United States Environmental Protection Agency
Office of Water
Washington, DC
EPA 841-B-16-004
National Lakes Assessment 2017
Laboratory Operations
Manual
Version 1.1, May 2017
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NOTICE
The intention of the National Lakes Assessment 2017 (NLA 2017) 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 2017: Quality Assurance Project Plan (QAPP) (EPA 841-B-16-003)
National Lakes Assessment 2017: Site Evaluation Guidelines (SEG) (EPA 841-B-16-001)
National Lakes Assessment 2017: Field Operations Manual (FOM) (EPA 841-B-16-002)
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, bacteria
(E. coli), benthic macroinvertebrates, phytoplankton, sediment chemistry (contaminants, total organic
carbon, and grain size), water chemistry and chlorophyll a, and zooplankton) to be collected during the
project, quality assurance objectives, sample handling, and data reporting. Dissolved gases and fish
eDNA analysis are also included as part of the NLA 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 2017. 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. 2017. National Lakes Assessment 2017. Laboratory Operations Manual. V.l.l. EPA 841-B-16-
004. U.S. Environmental Protection Agency, Washington, DC.
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BITS
TITLE PAGE COVER PAGE
NOTICE Ill
TABLE OF CONTENTS IV
LIST OF TABLES VIII
LIST OF FIGURES VIII
LIST OF EQUATIONS VIII
LIST OF ACRONYMS 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: CYLINDROSPERMOPSIN IMMUNOASSAY PROCEDURE 7
3.1 Definitions and Required Personnel Qualifications 7
3.1.1 Definitions 7
3.1.2 Personnel Qualifications 8
3.2 Precautions 9
3.3 Equipment/Materials 9
3.4 Sample Receipt 10
3.5 Procedure 11
3.5.1 Sample Preparation 11
3.5.2 Kit Preparation 11
3.5.3 Insertion of Contents into Wells 12
3.5.4 Dilutions (if needed) 15
3.6 Pertinent QA/QC Procedures 15
3.6.1 QC Samples 15
3.6.2 Summary of QA/QC Requirements 16
4.0 ALGAL TOXIN: MICROCYSTIN IMMUNOASSAY PROCEDURE 19
4.1 Definitions and Personnel Qualifications 19
4.1.1 Definitions 19
i/i 4.1.2 Personnel Qualifications 20
z 4.2 Precautions 21
^ 4.2.1 Equipment/Materials 21
O 4.3 Sample Receipt 22
li_ 4.4 Procedure 23
2 4.4.1 Sample Preparation 23
^ 4.4.2 Kit Preparation 23
^ 4.4.3 Insertion of Contents into Wells 24
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4.4.4 Dilutions (if needed) 27
4.5 Pertinent QA/QC Procedures 28
4.5.1 QC Samples 28
4.5.2 Summary of QA/QC Requirements 28
5.0 BACTERIA (£. COLI) METHOD 31
5.1 Responsibility and Personnel Qualifications 32
5.2 Precautions 32
5.2.1 Storage and Stability 32
5.3 Equipment/Reagents/Standards 32
5.4 Sample Receipt 32
5.5 Procedure 34
5.5.1 Test preparation 34
5.5.2 Test Procedure 34
5.5.3 Results 34
5.5.4 Data Entry 35
5.6 Pertinent QA/QC Procedures 35
5.6.1 Internal QC 35
5.6.2 External QC 36
6.0 BENTHIC MACROINVERTEBRATE METHODS 39
6.1 Responsibility and Personnel Qualifications 39
6.2 Precautions 39
6.2.1 Sorting and Subsampling Precautions 39
6.2.2 Taxonomy Precautions 39
6.3 Equipment/Materials 40
6.3.1 Sorting and Subsampling Equipment/Materials 40
6.3.2 Taxonomy Equipment/Materials 40
6.4 Sample Receipt 41
6.5 Procedure 42
6.5.1 General 42
6.5.2 Subsampling 42
6.5.3 Sorting 44
6.5.4 Taxonomy Procedures 46
6.5.4.1 Taxonomic Level of Effort 47
6.6 Pertinent QA/QC Procedures 49
6.6.1 Sorting and Subsampling QC 49
6.6.2 Taxonomic QC 49
6.6.2.1 Internal Taxonomic QC 49
6.6.2.2 External Taxonomic QC 49
6.6.2.3 Taxonomic QC Review & Reconciliation 50
7.0 PHYTOPLANKTON METHODS 52
7.1 Responsibility and Personnel Qualifications 52
7.2 Precautions 52
7.3 Equipment/Materials 52
7.4 Sample Receipt 52
7.5 Procedure 54
7.5.1 Prepare Utermohl Sedimentation Chamber 54
7.5.2 Choose Count Method 54
7.5.2.1 Determine random fields 54 O
7.5.2.2 Determine transects 54 ^
7.5.3 Identify and Enumerate 400 Natural Algal Units 54 ^
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7.5.4 Identify and Enumerate Larger, Rarer Taxa 55
7.5.5 Measure Cell Biovolumes 55
7.6 Calculation and Reporting 56
7.7 Pertinent QA/QC Procedures 56
7.7.1 Internal Taxonomic QC 56
7.7.2 External Taxonomic QC 56
7.7.2.1 Plankton Re-identification 56
7.7.3 Taxonomic QC Review & Reconciliation 57
8.0 SEDIMENT CONTAMINANTS, GRAIN SIZE, AND TOC 58
8.1 Definitions and Required Resources (Personnel, Laboratories, and Equipment) 58
8.1.1 Definitions 58
