted like all other "Unknown" samples.
Up to four replicate filter concentrates (retentates) derived from the field filtration of 50-mL (in some
cases 10-mL and 25-mL) sample volumes of every sample will be received by NERL and stored at -20 to -
80°C. One filter retentate of each sample (and duplicates for 10% of samples) will be extracted to obtain
DNA lysates for Enterococcus qPCR analysis. The remaining filter concentrates will be archived for
possible extraction and analysis at a later time if needed.
Enterococcus and Sketa qPCR analysis will be performed upon 5-^L volumes of the non-diluted and 5-
fold diluted (in AE buffer) extracts which will be added to 20-^L qPCR Master Mix volumes and analyzed
in the Cepheid SmartCycler qPCR instrument in accordance with draft EPA Method 1606.
Duplicate Enterococcus and Sketa qPCR assays will be performed upon 10% of the sample extracts
(diluted and un-diluted) each week (batch) to determine qPCR assay variance.
6.12 Method Performance
Method Performance will be determined by the use of Performance Testing (PT)/Performance
Evaluation Samples (PES). Calibrator samples (filters spiked with frozen stocks of E. faecalis grown in
Brain Heart Infusion Broth) and Lab-Fortified Matrices (LFMs; duplicate sample filters spiked with frozen
stocks of E. faecalis grown in Brain Heart Infusion Broth) will be extracted and assayed by EPA Method
1606 Enterococcus and Sketa qPCR assays in order to ascertain method performance. The LFMs are
performed upon several samples (approx. 5% frequency) per batch, typically samples exhibiting non-
detection of Enterococcus, in order to determine method performance and also to insure that non-
detects are not due to poor DNA recovery caused by matrix effects.
6.13 Record Keeping & Data Management
Laboratory analysts shall follow the EPA OEME Laboratory Data Management SOP. Each lab analyst shall
record all details pertaining to sample processing and analysis in a designated, bound laboratory
notebook. Pertinent sample collection and analysis data shall be entered into the Laboratory
Information Management System (LIMS) and SeaGate Crystal Reports shall be generated as required by
the EPA (TOPO).
An EXCEL spreadsheet of sample analysis data and associated calculations used to derive a field sample's
or control sample's Enterococcus genomic DNA (GEQ) and Cell Equivalent (CEQ) concentration shall be
uploaded to the NRSA 2018-2019 database stored on a computer server in Corvallis, Oregon.
6.14 Waste Management & Pollution Prevention
During the sample processing procedures there may be hazardous waste produced. The waste must be
handled and disposed of in accordance with federal, state, and municipal regulations. All recyclable and
non-recyclable materials for disposal will be properly sorted for their respective waste streams and
placed into proper containers for janitorial staff to collect and process according to EPA guidelines.
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All ethanol used shall be consumed by ignition or evaporation. Volumes of ethanol remaining at the end
of the project can be stored for later use in a flammable cabinet or disposed of through appropriate
hazardous waste disposal vendors. Reagent ethanol shall be contained in screw cap tubes along with the
filter forceps to sterilize the latter and to prevent ethanol spillage during transport between sampling
sites.
After the DNA extract is recovered from the sample filter after bead-beating in buffer and
centrifugation, the filter and bead-tube will be discarded in autoclave bags and sterilized for 30-min at
121°C/30 psi to inactivate any potential pathogens that may be associated with the samples.
6.15 Tables, Diagrams, Flowcharts, Checklists, and Validation Data
Table 6.1 Enterococci: PCR assay mix composition (according to draft EPA method 1606)
Volume/Sample (multiply by #
Reagent
samples to be analyzed per day)
Sterile H 0
2
1.5 nL
Bovine Serum Albumen (20 mg/mL)
2.5 nL
TaqMan® master mix
12.5 nL
Primer/probe working stock solution
3.5 nL*
Note: This will give a final concentration of 1 nM of each primer and 80 nM of probe in the reactions. Prepare
sufficient quantity of assay mix for the number of samples to be analyzed per day including calibrators and negative
controls plus at least two extra samples. It is strongly recommended that preparation of assay mixes be performed
each day before handling of DNA samples.
Table 6.2 Enterococci: batch calibrator & enterococcus standards PCR run - 7 samples
Quantity
Quantity PCR
Sample Description*
Samples
PCR Assay Master Mix
Reactions
3 Calibrators (5- and/or 25-fold dilution)
3
Salmon DNA (Sketa)
6
3 Calibrators (5- and/or 25-fold dilution)
3
Enterococcus
6
4 Enterococcus faecalis DNA Standards
4
Enterococcus
8
No template control (reagent blank)
1
Enterococcus
1
* Diluted equivalently to the water samples
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Table 6.3 Enterococci: sub batch test sample PCR run - 26 samples & 1 method blank
Quantity Quantity PCR
Sample Description* Samples PCR Assay Master Mix Reactions
Water samples, (5-fold dilution)
26
Enterococcus
26
Method blank or Sample PCR Reaction Duplicate,
(1- or 5-fold dilution)
1
Enterococcus
1
Non-diluted SAE Buffer
1
Enterococcus
1
Water samples, (1- or 5-fold dilution)
26
Salmon DNA
26
Method blank or Sample PCR Reaction Duplicate,
(1- & 5-fold dilution)
1
Salmon DNA
1
* Use of 5-fold diluted samples for analysis is currently recommended if only one dilution can be analyzed. Analyses
of undiluted water sample extracts have been observed to cause a significantly higher incidence of PCR inhibition
while 25-fold dilutions analyses may unnecessarily sacrifice sensitivity.
Table 6.4 Enterococci: laboratory methods
Expected
Variable or QA Range and/
Measurement Class or Units Summary of Method References
Sample
Collection
C
NA
Sterile sample bottle submerged to collect
250-mL sample 6-12" below surface at 1-m
from shore
NRSA Field
Operations Manual
2008
Sub-sampling
N
NA
4 x 50-mL sub-samples poured in sterile 50-
mLtube after mixing by inversion 25 times.
NRSA Laboratory
Methods Manual
2008
Sub-sample
(& Buffer Blank)
Filtration
N
NA
Up to 50-mL sub-sample filtered through
sterile polycarbonate filter. Funnel rinsed
with minimal amount of buffer. Filter folded,
inserted in tube then frozen.
NRSA Laboratory
Methods Manual
2008
Preservation &
Shipment
C
-40Cto+40 C
Batches of sample tubes shipped on dry ice
to lab for analysis.
NRSA Laboratory
Methods Manual
2008
DNA Extraction
(Recovery)
C
10-141%
Bead-beating of filter in buffer containing
Extraction Control (SPC) DNA. DNA recovery
measured
EPA Draft Method
1606 Enterococcus
qPCR
Method 1606
(Enterococcus &
SPCqPCR)
C
<60 (RL) to
>100,000 ENT
CCEs/100-mL
5-nL aliquots of sample extract are analyzed
by ENT & Sketa qPCR assays along with
blanks, calibrator samples & standards. Field
and lab duplicates are analyzed at 5%
frequency. Field blanks analyzed along with
test samples.
EPA Draft Method
1606 Enterococcus
qPCR
NERL NRSA 2008
qPCR Analytical SOP
C = critical, N = non-critical quality assurance classification. 2
I
<
u
LU
U_
60
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Table 6.5 Enterococci: parameter measurement data quality objectives
Expected
Variable or QA Range and/or
Measurement Class Units Summary of Method References
DNA Extraction
(Recovery)
C
10-141%
Bead-beating of filter in buffer
containing Extraction Control (SPC)
DNA. DNA recovery measured
EPA Draft Method
1606 Enterococcus
qPCR
Enterococcus & SPC
qPCR
C
<60 to
>10,000 ENT
CEQs/100-mL
5-nL aliquots of sample extract are
analyzed by ENT & Sketa qPCR assays
along with blanks, calibrator samples &
standards. Field and lab duplicates are
analyzed at 5% frequency. Field blanks
analyzed at end of testing only if
significant detections observed.
EPA Draft Method
1606 Enterococcus
qPCR; NERLNRSA
2008 2009 qPCR
Analytical SOP
(QAPP)
SPC & ENT DNA
sequence numbers
of Calibrators &
Standards by AQM
RSD =
30%
80%
95%
ENT CCEs by dCt
RQM
RSD =
55%
40%
95%
ENT CCEs by ddCt
RQM
RSD =
55%
50%
95%
C = critical, N = non-critical quality assurance classification.
*AQM = Absolute Quantitation Method; RQM = Relative Quantitation Method;
SPC = Sample Processing Control (Salmon DNA/Sketa); CCEs = Calibrator Cell Equivalents
Table 6.6 Enterococci: laboratory QC procedures - enterococci DNA sequences
Check or Sample
Description Frequency Acceptance Criteria Corrective Action
SAMPLE PROCESSING
Re-process sub-
samples
(duplicates)
10% of all
samples
completed per
laboratory
Percent Similarity >70%
If <70%, re-process additional sub-
samples
qPCR ANALYSIS
Duplicate analysis
by different
biologist within
lab
10% of all
samples
completed per
laboratory
Percent Congruence <30% RSD
If >30%, determine reason and if
cause is systemic, re-analyze all
samples in question.
Independent
analysis by
external
laboratory
None
Independent analysis TBD
Determine if independent analysis
can be funded and conducted.
u
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Use single stock of
E. faecalis
calibrator
For all qPCR
calibrator
samples for
quantitation
All calibrator sample Cp (Ct)
must have an RSD <30%
If calibrator Cp (Ct) values exceed
an RSD value of 30% a batch's
calibrator samples shall be re-
analyzed and replaced with new
calibrators to be processed and
analyzed if RSD not back within
range.
DATA PROCESSING & REVIEW
100% verification
and review of
qPCR data
All qPCR
amplification
traces, raw and
processed data
sheets
All final data will be checked
against raw data, exported
data, and calculated data
printouts before entry into
LI MS and upload to Corvallis,
OR database.
Second tier review by contractor
and third tier review by EPA.
6.15.1 Enterococcus qPCR Analysis Decision Tree (ADT)
Created 10/25/07
Updated 1/2/08
Revised 11/05£I8
Figure 6.1 Enterococci: qPCR analysis decision tree (ADT)
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6.15.2 "Modified" MagNA Pure LC DNA Purification Kit III Protocol
26. Pre-warm the MagNA Pure LC DNA Isolation Kit III Lysis Buffer to 65 in waterbath. Quickly
pipette 260-nL of warm Lysis Buffer (un-amended) into each "Green Bead" tube with filter
(preserved after filtration temporarily on ice or during long-term storage in freezer). Shake tube
5 to 10 seconds to mix buffer with beads and filter. Let stand at RT until batch of 16 samples
(including positive control LFB or LFM and negative control LB samples) have all had Lysis Buffer
and had their caps sealed tight. Leave water bath on to use during 30-minute Proteinase K
treatment period.
27. Load the 16 samples into MagNA Lyser Rotor Plate and insert into MagNA Lyser. Tighten the
three handscrews of the locking mechanism. Close the lid tightly. Set controls to shake for 60-
sec at 5,000 rpm. Press the start button.
28. When the shake cycle has ended press the Open Lid Button. Open the lid and unlock the locking
mechanism screws. Remove tube plate and set on bench top MagNA Lyser tube ring hub.
Remove tubes, insert into tube styrofoam water bath float and cool tubes in ice water for 2-min.
or place directly into 24-place microfuge rotor, pre-chilled in freezer.
29. Insert tubes into centrifuge rotor symmetrically in order to balance rotor. Close lid of centrifuge.
Set spin parameters for 3,000 rpm for 1-min at 4^C. Press Start button. Centrifuge to collect
drops and foam off of cap down into tube.
30. When centrifuge stops, open lid and remove tubes from rotor. Uncap tubes in order and add 40-
piL of Proteinase K (dissolved in Lysis Buffer Elution Buffer). Re-cap tubes and mix lysate by
inversion. Do not vortex. Knock beads and filter down from cap into bottom of tube by tapping
tubes on bench countertop.
31. Insert tubes into styrofoam floating rack. Incubate tubes 30-min at 65^C in water bath. Set timer
for 15-min. At end of 15-min remove rack from water bath and inverts several times to mix
samples and tap beads and filter back down into tube. Re-place rack in 65^C waterbath for 15-
min. for total of 30-min.
32. Repeat steps 3 to 8 to process 16 more samples in parallel for loading MagNA Pure LC sample
cartridge with 32 DNA extracts for downstream processing in the robotic platform.
33. After 30-min in 65 ^c waterbath remove tubes from water bath and place in MagNA Lyser Bead
Beater for 15 seconds at 5,000 rpm. After 15 seconds of bead-beating, place in ice bath for 5-
min to cool.