8.1.2 Personnel 59
8.2 Precautions 59
8.3 Equipment/Materials 60
8.4 Sample Receipt 60
8.5 Laboratory Analysis: Requirements 61
8.5.1 Data Entry 65
8.6 Pertinent QA/QC Procedures 65
8.6.1 QC Samples 65
8.6.2 Summary of QA/QC Requirements 65
9.0 ATRAZINE PESTICIDE SCREEN 69
9.1 Responsibility and Personnel Qualifications 69
9.2 Precautions 69
9.2.1 Storage and Stability 69
9.3 Equipment 70
9.4 Sample Receipt 70
9.5 Procedure 71
9.5.1 Test preparation 71
9.5.2 Procedural notes and precautions 71
9.5.3 Assay procedure 72
9.5.4 Results 72
9.5.5 Data Entry 73
9.6 Pertinent QA/QC Procedures 73
9.6.1 Internal QC 73
9.6.2 External QC 74
10.0 WATER CHEMISTRY AND CHLOROPHYLLS 75
10.1 Analytical Parameters 75
10.2 Sample Receipt 76
10.3 Sample Processing and Preservation 77
10.3.1 Water Chemistry Samples 78
10.3.2 Chlorophyll-a Samples 79
10.4 Performance-based Methods 79
10.5 Pertinent QA/QC Procedures 81
10.5.1 Laboratory Performance Requirements 81
10.5.2 Laboratory Quality Control Samples 81
10.5.3 Data Reporting, Review, and Management 86
10.5.4 Data Entry 88
11.0 ZOOPLANKTON METHODS 89
11.1 Responsibility and Personnel Qualifications 89
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11.2 Precautions 89
11.3 Equipment/Materials 89
11.4 Sample Receipt 90
11.5 Procedure 91
11.5.1 Zooplankton Stratified Splitting 91
11.5.2 Taxon omy Procedures 92
11.5.2.1 Taxonomic Level of Effort 92
11.5.2.2 Macrozooplankton Identification and Enumeration (Excluding Rotifers and Nauplii) 92
11.5.2.3 Microzooplankton (Rotifers, Nauplii, and Crustaceans) 93
11.5.2.4 Measurement of Macrozooplankton and Microzooplankton 94
11.6 Calculating and Reporting 94
11.6.1 Volume of water filtered 94
11.6.2 Macrozooplankton Densities 95
11.6.3 Microzooplankton Densities 95
11.6.4 Zooplankton Biomass Estimates 95
11.6.5 Results of Laboratory Processing, Sample Archiving 95
11.7 Pertinent QA/QC Procedures 96
11.7.1 Sorting and Subsampling QC 96
11.7.2 Taxonomic QC 96
11.7.2.1 Internal Taxonomic QC 96
11.7.2.2 External Taxonomic QC 97
11.7.2.3 Taxonomic QC Review & Reconciliation 97
12.0 RESEARCH INDICATOR: DISSOLVED GASES 99
13.0 RESEARCH INDICATOR: FISH EDNA 100
14.0 LITERATURE CITED 101
APPENDIX A: LABORATORY REMOTE EVALUATION AND VERIFICATION FORMS 106
APPENDIX B: SAMPLE LABORATORY FORMS 112
Benthic Macroinvertebrate Laboratory Bench Sheet 114
Phytoplankton Measurement Datasheet 115
Zooplankton Sample Log In Form 116
Zooplankton Enumeration Datasheet 117
Zooplankton Measurement Datasheet 118
APPENDIX C: STATE SAMPLE TRACKING SPREADSHEET 120
APPENDIX D: REPORTING TEMPLATES 122
APPENDIX E: SUPPORTING METHODS
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LIST
Table 1-1 Contact information 2
Table 3.3-1 Cylindrospermopsin login: required data elements 10
Table 3-2 Cylindrospermopsin: sample and analysis quality control activities and objectives 16
Table 4-1 Microcystins login: required data elements 22
Table 4-2 Microcystin: sample and analysis quality control activities and objectives 28
Table 5-1 Bacteria: analytical methods 31
Table 5-2 Bacteria login: required data elements 33
Table 5-3 Bacteria: quality control activities for samples 36
Table 6-1 Benthic macroinvertebrate login: required data elements 41
Table 6-2 Required level of taxonomic identification for benthic macroinvertebrates 48
Table 6-3 Laboratory quality control: benthic indicator 50
Table 7-1 Phytoplankton login: required data elements 53
Table 7-2 Laboratory quality control: phytoplankton indicator 57
Table 8-1 Sediment chemistry, grain size, and TOC login: required data elements 60
Table 8-2 Sediment chemistry, grain size, and TOC: storage requirements and analytical methods 61
Table 8-3 Sediment chemistry, grain size, and TOC: required parameters 62
Table 8-4 Sediment chemistry, grain size, and TOC: quality control activities for samples 65
Table 9-1 Atrazine login: required data elements 70
Table 9-2 Test tube labeling for atrazine assay 72
Table 9-3 Atrazine: quality control requirements 73
Table 10-1 Water chemistry parameters measured for the National Lakes Assessment 2017 75
Table 10-2 Water Chemistry login: required data elements 76
Table 10-3 Acid preservatives added for various analytes 79
Table 10-4 Summary of analytical methods used by NLA 2017 (Central Laboratory, USEPA ORD-Corvallis) 80
Table 10-5 Laboratory method performance requirements for water chemistry and chlorophyll-o sample analysis.
82
Table 10-6 Laboratory quality control samples: water chemistry indicator 84
Table 10-7 Data validation quality control for water chemistry indicator 86
Table 10-8 Data reporting criteria: water chemistry indicator 86
Table 10-9 Constants for converting major ion concentration from mg/L to neq/L 87
Table 10-10 Factors to calculate equivalent conductivities of major ions 88
Table 11-1 Zooplankton login: required data elements 91
Table 11-2 Laboratory quality control: zooplankton indicator 98
LIST
Figure 3.1 Cylindrospermopsin: sample template 13
Figure 4.1 Microcystin: sample template 25
Figure 5.1 Bacteria (E.coli): disposable 97-well tray for use with the Quanti-Tray sealer 31
Figure 10.1 Water chemistry sample processing procedures 78
£ LIST OF EQUATIONS
¦Z.
£! Equation 3.1 Standard deviation 8
q Equation 3.2 Percent (%) coefficient of variation 8
u Equation 4.1 Standard deviation 20
O Equation 4.2 Percent (%) coefficient of variation 20
itl Equation 5.1 Precision 35
< Equation 6.1 Percent sorting efficiency (PSE) 49
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Equation 6.2 Percent difference in enumeration (PDE) 50
Equation 6.3 Percent taxonomic disagreement (PTD) 50
Equation 7.1 Phytoplankton abundance 56
Equation 7.2 Percent difference 57
Equation 8.1 Percent recovery 58
Equation 8.2 Relative percent difference 59
Equation 10.1 Percent ion difference (%IBD) 87
Equation 11.1 Volume of water filtered 94
Equation 11.2 Microcrustacean densities 95
Equation 11.3 Microzooplankton densities 95
Equation 11.4 Relative percent difference (RPD) 96
Equation 11.5 Root mean square error (RMSE) or standard error of estimate 96
Equation 11.6 Percent taxonomic disagreement (PTD) 97
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LIST OF ACRONYMS
A absorbance
Al Aluminum
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
Dl deionized
DO dissolved oxygen
DOC dissolved organic carbon
ELISA enzyme-linked immunosorbent assay
EMAP Environmental Monitoring and Assessment Program
USEPA United States Environmental Protection Agency
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 nagnesium
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
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N03 nitrate
N02 nitrite
PAH polycyclic aromatic hydrocarbon
PCB polychlorinated biphenyl
PD percent difference
PDE percent difference in enumeration
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's Contracting Officer's Representative
TP total phosphorus
USGS United States Geological Survey
WRS Willamette Research Station
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NATIONAL LAKES ASSESSMENT 2017
LABORATORY OPERATIONS MANUAL
The U.S. Environmental Protection Agency (USEPA), in partnership with state and tribal organizations,
has designed the National Lakes Assessment (NLA) 2017 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 and Alaska. The purposes of this manual are to:
1) document the standardized sample processing and analysis procedures used in the various
laboratories for the NLA 2017; 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, E. coli bacteria,
benthic macroinvertebrates, phytoplankton, sediment chemistry (contaminants, total organic carbon,
and grain size), atrazine screen, water chemistry, chlorophyll a, and zooplankton. Two indicators are
research indicators, dissolved gases and fish eDNA, and will be completed in collaboration with the
USEPA's Office of Research and Development.
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 2017. 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 2017 sample that involves laboratory processing. These coordinators will be
the laboratories main point of contact in regards to sample tracking.
Table 1-1 Contact information.
Title Name Contact Information
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QC
USEPA HQ Project Lead
Amina Pollard, OW
oollard.amina(® eoa.gov
202-566-2360
USEPA HQ Project QA
Coordinator
Sarah Lehmann, OW
lehmann.sarahPepa.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
Marlys Cappaert, SRA
International Inc.
caooaert.marlvs(® eoa.gov
541-754-4467
541-754-4799 (fax)
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 and/or paper 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
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and batching 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 2017 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 May 1, 2018 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: NLA17_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: 2017_11_01)
• Laboratory name (ex: MyLaboratory)
Combined, the file name would look as follows: Microcycstin_2017_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 2017 Project Lead. All samples and raw data
files (including logbooks, bench sheets, and instrument tracings) are to be retained by the laboratory for
3 years or until authorized for disposal, in writing, by the 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 2017, 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 2017 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 2017.