34. Insert tubes in centrifuge rotor and spin 3-min at 12,000 rpm and 4 ^C to pellet sediment and _
cell debris. When spinning is complete, open lid of centrifuge and rotor and mark side of outer u
O
side of cap where pellet should have formed. u
35. Carefully remove rotor from centrifuge and set on bench. Remove tubes one at a time from £5
rotor and use 200-nL pipettor and sterile aerosol-proof tips to transfer approximately 150nL
lysate supernatant from tube to wells in MagNA Pure LC Sample Cartridge in pre-designated g
order. ^
36. When all 16 sample supernatants transferred to sample cartridge put adhesive film over 5
cartridge to prevent contamination and evaporation. Put sample cartridge in ice water bath or ^
fridge to maintain 4 5C. u
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37. Repeat steps 9 to 13 for second batch of 16 samples (lysates). Re-cover sample cartridge with
adhesive film for storage. Centrifuge sample cartridge opposite a balance cartridge for 75-sec (1-
min, 15-sec) at 2800 rpm in IEC centrifuge (or equivalent) with rotor adaptors for microtiter
plates in place. Insert the film-covered sample cartridge in MagNA Pure LC platform.
38. Load the MagNa Pure LC platform with volumes of extraction kit reagents prescribed by MagNA
Pure LC computer software for the number of samples being extracted. Before closing the
platform' lid and starting the extraction process add 1.34nL of 9.3ng/mL Salmon DNA Stock (10
Hg/mL nominal concentration) per lmL Lysis Binding Buffer (blue soapy solution) as the Sample
Processing Control (SPC). If the amount of Salmon DNA stock to be added is less than lO-piL,
dilute the Salmon DNA stock so that a volume > 10-piL can be pipetted into the Lysis Binding
Buffer. Rinse pipette tip up and down three times in Lysis Binding Buffer.
39. Remove film from top of sample cartridge and re-insert in Roche MagNA Pure LC platform set up
with DNA Purification Kit III (Fungi; Bacteria) reagents in tubs, tips, tip holders, and processing/
elution cartridges. Close platform lid and after checking off checklist of loaded items (e.g.
reagents, tips) lock the lid and start the automated DNA III Extraction Protocol which purifies
each sample's DNA and elutes it into lOO-piL Elution Buffer.
40. When extraction process is complete, unlock the MagNA Pure LC platform lid and remove the
sample eluate cartridge. Cover the cartridge with adhesive film and store at 4 C until qPCR
analysis. Store cartridge at < -20 ^C for long term preservation.
41. Prepare Elution Buffer Control from 9.3ng/mL Salmon DNA Stock by diluting a small volume to
37.2pg/1000nL (1-mL). This control sample is only analyzed by the Sketa qPCR assay. The Ct
value obtained represents that value expected in Sketa qPCR assays of each MagNA Pure LC
purified sample if 100% of the Salmon DNA was recovered and detected. Vortex to mix on low
speed briefly prior qPCR analysis. Centrifuge for 1.5-min to coalesce droplets. Remove film to
aliquot sub-samples and re-place with new film cover to restore at cool temperatures.
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7.0 PHYTOPLANKTON METHODS
This method is adapted from protocols used for the U.S. Geological Survey National Water Quality
Assessment program (Charles et al. 2003) to identify and enumerate taxa in phytoplankton samples. The
method involves microscopic examination of preserved phytoplankton samples from integrated samples
collected from the euphotic zone of the water column.
Phytoplankton samples will be preserved in the field with Lugol's solution and shipped from field crews
to a contract batching laboratory. The contract batching laboratory will send the batched samples to the
analysis laboratory. Preserved samples will arrive in the analysis laboratory and can be held for several
months. Phytoplankton analysis laboratories will need to process samples in accordance with the time
frame outlined in contractual agreements. Contractual agreements for delivery of data do not supersede
indicator holding times.
7.1 Responsibility and Personnel Qualifications
This procedure may be used by any person who has received training in processing and/or identification
of phytoplankton samples. It is important that all taxonomists maintain contact with other taxonomists
through professional societies and other interactions and keep abreast with the pertinent literature,
because taxonomic groupings and nomenclatural basis for species identifications are updated
frequently. A second taxonomist will re-identify a randomly-selected 10% of the samples for QC, as
noted below, to quantify taxonomic precision, or consistency, as percent difference (PD), to help target
corrective actions, and ultimately to help minimize problems during data analysis. Samples are sent to
the laboratory from the field on a regular basis to avoid delays in processing and sample identification.
7.2 Precautions
Wear appropriate clothing for safety precautions, such as nitrile gloves, rubber apron, long pants, etc.
7.3 Equipment/Materials
• Compound microscope (with 10, 40, 100X objectives with 10 - 15X ocular, and
epifluorescence capability)
• Utermohl sedimentation chamber
• Pasteur pipette
• Volumetric cylinder
• Bench sheet
• Phytoplankton Sample Log-In Form
• Phytoplankton Laboratory Sheet
• Labels
7.4 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.
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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 1.2 of the
manual 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 sample tracking
form.
b. Record the information in Table 7.1 for the NARS IM Team, 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
c. 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 Chapter 2 of the
Manual).
3. Store samples until sample preparation begins.
4. Maintain the sample tracking forms with the samples.
Table 7.1 Phytoplankton login: required data elements.
FIELD FORMAT DESCRIPTION
LAB
text
Name or abbreviation for laboratory
DATE RECEIVED
MMDDYY
Date sample was received by laboratory
SITE ID
text
NLA 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
Q
Other quality concerns, not identified above
CONDITION
COMMENT
text
Comments about the condition of the sample.
LD
O
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7.5 Procedure
7.5.1 Prepare Utermohl Sedimentation Chamber
1. Use a light amount of vacuum grease to attach a cover glass to the bottom of an Utermohl
sedimentation chamber. It is critical that the cover glass be clean and grease free.
• For tubular varieties of settling chambers, seal a cover glass to the threaded end of the tube
and screw the tube into the base assembly.
• For a plate chamber type of settling chamber, attach the cover glass on the bottom of the
base, lock it into place with the metal ring and seal the cylinder on top of the base using a
light amount of vacuum grease.
2. Homogenize the concentrated samples by repeatedly inverting the sample bottle. Place a 10-mL
aliquot of the sample into the assembled settling chamber. Let the sample settle for at least 8
hours.
3. For the plate chamber type of Utermohl chamber, drain the volumetric cylinder by sliding over
the drainage hole. Slide the cover plate over the chamber without allowing air bubbles to form.
Analysis should proceed within a few hours of removing the cylinder.
7.5.2 Choose Count Method
7.5.2.1 Determine random fields
1. Using a high oil microscope objective (10-15X objective, 100-1500X total system magnification),
identify and enumerate algae in selected, random fields. Enumerate between 8 and 100 fields
from each Utermohl chamber. If necessary, use a second chamber.
2. Choose a random starting place in the upper left-hand quadrant of the counting chamber and
approximate the number of fields that must be analyzed (400 natural units [Section 7.5.3] need
to be counted with a minimum of 8 and maximum of 100 random fields).
3. Develop a pattern that allows for equal probability of landing in any area of the cell or chamber
with the exception of the edges and the center. A maximum pattern with 100 fields is made by
having an 8x8 grid, and then subtracting 3 or 4 fields in either direction of the center.
7.5.2.2 Determine transects
1. Using a high oil microscope objective (10-15x objective, 100-1500x total system magnification)
with a calibrated stage, identify and enumerate algae along transects, either horizontally or
vertically across the Utermohl plate chamber.
2. Without looking into the microscope, choose a location near the left edge in the upper third of
the chamber (if vertical transects are analyzed, choose a location near the top edge in the left
third of the chamber). Make a transect by moving only the horizontal stage control (or vertical
control for vertical transects) a measured distance.
3. Develop a pattern for the transects that will avoid the center and edges of the chamber. A
second Utermohl chamber can be used, if necessary (400 natural units need to be counted with
a minimum of one complete transect).
7.5.3 Identify and Enumerate 400 Natural Algal Units
1. Species-level resolution is the taxonomic requirement for phytoplankton which likely means
using a magnification of 1000X or higher.
2. Using the pattern developed above, move the microscope stage to a new position in the
pattern. Make all movements of the microscope stage without looking through the objectives.
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3. Identify and enumerate all algal forms in the field of view: enumerate algal forms using natural
counting units. Natural counting units are defined as one for each colony, filament, diatom cell
(regardless if colonial or filamentous) or unicell. With the exception of diatoms, identify algal
forms to species. Develop a method of selecting taxa that are only partially in view. For example,
only count taxa that are partially in the field of view if they are on the left side. If they are on the
right do not count.
4. Count only "living" diatoms at the time of collection. If there is any protoplast material in the
frustule, the diatom is considered to have been living when collected.
5. Differentiate diatoms to the lowest practical taxonomic level. This will usually be genus, but use
of categories such as naviculoid, cymbelloid, centric, nitzschoid is appropriate.
6. Count the number of algal cells comprising each multicellular counting unit.
7. Tabulate the data on a bench sheet APPENDIX B: SAMPLE LABORATORY FORMS, mechanical or
electronic tabulator.
8. Repeat steps 1-4 until 400 natural algal units have been enumerated. Again, count only "living"
diatoms as part of the required 400 natural algal units.
9. Add and record the tallies of each taxon on the bench sheet. Record the number of cells for
multicellular counting units in parentheses beside the tally of natural counting units.
10. Record the number of fields or the total transect length for the area that was observed.
7.5.4 Identify and Enumerate Larger, Rarer Taxa
There is an additional procedure that can be used for samples with low concentrations (less than five
natural counting units) of large cells or colonies (maximum dimension greater than 100 pim).
1. Using a low-power objective (10-15X), scan 20 fields or 4 transects. Count the larger, rarer taxa
(as defined above).
2. Enumerate as natural units and estimate the number of cells in each. Record the counts of each
of the taxa on the bench sheets, noting the scan area (i.e., total area for the 20 microscope
fields or 4 transects). Multiply the number of larger, rarer taxa by the ratio of the total area
scanned in the regular count to the area scanned in this count.
3. Record that number as the total count for that taxon.
7.5.5 Measure Cell Biovolumes
1. For each group of samples, measure the dimensions of the taxa that contribute most to sample
biovolume. Cell biovolumes of all identified taxa will be quantified on a per milliliter basis. Use
formulae for solid geometric shapes that most closely match the cell shape (Hillebrand et al., 1999)
to estimate biovolume. Base biovolume calculations on measurements of 10 organisms per
taxon for each sample where possible.
2. Biovolumes for each abundant taxon (i.e., occurring in more than 5% in any one sample) should
be based on measurements of 10 cells or more
3. Biovolumes for each common taxon (i.e., occurring 2 - 5% in any one sample) should be based
on measurements of one or more cells.
4. Biovolumes for each rare taxon (i.e., occurring in 0.1 - 2% in any one sample) should be based
on measurements from literature descriptions of taxa, previous measurements of the taxon, or
measurements of one or more cells.
5. For taxa with substantial size variation (e.g., diatoms), designate size classes based on sample
quality to determine average cell size (biovolume). For each taxon, measure 10 cells from each size
class (assuming that sufficient numbers are available). Use mean biovolumes within each size class
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to calculate the total biovolume contributed by the taxon to its representative sample (Burkholder
and Wetzel, 1989).
7.6 Calculation and Reporting
1. The calculation of phytoplankton abundance depends on the apparatus used during analysis.
Biovolume values are determined by multiplying the abundance (cells/mL) by the average
biovolume of each cell (nm3). Phytoplankton abundance (cells/mL) is calculated as follows:
Equation 7.1 Phytoplankton abundance.
cells/
' count x chamber xlOOOmL ^
_ numfields x field x mlsettled
mL
where count = number of cells counted, chamber = chamber area (in mm2), numfields = number
of microscope fields, field = microscope field area (in mm2), and ml settled = number of ml
settled in Utermohl chamber.
2. Prepare a spreadsheet file containing the count data, using the columns (fields) as shown in the
(APPENDIX B: SAMPLE LABORATORY FORMS: Phytoplankton Measurement Data Sheet.
Submit the file electronically to the USEPA.
7.7 Pertinent QA/QC Procedures
Table 7.2 provides a summary of quality control procedures for the phytoplankton indicator.
7.7.1 Internal Taxonomic QC
An in-house QC Analyst will randomly select 5 of the samples counted and identified by individual
taxonomists to ensure that each meets the acceptable criteria for percent identification efficiency which
is 90%. If the individual fails to maintain a > 90% identification as determined by QC checks, previous
samples will be re-counted and identified.
7.7.2 External Taxonomic QC
EPA may implement an external taxonomic QC review process for phytoplankton. If EPA implements an
external QC process, upon receipt of the data after initial identification, the Indicator QC Coordinator for
phytoplankton randomly selects 10% of the samples for external QC analysis. The Indicator QC
Coordinator will direct the original laboratory to send those samples to a QC taxonomist, a second
experienced taxonomist who did not participate in the original identifications. The original laboratory
will complete a sample tracking form and send it with the samples.
7.7.2.1 Plankton Re-identification
Duplicate processing (duplicate the processing steps presented in Section 7.5.1 - 7.5.5).
The remaining concentrated sample will be sent to the QC taxonomist.
1. Using the same volume as the original Utermohl chamber, prepare a duplicate Utermohl
chamber cell and enumerate 400 natural algal units. Complete another copy of the Taxonomic
Bench Sheet for each sample. Label each bench sheet with the term "QC Dup-ID." As each bench
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sheet is completed, the laboratory sends it (through email or fax) to the Indicator QC
Coordinator.
2. The Indicator QC Coordinator compares the taxonomic results generated by the primary and QC
taxonomists for each sample and calculate percent difference using:
Equation 7.2 Percent difference.
PctDiff = 100 - ^ min (a, b)
where a and b are the relative proportions recorded for a given taxon by the primary taxonomist
(a) and the QC taxonomist (b).
3. Values will be a combination of subsampling error and taxonomic error; the MQO is that the two
counts will have a percent difference of < 50.
4. If it appears that high percent difference for soft-bodied phytoplankton are due to subsampling
inconsistency, then determine and implement appropriate corrective actions working with the
Indicator QC Coordinator. In addition, disagreements resulting from identification to a specific
taxonomic level, creating the possibility to double-count "unique" or "distinct" taxa shall be
rectified through corrective actions working with the Indicator QC Coordinator.
7.7.3 Taxonomic QC Review & Reconciliation
The Indicator QC Coordinator prepares a report or technical memorandum to quantify aspects of
taxonomic precision, assess data acceptability, highlight taxonomic problem areas, and provide
recommendations for improving precision. This report is submitted to the HQ Project Management
Team, with copies sent to the primary and QC taxonomists. Another copy is maintained in the project
file. Significant differences may result in the re-identification of samples by the primary taxonomist and
a second QC check by the secondary taxonomist.
All samples are stored at the laboratory until the Project Lead notifies the laboratory regarding
disposition.
Table 7.2 Laboratory quality control: phytoplankton indicator.
Check or Sample
Frequency
Acceptance Criteria
Corrective Action
Description
IDENTIFICATION
Independent
identification by
outside taxonomist
All uncertain taxa
Uncertain identifications to be
confirmed by expert in
particular taxon
Record both tentative and
independent IDs
Use standard taxonomic
references
For all
identifications
All keys and references used
must be on bibliography
prepared by another
laboratory
If other references desired,
obtain permission to use from
Project Facilitator
Prepare reference
collection
Each new taxon
per laboratory
Complete reference collection
to be maintained by each
individual laboratory
Laboratory Manager periodically
reviews data and reference
collection to ensure reference
collection is complete and
identifications are accurate
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External QC (if
implemented)
10% of all samples
completed per
laboratory
Efficiency (PD) < 50%
If PD > 50%, implement
recommended corrective
actions
DATA VALIDATION
Taxonomic "reasonable-
ness" checks
All data sheets
Genera known to occur in
given lakes or geographic area
Second or third identification by
expert in that taxon
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8.0 PESTICIDE SCREEN: ATRAZINE
This method describes the application of enzyme linked immunosorbent assay (ELISA) to the
determination of atrazine and related atrazine occurrence and concentration in surface water samples.
The Eurofins Technologies (formerly Abraxis) magnetic particle atrazine kit is used for this analysis.
You will filter the lake water sample, add the filtered water to a disposable test tube with an enzyme
conjugate, and then add paramagnetic particles with atrazine-specific antibodies. After allowing for a
15-minute reaction between the sample and reagents, you apply a magnetic field to the test tube that
retains the paramagnetic particles (with atrazine and labeled atrazine bound to the antibodies on the
particles in proportion to their original concentration) and allow the unbound reagents to be decanted.
After decanting, wash the particles with the washing solution. You will detect the presence of atrazine
and related atrazines by adding the color solution. After an incubation period, the reaction is stopped
and stabilized by the addition of a dilute acid (Stopping Solution). Because the labeled atrazine
(conjugate) was in competition with any unlabeled atrazine in the sample for the antibody sites, the
color developed is inversely proportional to the concentration of atrazine in the sample. The detection
limit for this method is 0.03 ng/L and the reporting limit is 0.05 ng/L.
The field crews will ship chilled atrazine pesticide screen samples to the contract batching laboratory as
part of the batch frozen shipment. The chilled samples will be placed in an insulated tube in a separate
component of the dry ice liner to prevent freezing during shipment. The contract batching laboratory
will store samples in the refrigerator and send the batched samples to the analysis laboratory in coolers
on wet ice. Samples will arrive in the analysis laboratory chilled, and they can be held in a refrigerator or
cold room for several weeks. Atrazine pesticide screen analysis laboratories will need to process samples
within the 90-day holding time and in accordance with the time frame outlined in contractual
agreements.
The methods listed below follow the methods used by Minnesota Pollution Control Agency (MPCA)
based on the ELISA kit instructions.
8.1 Responsibility and Personnel Qualifications
All laboratory personnel are trained in advance in the use of equipment and procedures used during the
implementation of this SOP. All personnel are responsible for complying with all of the QA/QC
requirements that pertain to this indicator.
8.2 Precautions
The stopping solution contains diluted sulfuric acid (H2S04). Avoid contact of the stopping solution with
skin and mucous membranes. If this reagent comes in contact with the skin, wash with water. Consult
state, local, and federal regulations for proper disposal of all reagents.
8.2.1 Storage and Stability
Store all reagents at 2-8°C. Do not freeze reagents. Before use, allow the solutions to reach room
temperature (20-25°C). Reagents may be used until the expiration date on the box. The test tubes and
the washing solution require no special storage condition and may be stored separately from the
reagents.
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8.3 Equipment
• Abraxis Atrazine Kit (each kit contains Atrazine Antibody Coupled Paramagnetic
Particles, Atrazine Enzyme Conjugate, Atrazine Standards, Control, Diluent/ Zero
Standard, Color Solution, Stopping Solution, Washing Solution, and test tubes)
• Precision pipets capable of delivering 250 and 500 piL and a 1.0 mL repeating pipet
• Vortex mixer
• Magnetic separation system
• Photometer capable of readings at 450 nm
8.4 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 upon receiving a
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 1.2 of the
manual 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 sample tracking
form
b. Record the information in Table 8.1 for the NARS IM Team, 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. W: Sample is warm (>4°), record the temperature in the comment field, and perform the
c. 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 Chapter 2 of the
Manual).
3. Store samples in the refrigerator until sample preparation begins.
delivery.
assay
4. Maintain the sample tracking forms with the samples.
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Table 8.1 Atrazine login: required data elements.
FIELD FORMAT DESCRIPTION
LABORATORY ID
text
Name or abbreviation for laboratory
DATE RECEIVED
MMDDYY
Date sample was received by laboratory
SITE ID
text
NLA 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
NF
Sample is not frozen
Q
Other quality concerns, not identified
above
CONDITION
COMMENT
text
Comments about the condition of the sample. If the
condition code='W' then provide the temperature
8.5 Procedure
8.5.1 Test preparation
1. Filter all lake water samples with a 0.2 pirn filter (e.g., Anotop or Arcodisc) to remove particles.
2. If the atrazine concentration of a sample exceeds 5 ppb, you will need to repeat the test with a
diluted sample. A ten-fold or greater dilution of the sample is recommended with an
appropriate amount of Diluent/ Zero Standard or Sample Diluent (e.g., make a ten-fold dilution
by adding 100 piL of the sample to 900 piL if Diluent/ Zero Standard). Mix the dilution thoroughly
before assaying. Perform the assay according to the Assay Procedure and calculate the final
results by multiplying the value obtained by the dilution factor.
3. Bring reagents to room temperature and thoroughly mix the antibody coupled paramagnetic
particles before use. 2
M
8.5.2 Procedural notes and precautions ^
• A consistent technique is important for optimal performance. For the greatest precision, treat
each tube in an identical manner. IS
LU
• Add reagents directly to the bottom of the tube while avoiding contact between the reagents g
already added to the tube and the pipet tip. This will help assure consistent quantities of
reagent in the test mixture.
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• Avoid cross contamination and carryover of reagents by using clean pipets for each sample
addition and by avoiding contact between reagent droplets on the tubes and the pipet tips.
• Avoid foam formation during vortexing.
• Mix the antibody coupled paramagnetic particles just prior to pipeting.
8.5.3 Assay procedure
1. Label test tubes for standards, controls, and samples (Table 8.2).
2. Add 200 or 250 piL of the appropriate standard, control, or sample to the test tube.
3. Add 250 piL of Atrazine Enzyme Conjugate to each tube.
4. Mix the Atrazine Antibody Coupled Paramagnetic Particles thoroughly and add 500 piL to each
tube.
5. Vortex for 1 to 2 seconds minimizing foaming.
6. Incubate for 15 minutes at room temperature.
7. Separate in the Magnetic Separation System for two minutes.
8. Decant and gently blot all tubes briefly in a consistent manner.
9. Add lmL of washing solution to each tube and allow them to remain in the magnetic separation
unit for two minutes.
10. Decant and gently blot all tubes briefly in a consistent manner.
11. Repeat steps 9 and 10 one additional time.
12. Remove the rack from the separator and add 500 piL of Color Solution to each tube.
13. Vortex for 1 to 2 seconds minimizing foaming.
14. Incubate for 20 minutes at room temperature.
15. Add 500 piL of Stopping Solution to each tube.
16. Add 1 mL Washing Solution to a clean test tube. Use as a blank in Step 17.
17. Within 15 minutes after the addition of the stopping solution, read the absorbance at 450 nm
with a photometer.
8.5.4 Results
1. Calculate the mean absorbance value for each of the standards.
2. Calculate the %B/B0 for each standard by dividing the mean absorbance value for the standard
by the mean absorbance value for the Diluent/ Zero Standard.
3. Construct a standard curve by plotting the %B/B0 for each standard on the vertical logit (Y) axis
versus the corresponding atrazine concentration on the horizontal logarithmic (X) axis.
4. %B/B0 for controls and samples will then yield levels in ppb of atrazine by interpolation of the
standard curve.
Some instrument manufacturers make photometers that allow for automatic calculation of calibration
curves. Refer to instrument operating manuals for detailed instructions.
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Table 8.2 Test tube labeling for atrazine assay.
Tube Number Contents of Tube
1,2
Diluent/ Zero Standard, 0 ppb
3,4
Standard 1, 0.1 ppb
5,6
Standard 2,1.0 ppb
7,8
Standard 3 5.0 ppb
9
Control
10
Sample 1
11
Sample 2
12
Sample 3
8.5.5 Data Entry
Required data elements that laboratories must provide to the USEPA, are identified in the USEPA's data
template, available separately from the USEPA. If the laboratory applies its own QC codes, the data
transmittal must define the codes.
8.6 Pertinent QA/QC Procedures
8.6.1 Internal QC
1. A control solution at approximately 3 ppb of atrazine is provided in the atrazine kit. Include a
control in every run and treat it in the same manner as an unknown sample.
2. Prepare and incubate one duplicate sample for every 10 samples analyzed.
3. Table 8.3 provides a summary of the quality control requirements.
Table 8.3 Atrazine: quality control requirements.
Quality Control Description and Requirements
Corrective Action
Activity
Kit - Shelf Life
Is within its expiration date listed on kit
box.
If kit has expired, then discard or set aside for
training activities.
Kit - Contents
All required contents must be present
and in acceptable condition. This is
important because Abraxis has calibrated
the standards and reagents separately
for each kit.
If any bottles are missing or damaged, discard
the kit.
Calibration
All of the following must be met:
o Standard curve must have a
correlation coefficient of >0.99;
o Average absorbance value, Ao,
for SO must be >0.80; and
o Standards S0-S3 must have
decreasing average absorbance
values. That is, if Ai is the
average of the absorbance
values for Si, then the
absorbance average values must
be: Ao > Ai > A2> A3
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.
N
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QC
QC
u
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Q
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Quality Control Description and Requirements
Corrective Action
Activity
Kit Control
The average concentration value of the
duplicates (or triplicate) must be within
the range of 3 ppb +/-10%.
If the 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:
o All concentration values must be
< 0.05 ng/L (i.e., the reporting
limit)
Sample
Evaluations
Samples are run in duplicate: requires 1
in 10 duplication of samples; Each
duplicate pair must have %CV<10%
between its absorbance values.
If the requirement fails when applying the:
All samples in the kit, not just the sample that
failed, must be run a second time.
No samples are to be run more than twice.
ResuIts Within
Calibration
Range
If the result is less than the upper
calibration range (i.e., 5.0 ng/Lfor
undiluted samples), then the
requirement is met.
If a result registers as "HIGH", then record the
result with a data flag of "HI." If the result
registers as 'HIGH/ then the sample must be
diluted and re-run. If the sample is evaluated
using a duplicate pair, if one or both results
register as 'HIGH', then the sample must be
diluted and re-run. No samples are to be run
more than twice. The lab reports both the
original and diluted sample results.
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.
8.6.2 External QC ^
~z.
1. For the national lab, analyze 10 provided spiked samples (blind sample) provided by the USEPA n
<
HQ Laboratory Review Coordinator. For the state labs, analyze 5 provided spiked samples (blind
sample) provided by the USEPA HQ Laboratory Review Coordinator. After processing the "f;
samples, the laboratory will send the results to the USEPA HQ Laboratory Review Coordinator. ^
l i l
The results will be compared to the known concentrations and a determination made.