Given the large number of laboratories participating in the NLA 2017, 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 2017 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
O
u
method detection limits, accuracy, and precision targets.
Zi Quality assurance and quality control requirements.
<
d To demonstrate its competency in quality assurance and quality control procedures, each laboratory
^ shall provide the USEPA with copies of the quality-related documents relevant to the procedure.
° Examples include Quality Management Plans (QMP), QAPPs, and applicable SOPs.
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a The evaluation and verification of the laboratories is being considered as a form of an AV rather than an audit
because the evaluation and verification phase is designed for the laboratories to demonstrate competency of
performance and for the EPA HQ Project Management Team to provide guidance to the laboratories rather than as
"inspection" as in a traditional audit.
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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 2017 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 2017 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 2017 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 2017 Laboratory Operations Manual (or equivalent). If
using equivalent procedures, please provide procedural manual to demonstrate ability to meet
the required MQOs.
2. Read and abide by the NLA 2017 Quality Assurance Project Plan (QAPP) and related SOPs.
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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 2017 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, phytoplankton, and zooplankton data for the NLA 2017. 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: CYLINDROSPERMOPSIN IMMUNOASSAY
PROCEDURE
This chapter describes an immunoassay procedure that measures concentrations of total
cylindrospermopsin in water samples. In applying the procedure, the laboratory uses Abraxis'
Cylindrospermopsin Test Kits ("kits"). Each kit is an enzyme-linked immunosorbent assay (ELISA) for the
determination of cylindrospermopsin in water samples.
Cold cylindrospermopsin samples will be shipped on ice from the field crews to the contract batching
laboratory. The contract batching laboratory will freeze samples and send the batched samples to the
analysis laboratory in coolers on dry ice. Samples will arrive in the analysis laboratory frozen and they
can be held in a freezer for several weeks. Cylindrospermopsin analysis laboratories will need to
process samples in accordance with the time frame outlined in contractual agreements.
The procedure is an adaption of the instructions provided by Abraxis for determining total
cylindrospermopsin concentrations using its ELISA kits.b 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.
3.1 Definitions and Required Personnel Qualifications
This section provides definitions and required resources for using the procedure.
3.1.1 Definitions
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The following terms are used throughout the procedure: ^
Q
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. §
CL
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. $
Values outside the range are handled as follows. If the value is: O
-z.
• < 0.05 ng/L, then the laboratory reports the result as being non-detected ("<0.10 ng/L"). ^
• 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 (i.e., ^
DATA FLAG=J). O
• 2.0 ng/L, the laboratory must dilute and reanalyze the sample. oc
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l/l
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: §
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Equation 3.1 Standard deviation
S =
n
1/2
i = 1
where n is the number of replicate samples, Ais the absorbance measured for the replicate. Per
Section 3.5.3, samples are evaluated in duplicate (i=l or 2); controls are either evaluated in duplicate
or triplicate (i=l, 2,3). A is the average absorbance of the replicates. Then, calculate %CV as:
Equation 3.2 Percent (%) coefficient of variation
S
A
Dark or Dimly Lit: Away from sunlight, but under incandescent lighting is acceptable.
%CV =
x 100
Detection Limit is the minimum concentration at which the analyte can be defected with confidence
(0.05 ng/L). 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 0.1 ng/L, at which the
measured value of the analyte can be reported with confidence. Also see "Sample-Specific Detection
Limit" below.
Duplicates are defined as two aliquots of the same sample which are analyzed separately using
identical procedures. The results are used to evaluate the precision of the laboratory analyses. Per
Section 3.5.3, controls are evaluated in duplicate or triplicate (i.e., three aliquots).
Relative Standard Deviation (RSD) is the same as the coefficient of variation (%CV). Because many of
the plate reader software programs provide the CV in their outputs, the procedure presents the quality
control requirement in terms of %CV instead of RSD.
LLJ
Efj Reporting Limit: For undiluted freshwater samples, the reporting limit is 0.1 ng/L. A reporting limit is
g the point at which the measured value of the analyte can be reported with confidence,
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§ Standard Deviation (S) shows variation from the average
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> Sample-Specific Detection Limit: Most samples will have a sample-specific detection limit equal to the
$ method's detection limit of 0.05 ng/L. For diluted samples, the sample-specific detection limit will be
O the product of the method's detection limit of 0.05 ng/L and the dilution factor. Typical values for the
3 dilution factor will be 10 or 100.
^ 3.1.2 Personnel Qualifications
p Laboratory Technician: This procedure may be used by any laboratory technician who is familiar with
O the NLA QAPP, and this procedure in the NLA LOM (which differs from the Abraxis instructions). The
^ laboratory technician also must be familiar with the use of a multichannel pipette and plate readers.
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Si External QC Coordinator is an USEPA staff person who is responsible for selecting and managing the
o
QC
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§ "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 the 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
h summary reports.
i
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3.2 Precautions
Laboratory Operations Manual
Page 9 of 124
The laboratory must require its staff to abide by appropriate health and safety precautions, because
the kit substrate solution contains tetramethylbenzidine (TMB) and the stop solution contains diluted
sulfuric acid. In addition to the laboratory's usual requirements such as a Chemical Hygiene Plan, the
laboratory must adhere to the following health and safety procedures:
1. Laboratory facilities must properly store and dispose of solutions of weak acid.
2. Laboratory personnel must wear proper personal protection clothing and equipment (e.g.,
laboratory coat, protective eyewear, gloves).
3. When working with potential hazardous chemicals (e.g., weak acid), laboratory personnel must
avoid inhalation, skin contact, eye contact, or ingestion. Laboratory personnel must avoid
contacting skin and mucous membranes with the TMB and stopping solution. If skin contact
occurs, remove clothing immediately. Wash and rinse the affected skin areas thoroughly with
large amounts of water.
3.3 Equipment/Materials
The procedures require the following equipment and information:
• Abraxis Cylindrospermospin ELISA (Microtiter) Test Kit, Product # 522011 (see items in Section
3.5.2).
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, LC, vials (20 mL). 3
Multichannel Pipette & Tips: An 8-channel pipette is used for this method. Familiarity of the y
use of the multichannel pipette is necessary to achieve reliable results. Practice with water if O
you have never used this before. ^
Norm-ject syringes (or equivalent).
to
Paper Towels: For blotting the microtiter plates dry after washing. <
Permanent Marker (Sharpie Fine Point): For labeling samples, bottles, plates and covers. z
Plate Reader (such as Metertech, Model M965 AccuReader): Complete with Metertech PC ^
Mate software for operation of machine. This machine reads the microtiter plates. ^
Project Quality Control Samples. ?
Reagent Reservoirs (Costar Cat Number 4870): Plain plastic reservoir for reagents that q
accommodate the use of a multi-channel pipette.
Test tubes: For dilutions, if needed.
Timer: For measuring incubation times. O
QC
Vortex Genie: For mixing dilutions. ~
Whatman Glass fiber syringe filter (25mm, GF 0.45 pim filter). =i
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3.4 Sample Receipt
Because USEPA initiates tracking procedures designed to recover any missing shipment, the laboratory
personnel responsible for tracking samples must start the following login steps within 24 clock hours of
receiving a delivery.
1. Report receipt of samples to the NARS IM Team by completing and emailing the sample
tracking spreadsheet with the sample login and sample condition information. (See Section 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 3.3-1 for the NARS IM Team, including the Condition Code
for each sample:
OK: Sample is in good condition
C: Sample container was cracked
in. L: Sample container is leaking
iv. ML: Sample label is missing
v. W: Sample is warm (>8°), 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).
QC
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3. Store samples in the freezer until sample preparation begins.
4. Maintain the sample tracking forms with the samples.
Table 3.3-1 Cylindrospermopsin login: required data elements.
FIELD
FORMAT
DESCRIPTION
LABORATORY ID
text
Name or abbreviation for QC 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
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CONDITION
COMMENT
text
Laboratory Operations Manual
Page 11 of 124
ML Sample label is missing
W Sample is warm (>8°)
Q Other quality concerns, not identified
above
Comments about the condition of the sample. If
the condition code='W' then provide the
temperature
3.5 Procedure
The following sections describe the sample, kit preparation and analysis.
3.5.1 Sample Preparation
For each frozen sample (500 mL per sample), the laboratory technician runs it through a freeze-thaw
cycle three times to lyse the cells as follows:
1. All cycles: Keep the samples in dark or dimly lit areas (i.e., away from sunlight, but under
incandescent lighting is acceptable).
2. First freeze-thaw cycle:
a. Start with a frozen 500 ml sample.
b. Thaw the sample to room temperature (approximately 25° C). Swirl the sample to check for
ice crystals. At this temperature, no ice crystals should be present in the sample.
c. Shake well to homogenize the sample, then transfer 10 mL to an appropriately labeled
clean 20 mL glass vial. ^
3. Second freeze-thaw cycle: q
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a. Freeze the vial. ^
b. Keep the large sample bottle (from the 500 mL initial sample) frozen for future use. cl
c. Thaw the sample vial contents to room temperature. <
4. Third freeze-thaw cycle: <
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a. Freeze the vial. z
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, g
GF 0.45 pirn filter) or other similar alternative are acceptable. One new syringe and filter ^
should be used per sample. ^
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3.5.2 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, x
O
consider keeping it for training activities.) ^
2. Verify that each kit contains all of the required contents: ^
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• Microtiter plate
• Standards (7) referenced in this procedure as follows with the associated concentration:
SO:0 ng/L
SI: 0.05 ng/L
S2: 0.1 |J.g/L,
S3: 0.25 ng/L
S4: 0.5 ng/L
S5: 1.0 ng/L
S6: 2.0 ng/L
Kit Control (KC): 0.75 ng/L
Sample Diluent (distilled or deionized water)
Cylindrospermospin-HRP conjugate Soultion (vortex before use)
Antibody solution (rabbit anti-Cylindrospermopsin)
Wash Solution 5X Concentrate
Substrate (Color) Solution
Stop Solution
QC
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3. If any bottles are missing or damaged, discard the kit. This step is important because Abraxis
has calibrated the standards and reagents separately for each kit.