CO
LU
Q
u
I—
CO
LU
Q_
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9.0 FISH TISSUE FILLET (Whole Fish Composite Sample)
Laboratory methods incorporated into EPA Office of Science and Technology QA documents.
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10.0 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 NLA 2022
and related studies.
10.1 Analytical Parameters
A total of 20 parameters are determined from each bulk water chemistry sample collected (Table 10.1). In
addition, chlorophyll a is determined from a separate, discrete sample following the same performance-based
methods approach as proposed for water chemistry analytes.
Table 10.1 Water chemistry parameters measured for the National Lakes Assessment 2022.
Analyte
Units
Comments
Conductivity
|j.S/cm at 25°C
PH
Standard (Std) Units
Turbidity
NTU
Acid Neutralizing Capacity (ANC)
M-eq/L
(20 neq/L=l mg as CaCOs)
Dissolved Organic Carbon (DOC)
mg/L
Ammonia-N (NH3-N)
mg/L
The method measures ammonia and ammonium;
the relative proportion between these two
analytes depends on pH. Typically, NLA (and other
NARS) samples consist of mostly ammonium
Nitrate-Nitrite (NO3-NO2)
mg/L
Note different preservation methods and holding
times depending on whether the lab is using ion
chromotography (IC) or flow injection analysis (FIA)
Total Nitrogen (TN)
mg/L
Total Phosphorus (TP)
Hg/L
Total Dissolved Nitrogen (TDN)
mg/L
Supplemental analysis in NLA 2022
Total Dissolved Phosphorous (TDP)
Hg/L
Supplemental analysis in NLA 2022
Sulfate (SO4)
mg/L
Chloride (CI)
mg/L
Nitrate (NO3)
mg/L
May be obtained as part of nitrate-nitrite
determination (use FIA to obtain nitrate-nitrite and
nitrite separately, then calculate difference for
nitrate), or as a direct measurement (e.g., ion
chromatography)
Calcium (Ca)
mg/L
Magnesium (Mg)
mg/L
Sodium (Na)
mg/L
Potassium (K)
mg/L
Silica (Si02>
mg/L
True Color
PCU
Performance objectives based on use of visual
estimation method
Chlorophyll a
]ug/L (in extract)
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10.2 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 upon 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 1.2 of the manual 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 sample tracking form
b. Record the information in Table 10.2 for the NARS IM Team, 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. W: Sample is warm (>7°), record the temperature in the comment field, and perform the assay
c. 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 Chapter 2 of the Manual).
3. Store samples in the refrigerator until sample preparation begins.
4. Maintain the sample tracking forms with the samples.
Table 10.2 Water Chemistry login: required data elements.
FIELD FORMAT DESCRIPTION
LABORATORY ID
text
Name or abbreviation for laboratory
DATE RECEIVED
MMDDYY
Date sample was received by laboratory
SITE ID
text
NLA site id as used on sample label
VISIT NUMBER
numeric
Sequential visits to site (1 or 2)
SAMPLE ID
numeric
Sample id as used on field sheet (on sample label)
DATE COLLECTED
MMDDYY
Date sample was collected
CONDITION CODE
text
Condition codes describing the condition of the sample upon arrival
at the laboratory.
Flag
Definition
OK
Sample is in good condition
C
Sample container is cracked
L
Sample or container is leaking
ML
Sample label is missing
W
Sample is warm (>7°)
Q
Other quality concerns, not identified above
CONDITION
COMMENT
text
Comments about the condition of the sample. If the condition
code='W' then provide the temperature
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10.3 Sample Processing and Preservation
Due to the short holding time of these samples, samples will be shipped overnight by the field crews and must
be preserved by close of business (COB) the day after sample collection. If expected samples do not arrive or
arrive after the acceptable time frame (24 hours after the samples were collected), laboratories must flag those
samples on the sample check-in spreadsheet provided by the NARS IM Team and notify the NARS IM
Coordinator (see Section 1.2).
Upon receipt of samples, inspect each sample and review the tracking form that was included with the samples.
Samples damaged during the shipping process are flagged by the laboratory on the sample check-in spreadsheet
upon receipt and inspection. Store samples at 4°C in darkness until aliquots are ready to be prepared. If possible,
prepare aliquots the same day as samples are received, but no later than 48 hours after receipt. Laboratories
should be familiar with and ensure that samples meet all defined target holding times. Any sample that does not
meet holding time requirements is flagged and evaluated to determine if the exceedance impacts either sample
integrity or any potential end uses of the data (USEPA 2002). Results from samples that exceeded target holding
times are not rejected outright.
10.3.1 Water Chemistry Samples
Figure 10.1 illustrates sample preparation processing for the water chemistry indicators, including filtering and
acidifying, for the various analytes.
1. Use 0.4nm pore size polycarbonate filters for all filtration.
2. Rinse vacuum filter funnel units thoroughly with reverse-osmosis (RO) or deionized (Dl) water (ASTM
Type II reagent water) five times before each use and in between samples. After placing a filter in the
funnel unit, run approximately 100 mL of RO or Dl water through the filter, with vacuum pressure, to
rinse the filter. Discard the rinse water.
3. Place the appropriate sample bottle under the funnel unit and filter sample directly into the bottle. If a
new filter is needed, remove the sample bottle, and rinse the new filter with 100 mL of RO or Dl water
before continuing.
4. After all filtered and unfiltered aliquots are collected, add ultra-pure acid (HN03 or H2S04, depending on
the analyte, see Table 10.3) to the sample in the aliquot container. Cap tightly and invert the bottle
several times to mix.
5. Store all aliquots except the cation aliquot (filtered, acidified with HN03) in a refrigerator at 4°C. <
I
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Sample Receipt
4 L Bulk Sample
Inspect samples and complete
tracking form
Store at 4°C in darkness
r
Process Sample
Within 24 hours
Filtration (0.4um)
Not Filtered
HDPE
bottle
Acid
washed
Preserve
with
HNO
Analyses
Calcium (180 d)
Magnesium (180 d)
Sodium (180 d)
Potassium (180 d)
HDPE
bottle
Not acid
washed
Store at
4 °C in
J
Analyses
Chloride (28 d)
Nitrate (7 d)
Sulfate (28 d)
Silica (28 d)
Nitrate-Nitrite
(with IC) (7 d)
Nitrate (with IC)
(7 d)
True Color (3 days)
HDPE
bottle
Acid
washed
Preserve
with
_L
Analyses
Ammonia-N (28 d)
Dissolved Organic
Carbon (28 d)
Nitrate-Nitrite (with
FIA) (28 d)
Nitrate (with FIA) (28 d)
Dissolved TN (28 d)
Dissolved TP (28d)
-SZZZ-
HDPE
bottle
Acid
washed
Preserve
with
H2S0F
Analyses
Total Phosphorus
(28 d)
Total Nitrogen
(28 d)
T7T»—(
HDPE
bottle
Not acid
washed
Store at
4 °C in
\
Analyses
pH (3 d)
ANC (7 d)
Conductivity
(7 d)
Turbidity (3 d)
/
Figure 10.1 Water chemistry sample processing procedures.
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Table 10.3 Acid preservatives added for various analytes.
Preservatives
H2SO4
HNO3
DOC
Ca
NHs-N
Mg
TN
Na
TP
K
TDN
TDP
NO2-NO3
National Lakes Assessment 2022
Version 1.1, May 2022
10.3.2 Chlorophyll a Samples
Chlorophyll a samples are filtered in the field, placed in a labeled centrifuge tube in a dark cooler, and stored on
ice until arrival at the laboratory. Store the filter in the centrifuge tube in the freezer at -20 ± 2°C for no more
than thirty days before analysis.
10.4 Performance-based Methods
As an alternative to specifying laboratory methods for sample analysis, a performance-based approach that
defines a set of laboratory method performance requirements for data quality is utilized for this survey. Method
performance requirements for this project identify lower reporting limit (LRL), precision, and bias objectives for
each parameter (Table 10.5). The LRL is the lowest value that needs to be quantified (as opposed to just
detected), and is equal to or above the value of the lowest non-zero calibration standard. It is set to double the
long-term method detection limit (LT-MDL), following guidance presented in Oblinger, Childress et al. (1999).
Precision and bias objectives are expressed in both absolute and relative terms following Hunt and Wilson
(1986). The transition value is the value at which performance objectives for precision and bias switch from
absolute (< transition value) to relative (> transition value). For pH, the objectives are established for samples
with lower H+ (or OH") concentrations (pH between 5.75 and 8.25) and higher H+ (or OH") concentrations (pH <
5.75 or >8.25).
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 (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. Bias (i.e., systematic error) is estimated as the difference between the mean measured O
value and the target value of a performance evaluation and/or internal reference samples at the lower O
concentration range measured across sample batches, and as the percent difference at the higher concentration J
range. "g
tc
Analytical methods used for past surveys (USEPA ORD-Corvallis) are summarized in Table 10.4. Participating ^
laboratories may use alternative analytical methods for each target analyte if they can satisfactorily
demonstrate the alternative method can achieve the performance requirements as listed in Table 10.5. ^
Information is provided by the laboratory to the NLA Quality Team. The team reviews the information to J
determine whether the laboratories meet the necessary requirements. The information from this process is g
maintained in the NLA 2022 QA files by the USEPA HQ Laboratory Review Coordinator. !<
CL
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Table 10.4 Summary of analytical methods used by NLA 2022 (PESD Laboratory, USEPA ORD-Corvallis).
Analyte
Summary of Method6
References'
WRS SOPg
pH (laboratory)
Automated, using ManSci PC-Titrate w/Titra-Sip autotitrator and Ross
combination pH electrode. Initial pH determination for ANC titration
EPA 150.1 (modified),
APHA 4500H
WRS 16A.2 (April
2021)
Specific conductance @
25°C
Electrolytic, Man-Tech TitraSip automated analysis
OR manual analysis, electrolytic
EPA 120.1, APHA 2510
WRS 16A.2 (April
2021)
WRS 11A.5 (April
2021)
Acid neutralizing
capacity (ANC)
Automated acidimetric titration to pH<3.5, with modified Gran plot
analysis
EPA 310.1, APHA 2320
WRS 16A.2 (April
2021)
Turbidity
Nephelometric; Man-Tech TitraSip automated analysis,
OR
Manual analysis using Hach turbidimeter (high turbidity samples)
EPA 180, APHA 2130
WRS 16A.2 (April
2021)
WRS 13A.4 (April
2021)
True color (Hach Kit)
Visual comparison to calibrated glass color disk.
EPA 110.2 (modified),
APHA2120
WRS 15A.4 (April
2021)
Dissolved Organic
Carbon (DOC)
UV promoted persulfate oxidation to CO2 with infrared detection
APHA 5310-C, EPA 415.3
WRS 21A.5 (May
2021)
Nitrate+Nitrite, as N
(fresh waters)
Ion Chromatography
OR
FIA automated colorimetric (cadmium reduction)
EPA 300.1; SW-846
9056A; APHA 4110B
EPA 353.2
APHA 4500-N03-N-E
Lachat 10-107-04-1-C
WRS 36A.2 (April
2021
WRS 40A.7
(February 2021)
Nitrate
Measured as part of Nitrate-Nitrite when using IC or calculated after
measuring Nitrate+Nitrite using FIA.
See above
See above
Ammonia, as N (fresh
waters)
FIA automated colorimetric (salicylate, dichloroisocyanurate)
Lachat 10-107-06-3-D
WRS 30A.5 (April
2021)
Silica, dissolved (SiOz)
(fresh waters)
FIA automated colorimetric (molybdate, stannous chloride)
EPA366.0,APHA 4500-
Si02 F,
Lachat 10-114-27-1-B
WRS 32A.6
(February 2021)
Total nitrogen (TN)
Persulfate Digestion; FIA Automated Colorimetric Analysis (Cadmium
Reduction, sulfanilamide)
EPA353.2 (modified)
APHA 4500-N-C
(modified)
ASTM WK31786
Lachat 10-107-04-1-C
(modified)
WRS 34A.6
(February 2021)
Total phosphorus (TP)
Persulfate Digestion; Automated Colorimetric Analysis (molybdate,
ascorbic acid)
EPA 365.1 (modified),
APHA 4500-P-E
USGS 1-4650-03
Lachat 115-01-1-B
(modified)
WRS 34A.6
(February 2021)
Dissolved nitrogen
Persulfate Digestion; FIA Automated Colorimetric Analysis (Cadmium
Reduction, sulfanilamide)
EPA353.2 (modified)
APHA 4500-N-C
(modified)
ASTM WK31786
WRS 34A.6
(February 2021)
e FIA=Flow injection analysis.
f APHA= American Public Health Association, Standard Methods for the Examination of Water and Wastewater,. EPA=Methods for
Chemical Analysis of Water and Wastes.
g WRS= Willamette Research Station. References are to laboratory SOP. Available upon request, (contact the Project Lead). The WRS
laboratory is now known as the Pacific Ecological Surveys Division but SOP names have not yet been changed.)
U
oc
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Analyte
Summary of Method6
References'
WRS SOPg
Lachat 10-107-04-1-C
(modified)
Dissolved phosphorus
Persulfate Digestion; Automated Colorimetric Analysis (molybdate,
ascorbic acid)
EPA 365.1 (modified),
APHA 4500-P-E
USGS 1-4650-03
Lachat 115-01-1-B
(modified)
WRS 34A.6
(February 2021)
Major anions, dissolved
chloride, nitrate,
nitrite, sulfate
Ion Chromatography
EPA 300.1; SW-846
9056A; APHA 4110B
WRS 40A.7
(February 2021)
Major cations,
dissolved
calcium, sodium,
potassium,
magnesium,
Inductively-coupled Plasma Atomic Emission Spectroscopy (ICP-AES)
EPA 200.7; SW6010D
WRS 52A.14
(January 2017)
Chlorophyll a
(Chl-a)
Extraction 90% acetone analysis by fluorometry
EPA 445.0, EPA 446.0
WRS 71A.5
(February 2021)
10.5 Pertinent QA/QC Procedures
A single analytical laboratory and some State laboratories will analyze the water chemistry samples. The specific
quality control procedures used by each laboratory are implemented to ensure that:
• Objectives established for various data quality indicators being met.
• Results are consistent and comparable among all participating laboratories.
The analytical laboratory demonstrated in previous studies that it can meet the required LRL (USEPA 2004).
QA/QC procedures outlined in this manual and the NLA 2022 QAPP will be followed to ensure these LRLs are
met for the NLA 2022.
10.5.1 Laboratory Performance Requirements
Table 10.5 summarizes the pertinent laboratory performance requirements for the water chemistry and
chlorophyll a indicators.
10.5.2 Laboratory Quality Control Samples
Table 10.6 summarizes the pertinent laboratory quality control samples for the water chemistry and chlorophyll
a indicators.
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Table 10.5 Laboratory method performance requirements for water chemistry and chlorophyll a sample analysis.
Analyte Units Potential Range
of Samples'1
Lower Transition Precision Bias
Reporting Value' Objectivek Objective1
Limit1
Conductivity
l_iS/cm at
25°C
2-34,000
2.0
20
±1 or ±10%
± 1 or 5%
pH (laboratory)
Std Units
3.5 to 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: ±0.05
5.75- 8.25:
±0.10
Turbidity
NTU
0 to 1,000
2.0
10
±1 or ±10%
± 1 or ±10%
Dissolved Organic
Carbon (DOC)
mg/L
0.2 to 160
0.20
< 1
> 1
±0.10 or ±10%
±0.10 or ±10%
Ammonia as
N(NHs-N)
mg/L
0 to 3
0.02
0.02
± 0.002 or
±10%
± 0.002 or
±10%
Nitrate-Nitrate
(NO3-NO2)
mg/L
0 to 360 (as
nitrate)
0.02
0.10
± 0.01 or ±10%
± 0.01 or ±10%
Total Nitrogen (TN)
mg/L
0.01 to 36
0.02
0.10
± 0.01 or ±10%
± 0.01 or ±10%
Total Phosphorus
(TP)
Hg/L
0 to 11,000
4
20
± 2 or ±10%
± 2 or±10%
Dissolved Nitrogen
mg/L
NA
0.02
0.10
± 0.01 or ±10%
± 0.01 or ±10%
h Estimated from samples analyzed at the PESD-Corvallis laboratory between for NLA 2017.
' The lower reporting limit is the lowest value that needs to be quantified (as opposed to just detected) and is equal to or
above the value of the lowest nonzero calibration standard. It is set to 2 times the long-term detection limit, following USGS
Open File Report 99-193 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 (USGS —j
1999). ^
CL
J Value at which performance objectives for precision and bias switch from absolute (< transition value) to relative > §
transition value). Two-tiered approach based on Hunt, D.T.E. and A.L. Wilson. 1986. The Chemical Analysis of Water: O
General Principles and Techniques. 2nd ed. Royal Society of Chemistry, London, England. J
~o
k For duplicate samples, precision is estimated as the pooled standard deviation (calculated as the root-mean square) of all c
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 H
measurements across batches at the lower concentration range, and as percent relative standard deviation of repeated ^
measurements across batches at the higher concentration range. ^
. u
1 Bias (systematic error) is estimated as the difference between the mean measured value and the target value of a cc
LU
performance evaluation and/or internal reference samples at the lower concentration range measured across sample ^
batches, and as the percent difference at the higher concentration range. ^
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Analyte
Units
Potential Range
of Samples'1
Lower
Reporting
Limit1
Transition
Value1
Precision
Objectivek
Bias
Objective1
Dissolved
Phosphorus
Hg/L
NA
4
20
± 2 or±10%
± 2 or ±10%
Sulfate (SO4)
mg/L
0 to 7,000
0.50
2.5
± 0.25 or ±10%
± 0.25 or ±10%
Chloride (CI)
mg/L
0 to 12,000
0.20
1
±0.10 or ±10%
±0.10 or ±10%
Nitrate (NO3)
mg/L
Oto 30
0.02
0.1
± 0.01 or ±10%
± 0.01 ±10%
Calcium (Ca)
mg /L
0.04 to 550
0.10
0.5
± 0.05 or ±10%
± 0.05 or ±10%
Magnesium (Mg)
mg/L
0.0.3 to 1000
0.10
0.5
± 0.05 or ±10%
± 0.05 or ±10%
Sodium (Na)
mg/L
0.08 to 8,000
0.10
0.5
± 0.05 or ±10%
± 0.05 or ±10%
Potassium (K)
mg/L
0.01 to 440
0.10
0.5
± 0.05 or ±10%
± 0.05 or ±10%
Silica (SiC>2)
mg/L
0.02 to 70
0.10
0.5
± 0.05 or ±10%
± 0.05 or ±10%
True Color
PCU
0 to 250
5
50
±5 or ±10%
±5 or ±10%
Chlorophyll a
M-g/L (in
extract)
3.5 to 4,000
0.5
15
± 1.5 or ±10%
± 1.5 or ±10%
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Table 10.6 Laboratory quality control samples: water chemistry indicator.
QC Sample
Analytes
Description
Frequency
Acceptance
Corrective Action
Type and
Criteria
Description
Laboratory/
Reagent
Blank
All
Once per
analytical batch
prior to sample
analysis
Control limits <
LRL
Prepare and analyze new
blank. Determine and
correct problem (e.g.,
reagent contamination,
instrument calibration, or
contamination introduced
during filtration) before
proceeding with any sample
analyses. Reestablish
statistical control by
analyzing three blank
samples.
Filtration
Blank
All dissolved
analytes
ASTM Type II
reagent water
processed
through
filtration unit
Prepare once per
week and archive
Prepare filter
blank for each box
of 100 filters, and
examine the
results before any
other filters are
used from that
box.
No analytes
>LRL
Measure archived samples if
review of other laboratory
blank information suggest
source of contamination is
sample processing.
Method
Detection
Limit Check
Standard
(MDL-C)
All analyses
except true
color and
turbidity
Prepared so
concentration
is four to six
times the LT-
MDL objective
Once per day
Target LT-MDL
value (which is
calculated as a
99% confidence
interval)
Confirm achieved LRL by
repeated analysis of LT-MDL
QCCS. Evaluate affected
samples for possible re-
analysis.
Initial and
Continuing
Calibration
Verification
Analyze ICV after
calibration.
Analyze CCV after
every 10 samples
and at the end of
analytical batch.
±10% or
method criteria
Perform corrective action
and repeat all associated
samples since last successful
CCV. Alternatively,
recalibrate and re-analyze all
samples since last successful
CCV.
Analytical
Duplicate
Sample
All analyses
One per 10
samples
Within
precision
objective
If results are below LRL:
Prepare and analyze
duplicate from different
sample (volume permitting).
Review precision of batch.
Check preparation of
duplicate sample.
Standard
Reference
Material
(SRM)
When
available for a
particular
analyte
One analysis in a
minimum of five
separate batches
Manufacturers
certified range
Analyze standard in next
batch to confirm suspected
imprecision or bias. Evaluate
calibration standards for
contamination and
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QC Sample
Analytes
Description
Frequency
Acceptance
Corrective Action
Type and
Criteria
Description
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.
Matrix Spike
Samples
Only prepared
when samples
with potential
for matrix
interferences
are
encountered
One per batch
Control limits
for recovery
cannot exceed
100±20%
Select two additional
samples and prepare
fortified subsamples.
Reanalyze all suspected
samples in batch by the
method of standard
additions. Prepare three
subsamples (unfortified,
fortified with solution
approximately equal to the
endogenous concentration,
and fortified with solution
approximately twice the
endogenous concentration).
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10.5.3 Data Reporting, Review, and Management
Checks made of the data in the process of review and verification are summarized in Table 10.7. Data reporting
units and significant figures are given in Table 10.8. The NLA 2022 Project QA Officer is ultimately responsible for
ensuring the validity of the data, although performance of the specific checks may be delegated to other staff
members.
Table 10.7 Data validation quality control for water chemistry indicator.
Activity or Procedure
Requirements and Corrective Action
Range checks, summary statistics, and/or
exploratory data analysis (e.g., box and
whisker plots)
Correct reporting errors or qualify as suspect or invalid.
Review holding times
Qualify value for additional review
Ion balance:
Calculate percent ion balance difference
(%IBD) using data from cations, anions,
pH, and ANC. See Equation 10.1.
• If total ionic strength <100 |aeq/L
- %IBD < ±25%.
• If total ionic strength > 100 |aeq/L
- %IBD <±10%.
• Determine which analytes, if any, are the largest
contributors to the ion imbalance. Review suspect
analytes for analytical error and reanalyze.
— Flag = unacceptable %IBD
• If analytical error is not indicated, qualify sample to
attribute imbalance to unmeasured ions. Reanalysis is
not required.
— Flag = %IBD outside acceptance criteria due to
unmeasured ions
Conductivity check:
Compare measured conductivity of each
sample to a calculated conductivity based
on the equivalent conductance of major
ions in solution (Hillman et al., 1987)
• If measured conductivity < 25 |j.S/cm,
— ([measured - calculated] -r- measured) < ±25%.
• If measured conductivity > 25 |j.S/cm,
— ([measured - calculated] -r- measured) < ±15%.
• Determine which analytes, if any, are the largest
contributors to the difference between calculated and
measured conductivity.
• Review suspect analytes for analytical error and
reanalyze.
• If analytical error is not indicated, qualify sample to
attribute conductivity difference to unmeasured ions.
Reanalysis is not required.
Review data from QA samples (laboratory
PE samples, and inter-laboratory
comparison samples)
Indicator QC Coordinator determines impact and possible
limitations on overall usability of data based on the specific
issue.
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Table 10.8 Data reporting criteria: water chemistry indicator.
Measurement
Units
No. Significant
Figures
Minimum No.
Decimal Places
DO
mg/L
2
1
Temperature
°C
2
1
PH
pH units
3
2
Carbon, dissolved organic
mg/L
3
1
ANC
M-eq/L
3
1
Conductivity
|j.S/cm at 25 °C
3
1
Calcium, magnesium, sodium, potassium, chloride,
nitrate, and sulfate
M-eq/L
3
1
Silica
mg/L
3
2
Total phosphorus
M-g/L
3
0
Total nitrogen
mg/L
3
2
Total dissolved phosphorus
M-g/L
3
0
Total dissolved nitrogen
mg/L
3
2
Nitrate-Nitrite
mg/L
3
2
Ammonia-N
mg/L
3
2
Turbidity
NTU
3
0
True color
PCU
2
0
Chlorophyll a
ug/l
3
2
The ion balance for each sample is computed using the results for major cations, anions, and the measured acid
neutralizing capacity. The percent ion difference (%IBD) for a sample is calculated as:
Equation 10.1 Percent ion difference (%IBD)
(V cations - \ anions)- ANC
%IBD = r—1
ANC + > anions + \ cations + 2\H J
where ANC is the acid neutralization capacity; cations are the concentrations of calcium, magnesium, sodium,
potassium, and ammonium (converted from mg/L to |a,eq/L); anions are the concentrations of chloride, nitrate,
and sulfate (converted from mg/L to |a,eq/L), and H+ is the hydrogen ion concentration calculated from the
antilog of the sample pH. Factors to convert major ions from mg/L to |a,eq/L are presented in Table 10.9.
For the conductivity check, equivalent conductivities for major ions are presented in Table 10.10.
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Table 10.9 Constants for converting major ion concentration from mg/Lto |ieq/L
Analyte Conversion from mg/Lto |ieq/Lm
Calcium
49.9
Magnesium
82.3
Potassium
25.6
Sodium
43.5
Ammonia-N
55.4
Chloride
28.2
Nitrate
16.1
Sulfate
20.8
Table 10.10 Factors to calculate equivalent conductivities of major ions."