4. Adjust the microtiter plate, samples, standards, and the reagents to room temperature.
5. Remove 12 microtiter plate strips (each for 8 wells) from the foil bag for each kit. The plates
contain 12 strips of 8 wells. If running less than a whole plate, remove unneeded strips from
the strip holder and store in the foil bag, ziplocked closed, and store in the refrigerator (4-8 °C).
6. Prepare a negative control (NC) using distilled or deionized water
7. The standards, controls, antibody solution, enzyme conjugate, color solution, and stop
solutions are ready to use and do not require any further dilutions.
8. Dilute the wash solution with distilled or deionized water. (The wash solution is a 5X
concentrated solution.) In a 1L container, dilute the 5X solution 1:5 (i.e., 100 mL of the 5X wash
solution plus 400 mL of distilled or deionized water). Mix thoroughly. Set aside the diluted
solution to wash the microtiter wells later.
9. Handle the stop solution containing diluted H2S04 (sulfuric acid) with care.
3.5.3 Insertion of Contents into Wells
This section describes the steps for placing the different solutions into the 96 wells. Because of the
potential for cross contamination using a shaker table, the following steps specify manual shaking of
the kits instead mechanized shaking.
1. While preparing the samples and kit, turn the plate reader on so it can warm up. The plate
reader needs a minimum of 30 minutes to warm up.
2. Turn on the computer so that it can control and access the plate reader.
3. Print the template (Figure 3.1) to use as reference when loading the standards, controls, and
samples as described in the next step. Templates contain rows, labeled with a marking pen, of
strips of 8 wells that snap into the blank frame. (If the laboratory wishes to use a different
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template, provide a copy to the USEPA HQ Laboratory Review Coordinator for approval prior to
first use. (See Section 1.2 of the manual for contact information.)
4. 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.
10 11 12
A
SO
S4
NC
U4
U8
U12
U16
U20
U24
U28
U32
U36
B
SO
S4
NC
U4
U8
U12
U16
U20
U24
U28
U32
U36
C
SI
S5
U1
U5
U9
U13
U17
U21
U25
U29
U33
U37
D
SI
S5
U1
U5
U9
U13
U17
U21
U25
U29
U33
U37
E
S2
S6
U2
U6
U10
U14
U18
U22
U26
U30
U34
U38
F
S2
S6
U2
U6
U10
U14
U18
U22
U26
U30
U34
U38
G
S3
KC
U3
U7
Ull
U15
U19
U23
U27
U31
U35
U39
H
S3
KC
U3
U7
Ull
U15
U19
U23
U27
U31
U35
U39
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);
U = Unknown (sample collected by the field crew);
5. Add 50 piL of the conjugate solution to each well using the multi-channel pipettor and a reagent
reservoir. Add 50 piL of the cylindrospermopsin antibody solution to each well using the multi-
channel pipettor and a reagent reservoir. Use dedicated reagent reservoirs for each reagent to
avoid contamination from one reagent to another.
6. Place the sealing Parafilm over the wells.
7. Manually mix the contents by moving the strip holder in a rapid circular motion on the
benchtop for 30 seconds. Be careful not to spill the contents.
8. Place the plate in an area away from light for 45 minutes.
9. After 45 minutes, carefully remove the Parafilm.
10. Empty the contents of the plate into the sink, pat inverted plate dry on a stack of paper towels,
and then wash the wells of the plate 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.
11. Add 100 piL of substrate/ color solution to all wells using the multi-channel pipettor.
12. Cover the wells with Parafilm.
13. Manually mix the contents by moving the strip holder in a rapid circular motion on the
benchtop for 30 seconds. Be careful not to spill the contents.
14. Place the strip holder in an area away from light for 30-45 minutes.
QC
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15. 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.
16. 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.
17. Dispose of solution in plates in a laboratory sink. Rinse plates and sink with water to dilute the
weak acid present.
18. 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-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:
iii. Ao > Ai > A2 > A3 > A4 >As
iv. The average absorbance of the standard SO less than 0.8 (i.e., A0 < 0.8).
v. 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.
vi. 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.
q b. If either, or both, of the following failures occur, then the sample must be reanalyzed
q (maximum of two analyses, consisting of the original analysis and, if necessary, one
cl reanalysis):
< i. The concentration value registers as HIGH (exceeds the calibration range). Dilute the
CO
< sample for the reanalysis per Section 3.5.4.
^ ii. The %CV > 15% between the duplicate absorbance values for a sample.