Ion
Equivalent Conductance per mg/L Ion
Equivalent Conductance per
(|4,S/cm at 25 °C)
mg/L (|iS/cm at 25 °C)
Calcium
2.60
Nitrate
1.15
Magnesium
3.82
Sulfate
1.54
Potassium
1.84
Hydrogen
3.5 x 105°
Sodium
2.13
Hydroxide
1.92 x 105
Ammonia-N
4.13
Bicarbonate
0.715
Chloride
2.14
Carbonate
2.82
10.5.4 Data Entry
Required data elements that laboratories must provide to the USEPA, are identified in the USEPA's data
template, available separately from the USEPA. If the laboratory applies its own QC codes, the data transmittal
must define the codes.
CL
O
QC
o
u
T3
C
tc
>
QC
m Measured values are multiplied by the conversion factor. J
" From Hillman et al. (1987). 2j
° Specific conductance per mole/L, rather than per mg/L. ^
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11.0 ZOOPLANKTON METHODS
This method is used to identify and enumerate species of lake zooplankton collected with vertical plankton net
tows using the NLA 2022 method. Macrozooplankton are counted from a sample using a course (150 pim) mesh
nets. Microzooplankton, especially rotifers, nauplii, copepodites <0.6 mm long, and cladocerans <0.2 mm long,
are counted from a sample collected using a fine mesh net (50 pim). Both the course and fine mesh nets include
a reducing collar with diameter of 20 cm (30 cm to 20 cm cowling).
Zooplankton samples will be preserved in the field with EtOH and shipped from field crews to a contract
batching laboratory. The contract batching laboratory will send the batched samples to the analysis laboratory.
Preserved samples can be held for several months, but zooplankton analysis laboratories will need to process
samples in accordance with the time frame outlined in contractual agreements. Contractual agreements for
delivery of data do not supersede indicator holding times.
11.1 Responsibility and Personnel Qualifications
This procedure may be used by any person who has received training in processing and/or identification of
zooplankton samples. It is also important that the taxonomist maintains contact with other taxonomists through
professional societies and other interactions, and keep abreast of the pertinent literature, because taxonomic
groupings and nomenclatural basis for taxonomy and nomenclature are updated frequently. A second
taxonomist will re-identify a randomly-selected 10% of the samples for QC, as noted below, to quantify
taxonomic precision, or consistency, as percent taxonomic disagreement (PTD), help target corrective actions,
and ultimately help minimize problems during data analysis. Samples are sent from the field to the laboratory on
a regular basis during the project to avoid delays in processing and specimen identification.
11.2 Precautions
Wear appropriate clothing for safety precautions, such as nitrile gloves, rubber apron, long pants, etc. Follow all
laboratory safety and waste disposal guidelines regarding the disposal of formalin (37% formaldehyde)
solutions.
11.3 Equipment/Materials
• Dissection microscope (magnifications: 10X-50X)
• Compound microscope (magnifications: 40X-400X with phase-contrast capability)
• Hensen-Stempel pipettes (1, 2, and 5 mL)
• Graduated cylinders (100-, 250-, and 500mL)
• Folsom plankton Splitter
• Ward counting wheel or other suitable counting chamber
• Utermohl counting chamber or Sedgwick-Rafter counting cell (1 mL vol) with cover slips
• Ring nets with 50, 500 and 1000 pim Nitex mesh
• Mechanical or electronic tally counters
• Microscope slides, 1 x 3 inch
• Cover slips
• Tubes for concentrating plankton samples (see below)
• Small sieves with 45 and 140-pim mesh
• 50-nm Nitex mesh Heavy duty rubber bulb Microprobe
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• 150-nm Nitex mesh Heavy duty rubber bulb Microprobe
• Micro-forceps
• 100- to 500-mL glass jars with split fractions written on labels
• Zooplankton Sample Log-In Form
• Zooplankton Laboratory Sheet
• Labels
Construct the first plankton concentrating tube by covering one end of a wide glass tube (such as a
chromatography tube) with 50-nm mesh. Secure the mesh with O-rings and attach a heavy-duty bulb to the
other end to provide suction. Construct the second plankton concentrating tube by covering one end of a wide
glass tube (such as a chromatography tube) with 150-nm mesh. Secure the mesh with O-rings and attach a
heavy-duty bulb to the other end to provide suction.
The following reagents are needed:
• Formalin (37% formaldehyde solution)
• 95% EtOH
• 5% Sodium hypochlorite solution (unscented bleach)
• Rose Bengal stain dissolved in EtOH
• Dilute solution of laboratory detergent
11.4 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 1.2 of the manual 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 sample tracking form.
b. Record the information in Table 11.1 for the NARS IM Team, 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
c. If any sample is damaged or missing, contact the USEPA HQ Laboratory Review Coordinator to
discuss whether the sample can be analyzed.
3. Store samples until sample preparation begins.
4. Maintain the sample tracking forms with the samples.
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Table 11.1 Zooplankton login: required data elements.
FIELD
FORMAT
DESCRIPTION
LAB
text
Name or abbreviation for laboratory
DATE RECEIVED
MMDDYY
Date sample was received by laboratory
SITE ID
text
NLA 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
Q
Other quality concerns, not identified
above
CONDITION
COMMENT
text
Comments about the condition of the sample.
11.5 Procedure
11.5.1 Zooplankton Stratified Splitting
1. Record all zooplankton samples received at the laboratory in a logbook or sample log form (See
APPENDIX B: SAMPLE LABORATORY FORMS: Zooplankton Sample Log In Form). Add approximately 1 to
3 mL of Rose Bengal stain solution to each sample bottle to aid in finding the smaller organisms. Process
samples one at a time. Shake jar to mix water sample. Under the hood, rinse the first sample jar taken
with the 50-nm mesh net through a 45-pim mesh sieve with deionized (Dl) water to remove the EtOH;
the second sample bottle, taken from the 150-nm mesh net, is rinsed through a 145-nm mesh sieve with
Dl water to remove the EtOH. The two mesh size samples are treated as individual samples for
processing and identification and are to be recorded on the laboratory bench sheet with the sample
number and corresponding mesh size.
2. Be sure to rinse the corresponding sample bottles thoroughly with reverse osmosis (RO)/DI/distilled
water into the 45-pim mesh and 145-^m mesh sieve to remove any residual organisms adhering to walls
of the bottle. Rinse all containers from which zooplankton are transferred thoroughly, including the q
Folsom splitter, glass jars, and counting chambers. Wash the sample into a glass jar. Add a small amount §
of dilute laboratory soap to each sample, at this time, to prevent organisms from sticking to the sides of lH
the containers and from floating at the surface of the sample. ^
3. Stir the sample gently to break up algal clumps and then pour the entire sample into the Folsom h
plankton splitter. Stir the sample again to distribute animals uniformly and split the sample by ^
immediately rotating the splitter before the organisms can settle. Rinse the inside of the splitter well to ^
remove organisms that may stick to the sides. Rinse one sub-sample from the splitter receiving trays and §
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save it in a labeled jar indicating the fraction of total original volume of sample bottle (1/2).
4. Place the second sub-sample from the split in the Folsom plankton splitter and divide again. Save one
sub-sample in a labeled jar indicating the fraction of the total original volume it contains (1/4).
5. Repeat Steps 3 and 4 as many times as necessary until the last 2 sub-samples contain at least total of
400, and a maximum of 480 (400+20%), macrozooplankton each (not including rotifers and nauplii).
These 2 sub-samples represent equal fractions of the original sample. Save one sub-sample in a jar
labeled "A", and save the other sub-sample in a jar labeled "B". This process may vary depending on the
density of organisms in the sample. If the minimum count is reached in the "A" subsample, then there is
no need to identify individuals from subsample "B". Write the final split factor used, on the identification
and enumeration bench sheets (see APPENDIX B: SAMPLE LABORATORY FORMS: Zooplankton
Enumeration Data Sheet in).
11.5.2 Taxonomy Procedures
11.5.2.1 Taxonomic Level of Effort
The USEPA will supply a list of taxa that have been collected from previous iterations of the NLA (provided
during laboratory initiation call). This list should be used as a guide for the appropriate taxa names to be used
while processing samples. However, this list will clearly not include all potential taxa that may be encountered,
but should assist in ensuring consistency between surveys. When possible the following resources should be
used to identify zooplankton to species: Edmondson (1959), Pennak (1978), Smith and Fernando (1978),
Stemberger (1979), the online Free-living and Parasitic Copepods (Including Branchiurans) of the Laurentian
Great Lakes: Keys and Details on Individual Species and the online Image-Based Key to the Zooplankton of the
Northeast, USA, produced by the University of New Hampshire Center for Freshwater Biology (cfb.unh.edu).
Other resources and keys can be utilized, but should be provided to USEPA before processing of samples begin.
11.5.2.2 Macrozooplankton Identification and Enumeration (Excluding
Rotifers and Nauplii)
Macrozooplankton are counted and identified from samples collected with the coarse mesh (150 pim) plankton
net.
1. Species-level resolution will be the taxonomic requirement for macrozooplankton.
2. The taxonomist must examine and enumerate as many sub-samples needed to reach the target count
of 400 to 480 organisms and record the information on the appropriate form (see APPENDIX B:
SAMPLE LABORATORY FORMS: Zooplankton Enumeration Data Sheet).
3. Concentrate the sub-sample by using the small sieve or the condensing tube and place in a circular (or
other suitable) counting chamber.
4. Identify all macrozooplankton under a dissecting microscope and enumerate using a mechanical or
electronic tally counter.
5. Count the first two sub-samples which likely contain 400 organisms (Section 11.5.1, step 5) first, and
count additional subsamples to reach enumeration target, if need. Examine and enumerate all
macrozooplankton. If the minimum of 400 organisms in the first of the two original subsamples, then
stop. There will be no need to examine the second of the first two subsamples. During identification
and enumeration, make measurements on selected individuals. For dominate taxa, measure a
minimum of 20 individuals. For subdominant taxa (taxa encountered less than 40 times during
enumeration), measure 10 individuals. For rare taxa (taxa encountered less than 20 times during
enumeration), measure 5 individuals. If rare taxa are in a position that makes it difficult to measure
(e.g. odd angle), then remove these individuals after identification and enumeration and measure them
separately. Additionally, while enumerating and identifying samples, especially note invasive species
such as Bythotrephes and Cercopagis.
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11.5.2.2.1 General Analysis and Guidelines
1. Mount organisms requiring higher magnification for identification on slides and examine at 100 - lOOOx
magnification under a compound microscope.
2. While counting macrozooplankton, make sure that all organisms are settled to the bottom. It is possible
to sink floating macrozooplankton by gently pressing them down using the microprobe or by adding a
drop of dilute laboratory detergent.
3. If a sample cannot be completely counted and archived within 2 days, keep the sample in the
refrigerator and add a few drops of formalin to the jars to prevent organisms from clumping. Sample
analysis should not extend beyond four days.
4. Place voucher specimens in a labeled vial and preserve with 95% EtOH. The label in the vial should
include genus/species name, date preserved, analyst initials, station number, and sample number. A
second taxonomist should confirm the voucher specimens.
11.5.2.2.2 Large Taxa Scan
Observe non-counted sample portion for the following: Leptodora, Chaoborus, Craspedacusta sowerbii.
Mysidae, Ostracoda, and Hydracarina. Spend minimal effort here, < ~l-2 minutes. If detected, enter "yes" in the
LARGE_RARE column on spreadsheet, and put the number counted in the L/R_AUND column.
11.5.2.3 Microzooplankton (Rotifers, Nauplii, and Crustaceans^
Microzooplankton are counted and identified from samples collected with the fine mesh (50 pim) plankton net.
1. Species-level resolution is the taxonomic requirement for rotifers, copepods <0.6 mm long, and
cladocerans <0.2 mm long. Nauplii will be identified to the lowest possible taxonomic unit.
2. Selection of the split level from which a sub-sample for rotifer enumeration is based on estimates made
during macrozooplankton enumeration (rotifers and small crustaceans are visible in the dissecting
microscope).
3. Take two separate 1-mL sub-samples from the appropriate split. Count and identify microzooplankton
from these two sub-samples (see Section 11.5.2.4). In cases where abundances are particularly low, use
more than one 1-mL sub-sample for each count (see step 6).
4. Mix the sample thoroughly, and withdraw a 1-mL sub-sample with a Hensen-Stempel pipette (or other
pre-calibrated large-bore pipette).
5. The 1-mL sub-sample should contain 400 rotifers, crustacean, and nauplii.
6. If the sub-sample contains less than 400 organisms, take a different sub-sample from a jar with a larger
fraction of the original sample volume. If the sub-sample contains more than 480 organisms, use another
sub-sample from a jar with a smaller fraction.
7. It is also permissible to use a second 1-mL aliquot if the original aliquot has less than 400 organisms.
Count this second aliquot in the same manner as the first and combine the results to make a final count.
8. In cases of extremely low microzooplankton densities, concentrate the sample prior to taking sub-
samples with the pipette. The maximum number of 1-mL aliquots counted at the lowest possible split
level is 3 per count (i.e., a total of 6 mL), even if the sum does not reach 400 organisms.