^ 19. Record the results, even if the data failed the quality control requirements in #18b, for each
~ well in the USEPA's data template. The required entries are for the following columns:
£ a. TYPE should be one of the following codes: S0-S6 for standards; KC or NC, for controls; U
for unknown sample.
b. CONC contains the numeric concentration value. Two special cases:
Q_
q i. Non-detected concentrations: If the sample is non-detected, then provide the sample-
q specific detection limit which is 0.05 ng/L if the sample is undiluted. See Section 3.5.4
z
for calculating the sample-specific detection limit for a diluted sample.
V! ii. If the result shows that it is "HI," this indicates that the sample value is outside of the
O
QC
>< calibration range and must be diluted and re-run using another analytical run. Leave
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1- the CONC column blank and record 'HI' in the DATA FLAG column.
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c. DATA FLAGS have codes for the following special cases:
I
< i. ND if the sample was non-detected;
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ii. J if the value is detected but at a level below the reporting limit of 0.1 ng/L (for
undiluted samples);
iii. HI if the concentration value registers as HIGH (exceeds the calibration range).
d. QUALITY FLAGS have codes for the following special cases:
i. QCF if there is a QC failure per step 18 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).
3.5.4 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 vial. (Note: Dilutions may also be made
using the kit's diluent rather than distilled or deionized 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 0.05 ng/L by 10 to obtain the
QC
sample-specific detection limit of .5 ng/L. 3
#2 §
1:100 dilution §
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a. Add 3.96 mL of distilled or deionized water to a clean, appropriately labeled glass vial. ^
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(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. ^
c. Multiply the final concentration and Abraxis' detection limit of 0.05 ng/L by 100 to obtain ^
o_
the sample-specific detection limit of 50 ng/L. O
Other dilutions can be calculated in the same manner as #1 and #2 if needed.
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3.6 Pertinent QA/QC Procedures a
This section describes the quality assurance and quality control measures used to ensure that the data >
will meet the NLA's requirements. -
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The External QC Coordinator will instruct the QC contractor to provide one or two identical sets of <
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freshwater 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, including no action, flagging the data, or excluding some or all of the
laboratory's data.
3.6.2 Summary of QA/QC Requirements
QC
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Table 4-2 provides a summary of the quality control requirements described in Sections 3.5.2 and
3.5.3. 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 3-2 Cylindrospermopsin: sample and analysis quality control activities and objectives.
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.
Calibration
All of the following must be met:
o Standard curve must have a
correlation coefficient of >0.99;
o Average absorbance value, A0, for SO
must be >0.80; and
o Standards S0-S6 must have decreasing
average absorbance values. That is, if
A, is the average of the absorbance
values for S,, then the absorbance
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 USEPAfor
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Kit Control
Negative Control
Sample
Evaluations
Results Within
Calibration
Range
average values must be: A0 > Ai > A2 >
A3 > A4 >As>As
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.
The values for the negative control replicates
must meet the following requirements:
o All concentration values must be < 0.1
Hg/L (i.e., the reporting limit); and
o One or more concentration results
must be nondetectable (i.e., <0.05
Hg/L)
All samples are run in duplicate. Each
duplicate pair must have %CV<15% between
its absorbance values.
All samples are run in duplicate. If both of the
values are less than the upper calibration
range (i.e., 2.0 ng/Lfor undiluted samples),
then the requirement is met.
guidance on persistent
difficulties with calibration.
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.
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%).
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
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twice. The laboratory reports both
the original and diluted sample
results.
External Quality
External QC Coordinator, supported by QC
Based upon the evaluation, the
Control Sample
contractor, provides 1-2 sets of identical
External QC Coordinator may
samples to all laboratories and compares
request additional information
results.
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.
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4.0 ALGAL TOXIN: MICROCYSTIN IMMUNOASSAY PROCEDURE
This chapter describes an immunoassay procedure that measures concentrations of total microcystins
in water samples. In applying the procedure, the laboratory uses Abraxis' Microcystins-ADDA Test Kits
("kits"). See also EPA Standard Method 546. Each kit is an enzyme-linked immunosorbent assay (ELISA)
for the determination of microcystins and other 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.
Cold microcystin samples will be shipped on ice from the field crews to the contract batching
laboratory. The contract batching laboratory will freeze samples and send the batched samples to the
analysis laboratory in coolers on dry ice. Samples will arrive in the analysis laboratory frozen and they
can be held in a freezer for several weeks. Microcystin analysis laboratories will need to process
samples in accordance with the time frame 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.c 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, microcystin
concentrations as low as 0.10 ng/L. For samples with higher concentrations of microcystins, the
procedure includes the necessary dilution steps.
4.1 Definitions and Personnel Qualifications
This section provides definitions and required resources for using the procedure.