11.5.2.3.1 Preparation and Microzooplankton Enumeration o
1. Place the sub-sample in an Utermohl counting chamber or Sedgwick-Rafter cell and cover with a glass §
cover slip. lH
2. Identify and enumerate all rotifers, microzooplankton, nauplii, and Dreissena veligers and post-veligers ^
under a compound microscope at lOOx magnification. Record results on the appropriate form. Make O
measurements on selected individuals at this time, and follow dominate, subdominant, and rare (Section
11.5.2.2). See measurement parameters for macro- and microzooplankton in Sections 11.5.2.4.1 and
CL
11.5.2.4.2 respectively. O
O
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3. After the counts are completed, measure the volume of the split used, including the volume of the
aliquots, and record this information.
11.5.2.4 Measurement of Macrozooplankton and Microzooplankton
11.5.2.4.1 Crustaceans
To determine size distribution, measure zooplankton by use of a calibrated eyepiece micrometer during the
identification and enumeration process.
Measure the first 20 encounters per species per sample as follows:
Cladocera: Length from the top of the head to the base of the caudal spine or to the end of the
carapace.
Copepoda: Length from tip of the head to the insertion of spines into the caudal ramus.
Mysis: Carapace length, or the length from the tip of the head to the cleft in the telson.
Bythotrephes: Body length, excluding the caudal process.
Cercopagis: Body length, from the top of the eye to the end of the caudal claws.
NOTE: If the organisms are curved or bent, make several straight line measurements and sum to obtain
total length.
11.5.2.4.2 Rotifers
Measure at least 20 encounters per species as follows:
1. Loricate forms: body length from corona to the opposite end at the base of spine (if present).
2. Non-loricate forms: body length from corona to the opposite end, excluding spines, paddles, "toes" or
other extensions.
11.6 Calculating and Reporting
Report zooplankton densities as number of organisms per cubic meter, which is calculated in the following
equations.
11.6.1 Volume of water filtered
Equation 11.1 Volume of water filtered.
V = Lx A
where:
V = Volume of water filtered (m3)
L = Length of vertical tow*
A = Area of the mouth of the net (m2) = 0.0314 m2 for 0.2-m diameter net
i/i
* Field crews are to collect a cumulative tow length of 5 m for each net. Therefore, the tow length should be 5 m o
unless otherwise noted by the field crew in the comments. h
' LU
¦Z.
o
I—
O.
o
O
N
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11.6.2 Macrozooplankton Densities
Equation 11.2 Microcrustacean densities.
V
where:
D = Density of organisms in number per cubic meter
N = Number of organisms
S = Spilt factor
V = Volume of water filtered (from above calculation)
11.6.3 Microzooplankton Densities
Equation 11.3 Microzooplankton densities.
D = Density of organisms in number per cubic meter
N = Number of organisms
Na = Number of lmL aliquots examined
Vs = Volume of sub-samples from which aliquots were taken
S = Spilt factor
V = Volume of water filtered (from above calculation)
Biomass estimates will be based on established length/width relationships (Dumont et al. 1975; McCauley, 1984;
Lawrence et al. 1987). The lengths or the lengths and widths of each species encountered will be measured and will
be equal to 20 for common species and lesser for more rare taxa. For cladocerans, the length will be measured from
the tip of the head to the end of the body (shell spines excluded). For copepods, the length will be determined from
the tip of the head to the insertion of the caudal ramus. The length of rotifers will be measured from the tip of the
head to the end of the body (spines, toes, etc. excluded). In accordance with McCauley (1984), biomass will be
computed for the appropriate number of individuals for each sample location and the arithmetic mean biomass will
be multiplied times the species abundance to produce a species biomass for each sample. More detailed discussion
of the methodology is given in Havens et al (2011), Beaver et al. (2010), and Havens & Beaver (2010).
Prepare a completed data sheet with list of taxa and number of individuals of each taxon for each sample. In
addition, you should organize and archive the full complement of specimens (in containers of preservative
and/or on permanent slide mounts), the "counted" sample (in jars, vials, or slide mounts), the concentrated split
sample, and the unused sample split/fraction. All sample components should be clearly-labeled to associate
multiple vials and slides as a single sample. Labels should be as Sample ID "A," jar/vial 1 of x, and Sample ID "A,"
slide 1 of x; and Sample ID "A," unused sample fraction (1/2 original volume).
Required data elements that laboratories must provide to the USEPA, are identified in the USEPA's data
template, available separately from the USEPA. If the laboratory applies its own QC codes, the data transmittal
must define the codes.
r (NxV.xS)
N, x V
where:
11.6.4 Zooplankton Biomass Estimates
11.6.5 Results of Laboratory Processing, Sample Archiving
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11.7 Pertinent QA/QC Pro cedures
Table 11.2 provides a summary of quality assurance/quality control procedures for the zooplankton indicator.
11.7.1 TaxonomicQC
11.7.1.1 Internal TaxonomicQC
As directed by the Indicator QC Coordinator, an in-house QC Analyst will randomly select 5 of the samples
counted and identified by individual taxonomists to ensure that each meets the acceptable criteria for percent
identification efficiency which is 90%.
If the individual fails to maintain a > 90% identification as determined by QC checks, previous samples will be re-
counted and identified. EPA may also calculate or work with the lab to calculate the proportional analysis found
in the External Taxonomic QC section below using internal QC information.
11.7.1.2 External Taxonomic QC
1. EPA may implement an external taxonomic QC review process for zooplankton. If EPA implements an
external QC process, upon receipt of the data after initial identification, approximately 10% of the
samples (for each laboratory) are randomly-selected for evaluation of taxonomic precision by the
Indicator QC Coordinator. Following primary identification and enumeration, the remaining sample from
each of the randomly selected samples are sent by the original laboratory to a QC taxonomist for
complete sample processing and re-enumeration/identification. The laboratory will send the remaining
field collected sample with a sample tracking form. Differences between the two samples are an
indication of taxonomic precision.
2. Because the laboratory and QC taxonomist will be looking at different subsamples from the original field
collected sample, the QC process will utilize the relative abundance of each taxon identified by both the
laboratory and QC for each sample to determine the precision of taxonomic identifications. To
determine the precision of taxonomic identifications the Indicator QC Coordinator will utilize a Bray-
Curtis Dissimilarity index to compare taxonomic results from two independent taxonomists, using the
formula:
Equation 11.4 Bray-Curtis Dissimilarity (BCd).
BLd ~ w—;—r
UXi+Xj)
where x, andXj are the specific counts from two different taxonomists for each taxon identified in each
subsample.
3. A BCd of 0.25 or less is recommended for taxonomic difference (overall mean < 0.25is acceptable).
Individual samples exceeding 0.25 are examined for taxonomic areas of substantial disagreement, and
the reasons for disagreement investigated. A reconciliation call between the primary and secondary
taxonomist will facilitate this discussion. Results greater than this value are investigated and logged for
indication of error patterns or trends. g
4. Corrective actions include determining problem areas (taxa) and consistent disagreements and ^
addressing problems through taxonomist interactions. These actions help to rectify disagreements ^
resulting from identification to a specific taxonomic level. z
O
I—
11.7.1.3 Taxonomic QC Review & Reconciliation =£
The Indicator QC Coordinator prepares a report or technical memorandum to quantify aspects of taxonomic
precision, assess data acceptability, highlight taxonomic problem areas, and provide recommendations for o
improving precision. This report is submitted to the HQ Project Management Team, with copies sent to the m
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primary and QC taxonomists. Another copy is maintained in the project file. Significant differences may result in
the re-identification of samples by the primary taxonomist and a second QC check by the secondary taxonomist.
Each laboratory prepares reference/voucher samples. These samples will be identified and digitally referenced
(a photograph with taxonomic information superimposed on the photograph and in the file name) and will be
included in an electronic file folder on the NARS SharePoint Site.
All samples are stored at the laboratory until the Project Lead notifies the laboratory regarding disposition.
Table 11.2 Laboratory quality control: zooplankton indicator.
Check or Sample Frequency Acceptance Criteria Corrective Action
Description
IDENTIFICATION
Independent
identification by
outside taxonomist
All uncertain taxa
Uncertain identifications to be
confirmed by expert in
particular taxa
Record both tentative and
independent IDs
Use standard
taxonomic references
For all identifications
All keys and references used
must be on bibliography
prepared by another laboratory
If other references desired, obtain
permission to use from Project
Facilitator
Prepare reference
collection
Each new taxon per
laboratory
Complete reference collection
to be maintained by each
individual laboratory
Laboratory Manager periodically
reviews data and reference collection
to ensure reference collection is
complete and identifications are
accurate
External QC
10% of all samples
completed per
laboratory
Efficiency (BCd) < 0.25
If BCd > 0.25, implement
recommended corrective actions.
DATA VALIDATION
Taxonomic
"reasonable-ness"
checks
All data sheets
Genera known to occur in given
lake or geographic area
Second or third identification by
expert in that taxon
CO
O
o
o
I—
Q_
o
o
M
101
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12.0 RESEARCH INDICATOR: Environmental DNA (eDNA)
Information on this indicator is contained in other research documents.
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13.0 LITERATURE CITED
Abraxis, "Cylindrospermopsin ELISA (Microtiter Plate)/' Product 522011, Undated. Retrieved January
2022 from http://www.abraxiskits.com/wp-content/uploads/2016/08/Cylindrospermopsin-User-
guide-522011.pdf.
Abraxis, "Cylindrospermopsin ELISA Kit, Detailed Procedure," Undated. Retrieved January 2022 from
http://www.abraxiskits.com/wp-
content/uploads/2015/08/Cylindrospermopsin_PN522011_PL.pdf.Abraxis, "Microcystins-ADDA
ELISA (Microtiter Plate): User's Guide R021412." Retrieved on January 14, 2014 from
http://www.abraxiskits.com/uploads/products/docfiles/278_Microcystin%20PL%20ADDA%20users
%20R120214.pdf.
Abraxis, "Microcystins-ADDA ELISA (Microtiter Plate)," Product 520011, R021412, Undated. Retrieved
January 2014 from
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%20R120214.pdf.
Abraxis, "Microcystin-ADDA ELISA Kit, Detailed Procedure," Undated. Retrieved January 2014 from
http://www.abraxiskits.com/uploads/products/docfiles/253_PN520011FLOW.pdf.
Beaver, J.R.,T.E. Tietjen, B.J. Blasius-Wert, J.E. Kirsch, T.C. Rosati, G.C. Holdren, E.M. Kennedy, R.M.
Hollis, C.E. Teacher, K.M. Buccier, & S.K. Evans. 2010. Persistence of Daphnia in the epilimnion of
Lake Mead, Arizona-Nevada, during drought and expansion of invasive quagga mussels (2000-2009).
Lake and Reservoir Management: 26: 273-282.
Burkholder, J.M. and R.G. Wetzel. 1989. Epiphytic microalgae on natural substrata in a hardwater lake:
seasonal dynamics of community structure, biomass and ATP content. Arch. Hydrobiological Journal
Suppl. 83:1-56.
Charles, D. F., C. Knowles, and R.S. Davis. 2003. Protocols for the analysis of algal samples collected as
part of the U.S. Geological Survey National Water-Quality Assessment program. Patrick Center For
Environmental Research, The Academy Of Natural Sciences. Report No. 02-06.
Dumont, H. J., I. V. D. Velde, and S. Dumont. 1975. The dry weight estimate of biomass in a selection of
Cladocera, Copepoda and Rotifera from the plankton, periphyton and benthos of continental
waters. Oecologia 19: 75-97.
Edmondson, W.T (ed). 1959. Fresh-water Biology. 2nd ed., Wiley, New York, pp 1248.
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.
Eurofins Technologies (Abraxis), "Cylindrospermopsin ELISA (Microtiter Plate)," Product
522011, Undated. Retrieved March 12, 2020 from: https://www.eurofins-
technologies.com/cvlindrospermopsin-elisa-96-tests.html
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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
Havens, K.E., J.R. Beaver, D.A. Casamatta, T.L. East, R.T. James, E. J. Phlips & A.J. Rodusky. 2011.
Hurricane effects on the planktonic food web of a large subtropical lake. Journal of Plankton
Research: published online 21 Feb 2011.
Havens, K.E. & J.R. Beaver. 2010. Composition, size and biomass of zooplankton in large productive Florida
lakes. Hydrobiologia: published online 27 Aug 2010.
Hillman, D.C., S.H. Pia, and S.J. Simon. 1987. National Surface Water Survey: Stream Survey (Pilot,
Middle Atlantic Phase I, Southeast Screening, and Episode Pilot) Analytical Methods Manual. EPA
600/8-87-005. U.S. Environmental Protection Agency, Las Vegas, Nevada.
Hillebrand et al., 1999. Biovolume calculation for pelagic and benthic microalgae. Journal of Phycology
35: 305-424.
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.
IDEXX, "Quanti-Tray" product instructions. Number 06-02030-07, undated. Retrieved January 2022 from
https://www.idexx.com/water/products/quanti-tray.html.
IDEXX, "Quanti-Tray Sealer Model 2X User Manual." Number 06-03128-02, undated. Retrieved January
from 2022. https://www.idexx.com/water/products/quanti-tray.html.
James, R., et al., "Environmental Technology Verification Report: Abraxis Cylindrospermopsin 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.USEPA.gov/Adobe/PDF/P100EL6B.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.USEPA.gov/Adobe/PDF/P100EL6B.pdf.
Kamp, L. (Abraxis) "Re: Abraxis CYL"; Email to D. Grunzke (EPA). February 14, 2019.
Lawrence, S. G., D.F. Malley, W.J. Findlay, M.A. Maclver & I.L. Delbaere. 1987. Method for estimating dry
weight of freshwater planktonic crustaceans from measures of length and shape. Can. J. Fish. Aquat. Sci. 44:
264-274.
Magdych. 1981. An efficient, inexpensive elutriator design for separating benthos from sediment
samples Hydrobiologia 85(2): 157-159.
McCauley, E. 1984. The estimation of the abundance and biomass of zooplankton in samples, pp. 228-265,
In: J.A. Downing & F.H. Rigler (eds.) A Manual for the Assessment of Secondary Productivity in Fresh
Waters. Blackwell Scientific Publishers.
Merritt, R.W. and K.W. Cummins (eds). 1996. An introduction to the aquatic insects of North America,
3rd ed. Kendall/Hunt Publishing Company, Dubuque, Iowa.
Oblinger Childress, C.J., W.T. Foreman, B.F. Connor, and T.J. Maloney. 1999. New Reporting Procedures
Based on Long-Term Method Detection Levels and Some Considerations for Interpretations of
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Water-Quality Data Provided by the U.S. Geological Survey National Water Quality Laboratory. Open
File Report 99-193. U.S. Geologic Survey, Reston, VA.
Pennak, R.W. 1978. Fresh-water Invertebrates of the United States, 2nd Ed. Wiley Intersci. Pub. 803 pp.
Smith, K. E., and C. H. Fernando. 1978. A guide to the freshwater calanoid and cyclopoid copepod
Crustacea of Ontario. University of Waterloo Biological Series No. 18. 74pp.
Standard Method, "9223 A. 5.2 precautions.
Standard Method, "9223 B. Enzyme Substrate Test.
Stemberger, R. S. 1979. A guide to rotifers of the Laurentian Great Lakes. EPA-600/4-79-021.
Environmental Monitoring and Support Laboratory, Office of Research and Development. U. S.
Environmental Protection Agency, Cincinnati, OH
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.
USEPA. 2004. Revised Assessment of Detection and Quantitation Approaches. EPA-821-B-04-005. U.S.
Environmental Protection Agency, Office of Science and Technology, Washington, D.C.
USEPA. 2002. Guidance on Environmental Data Verification and Data Validation (EPA QA/G-8). EPA
240/R-02/004, US Environmental Protection Agency, Office of Environmental Information,
Washington, DC.
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. Draft Method 1606: Enterococci in Water and Wastewater by TaqMan® Quantitative Polymerase
Chain Reaction (qPCR) Assay. December 2006 (12/15/06 a).
USEPA. Method 245.7 "Mercury in Water by Cold Vapor Atomic Fluorescence Spectrometry, Revision
2.0" (USEPA-821-R-05-001, February 2005), retrieved June 27, 2014 from
http://water.USEPA.gov/scitech/methods/cwa/bioindicators/upload/2007_07_10_methods_metho
d_245_7.pdf.
USEPA. Method 546 "Determination of Total Microcystins and Nodularins in Drinking Water and
Ambient Water by Adda Enzyme-Linked Immunosorbent Assay" retrieved May 24, 2017 from
https://www.epa.gov/sites/production/files/2016-Q9/documents/method-546-determination-total-
microcystins-nodularins-drinking-water-ambient-water-adda-enzyme-linked-immunosorbent-
assay.pdf.
USEPA. Method 3051a "Microwave Assisted Acid Digestion of Sediments, Sludges, Soils, And Oils"
retrieved June 27, 2014 from
http://www.epa.gov/osw/hazard/testmethods/sw846/pdfs/3051a.pdf.
USEPA. Method 3150A "Microwave Assisted Acid Digestion of Sediments, Sludges, Soils, and Oils,"
retrieved June 27, 2014 from
http://www.epa.gov/osw/hazard/testmethods/sw846/pdfs/3051a.pdf.
USEPA. Method 3540C Method 3540C "Soxhlet Extraction" retrieved June 27, 2014 from
http://www.epa.gov/osw/hazard/testmethods/sw846/pdfs/3540c.pdf.
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USEPA. Method 6020A "Inductively Coupled Plasma-Mass Spectrometry" retrieved June 27, 2014 from
http://www.epa.gov/osw/hazard/testmethods/sw846/pdfs/6020A.pdf.
USEPA. Method 8270D "Semivolatile Organic Compounds by Gas Chromatography/Mass Spectrometry
(GC/MS) retrieved June 27, 2014 from
http://www.epa.gov/osw/hazard/testmethods/sw846/pdfs/8270D.pdf.
USEPA. Method 9171B "n-Hexane Extractable Material (HEM) for Sludge, Sediment, And Solid Samples,"
retrieved June 27, 2014 from
http://www.epa.gov/osw/hazard/testmethods/sw846/pdfs/9071b.pdf.
USEPA. 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-Q5/documents/hh-rec-criteria-habs-
document-2019.pdf.
USEPA. Region 1 (New England). OEME NERL Standard Operating Procedure for the Collection of
Chemical & Biological Ambient Water Samples (ECASOP-Ambient Water Sampling 2; January 31,
2007).
USEPA. NERL. OEME Draft Bench SOP for Real-Time PCR Method Quantifying Enterococci in Recreational
Water Samples (August 2006).
USGS. 1999. Open File Report 99-193. 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
Vinson, M.R. and C.P. Hawkins. 1996. Effects of sampling area and subsampling procedure on
comparisons of taxa richness among streams. Journal of the North American Benthological Society
15(3): 392-399.
Zar, J.H. 1999. Biostatistical Analysis. 4th ed. Prentice Hall. Upper Saddle River, New Jersey. 663pp. +
appendices and literature cite
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APPENDIX A: LABORATORY REMOTE EVALUATION AND VERIFICATION
FORMS
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Document Request Form - Chemistry Laboratories
The USEPA and its state and tribal partners will conduct a survey of the nation's lakes, ponds, and
reservoirs. This National Lakes Assessment is designed to provide statistically valid regional and national
estimates of the condition of lakes. Consistent sampling and analytical procedures ensure that the
results can be compared across the country.
As part of the National Lakes Assessment (NLA) 2022, 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 the USEPA's NLA.
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): analysis of Proficiency Testing samples
and/or a site visit. All laboratories will need to complete the following form:
~ A signature on the attached Laboratory Signature Form indicates that your laboratory will follow
the quality assurance protocols required for chemistry laboratories conducting analyses for the
NLA 2022.
In order for us to determine your ability to participate as a laboratory in the NLA, we are requesting that
you submit the following documents (if available) for review:
~ Documentation of a successful quality assurance audit from a prior survey that occurred within
the last 5 years (if you need assistance with this please contact the individual indicated below)
~ A copy of your Laboratory's accreditations and certifications if applicable (i.e. NELAC, ISO, state
certifications, etc...)
If your laboratory can provide either documentation of a prior audit or accreditation, no other
documentation is needed. If neither of the above is complete, please provide the following information.
~ A copy of your Laboratory's Quality Manual
~ Standard Operating Procedures (SOPs) for your laboratory for each analysis to be performed (if
not covered in NLA 2022 Laboratory Manual)
~ Other documentation supporting your laboratory's ability to meet the required level of data
quality (if available)
This documentation may be submitted electronically via e-mail to forde.kendra@epa.gov. Questions
concerning this request can be submitted to guenzel.lareina@epa.gov (202-566-0455) or
forde.kendra@epa.gov (202-564-0417).
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Laboratory Signature Form - Chemistry Laboratories
I certify that the laboratory,
located in , will abide by the following standards in
performing chemistry data analysis and reporting for the National Lakes Assessment (NLA).
1.) Utilize procedures identified in the NLA 2022 Laboratory Operations Manual (or
equivalent). If using equivalent procedures, please provide procedures manual.
2.) Read and abide by the NLA 2022 Quality Assurance Project Plan (QAPP) and
related Standard Operating Procedures (SOPs).
3.) Have an organized IT system in place for recording sample tracking and analysis
data.
4.) Provide data using the template provided in the Laboratory Operations Manual.
5.) Provide data results in a timely manner. This will vary with the type of analysis
and the number of samples to be processed. Sample data must be received no
later than March 1, 2023 or as otherwise negotiated with the USEPA.
6.) Participate in a laboratory technical assessment or audit if requested by an
USEPA NLA staff (this may be a conference call or on-site audit).
Signature
Date
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Document Request Form - Biology Laboratories
The USEPA and its state and tribal partners will conduct a survey of the nation's lakes, ponds, and
reservoirs. This National Lakes Assessment is designed to provide statistically valid regional and national
estimates of the condition of lakes. Consistent sampling and analytical procedures ensure that the
results can be compared across the country.
As part of the National Lakes Assessment (NLA) 2022, 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 the USEPA's NLA 2022.
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 need to complete the following form:
~ A signature on the attached Laboratory Signature Form indicates that your laboratory will follow
the quality assurance protocols required for chemistry laboratories conducting analyses for the
NLA 2022.
In order for us to determine your ability to participate as a laboratory in the NLA, we are requesting that
you submit the following documents (if available) for review:
~ Documentation of a successful quality assurance audit from a prior survey that occurred within
the last 5 years (if you need assistance with this please contact the individual listed below)
~ A copy of your Laboratory's accreditations and certifications if applicable (i.e. NELAC, ISO, state
certifications, etc...)
If your laboratory can provide either documentation of a prior audit or accreditation, no other
documentation is needed. If neither of the above is complete, please provide the following information:
~ Documentation of NABS certification for the taxonomists performing analyses (if available)
~ A copy of your Laboratory's Quality Manual
~ Standard Operating Procedures (SOPs) for your laboratory for each analysis to be performed (if
not covered in NLA 2022 Laboratory Manual)
D Other documentation supporting your laboratory's ability to meet the required level of data
quality (if available)
This documentation may be submitted electronically via e-mail to forde.kendra@epa.gov. Questions
concerning this request can be submitted to guenzel.lareina@epa.gov (202-566-0455) or
forde.kendra@epa.gov (202-564-0417)
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Laboratory Signature Form - Biology Laboratories
I certify that the laboratory,
located in , will abide by the following standards in
performing biology data analysis and reporting for the National Lakes Assessment (NLA).
1.) Utilize procedures identified in the NLA 2022 Laboratory Operations Manual (or
equivalent). If using equivalent procedures, please provide procedures manual.
2.) Read and abide by the NLA 2022 Quality Assurance Project Plan (QAPP) and
related Standard Operating Procedures (SOPs).
3.) Have an organized IT system in place for recording sample tracking and analysis
data.
4.) Use taxonomic standards outlined in the NLA 2022 Laboratory Manual.
5.) Participate in taxonomic reconciliation exercises during the field and data
analysis season, which include conference calls and other laboratory reviews.
6.) Provide data using the template provided in the Laboratory Operations Manual.
7.) 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, 2023 or as otherwise negotiated with the USEPA.
8.) Participate in a laboratory technical assessment or audit if requested by USEPA
NLA staff (this may be a conference call or on-site audit).
Signature
Date
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APPENDIX B: SAMPLE LABORATORY FORMS
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Benthic Macroinvertebrate Laboratory Bench Sheet
Project Name/Number Sample ID
Waterbody Name Site ID
Sorter (initially spread sample) Sort Date Collection Date
Grid Order Sorter's Initials Random Number of Cumulative
Number Grid ID Individuals per Number of
Grid Organisms
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
QC
o
Ll_
>
QC
o
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Phytoplankton Measurement Data Sheet
Site ID Sample # Lake
Laboratory # Date Collected
Analyzed by
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Zooplankton Sample Log In Form
Date
Received
Sample
Type
Sample
Number
SiteJD
Lake Name
Tow
Depth
(m)
Laboratory
Tracking #
Notes
Q_
Q_
<
115
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Zooplankton Enumeration Data Sheet
Sample# SiteJD Laboratory#
Date Collected Depth of tow (m) Analyzed by
Working Volume (mL) Milliliters in subsample (rotifers) Split
^Taxa / Count-»
A
B
C
D
Total Mature Copepoda
Total Immature Copepoda
Total Cladocera
Total Rotifera
Total Other Organisms
Note: For Rotifers only A and B counts are made.
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Zooplankton Measurement Data Sheet
Sample# SiteJD Laboratory#
Date Collected Depth of tow (m) Analyzed by_
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APPENDIX C: STATE SAMPLE TRACKING SPREADSHEET
Provided on the NARS SharePoint site or from the Laboratory Review Coordinator.
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APPENDIX D: REPORTING TEMPLATES
Templates will be provided on the NARS SharePoint Site.
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