4.1.1 Definitions £
3
The following terms are used throughout the procedure: £
u
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 microcystins. ^
0.10 ng/L and <0.15 ng/L), the p
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. §
u
X
o
c Abraxis, "Microcystins-ADDA ELISA (Microtiter Plate): User's Guide R021412." Retrieved on January 14, 2014 from ^
http://www.abraxiskits.com/uploads/products/docfiles/278 Microcvstin%20PL%20ADDA%20users%20R120214.pdf. ^
19
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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 4.1 Standard deviation
1/2
1 V
S =
n
i = 1
where n is the number of replicate samples, Ais the absorbance measured for the replicate. Per
Section 4.4.3, samples are evaluated in duplicate (i=l or 2); controls are either evaluated in duplicate
or triplicate (i=l, 2,3). A is the average absorbance of the replicates. Then, calculate %CV as:
Equation 4.2 Percent (%) coefficient of variation
S
A
Dark or Dimly Lit: Away from sunlight, but under incandescent lighting is acceptable.
%CV =
x 100
Detection Limit is the minimum concentration at which the analyte can be defected with confidence
(0.1 ng/L).. In other words, the outcome can be reported with confidence that it is greater than zero
(i.e., present in the sample). The detection limit is less than the reporting limit of 0.15 ng/L at which the
measured value of the analyte can be reported with confidence. Also see "Sample-Specific Detection
Limit" bBelow.
Duplicates are defined as two aliquots of the same sample which are analyzed separately using
identical procedures. The results are used to evaluate the precision of the laboratory analyses. Per
Section 4.4.3, controls are evaluated in duplicate or triplicate (i.e., three aliquots).
Relative Standard Deviation (RSD) is the same as the coefficient of variation (%CV). Because many of
the plate reader software programs provide the CV in their outputs, the procedure presents the quality
control requirement in terms of %CV instead of RSD.
lu Reporting Limit: For undiluted freshwater samples, the reporting limit is 0.15 ng/L. A reporting limit is
3 the point at which the measured value of the analyte can be reported with confidence.
Q
u Standard Deviation (S) shows variation from the average,
o
cl Sample-Specific Detection Limit: Most samples will have a sample-specific detection limits equal to the
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participating laboratories; comparing immunoassay results from the laboratories; and preparing brief
summary reports.
4.2 Precautions
The laboratory must require its staff to abide by appropriate health and safety precautions, because
the kit substrate solution contains tetramethylbenzidine (TMB) and the stop solution contains diluted
sulfuric acid. In addition to the laboratory's usual requirements such as a Chemical Hygiene Plan, the
laboratory must adhere to the following health and safety procedures:
1. Laboratory facilities must properly store and dispose of solutions of weak acid.
2. Laboratory personnel must wear proper personal protection clothing and equipment (e.g.
laboratory coat, protective eyewear, gloves).
3. When working with potential hazardous chemicals (e.g., weak acid), laboratory personnel must
avoid inhalation, skin contact, eye contact, or ingestion. Laboratory personnel must avoid
contacting skin and mucous membranes with the TMB and stopping solution. If skin contact
occurs, remove clothing immediately. Wash and rinse the affected skin areas thoroughly with
large amounts of water.
4.2.1 Equipment/Materials
The procedures require the following equipment and information:
Abraxis ADDA Test Kit, Product #520011 (see items in Section 4.4.2)
Adhesive Sealing Film (Parafilm) for Micro Plates): Used to cover plates during incubation.
Data Template - See Figure 4.1.
Distilled or Deionized Water: For diluting samples when necessary.
ELISA evaluation software
2 glass scintillation, LC, vials (20 mL)
LU
Multichannel Pipette & Tips: An 8-channel pipette is used for this method. Familiarity of the gc
use of the multichannel pipette is necessary to achieve reliable results. Practice with water if Q
you have never used this before. ^
QC
CL
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4.3 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 1.2
of the manual for contact information).
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 4-1 for the NARS IM Team, including the Condition Code
for each sample:
OK: Sample is in good condition
C: Sample container was cracked
in. L: Sample container is leaking
iv. ML: Sample label is missing
v. W: Sample is warm (>8°), 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).
QC
3
Q
LU
U
o
QC
CL
u
o
QC
u
X
o
<
3. Store samples in the freezer until sample preparation begins.
4. Maintain the sample tracking forms with the samples.
Table 4-1 Microcystins login: required data elements.
FIELD
FORMAT
DESCRIPTION
LABORATORY ID
text
Name or abbreviation for QC 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
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CONDITION
COMMENT
text
Laboratory Operations Manual
Page 23 of 124
L Sample or container is leaking
ML Sample label is missing
W Sample is warm (>8°)
Q Other quality concerns, not identified
above
Comments about the condition of the sample. If
the condition code='W' then provide the
temperature
4.4 Procedure
The following sections describe the sample, kit preparation and analysis.
4.4.1 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:
20. All cycles: Keep the samples in dark or dimly lit areas (i.e., away from sunlight, but under
incandescent lighting is acceptable).
21. First freeze-thaw cycle:
a. Start with a frozen 500 ml sample.
b. Thaw the sample to room temperature (approximately 25° C). Swirl the sample to check for
ice crystals. At this temperature, no ice crystals should be present in the sample.
c. Shake well to homogenize the sample, then transfer 10 mL to an appropriately labeled
clean 20 mL glass vial.
22. Second freeze-thaw cycle:
a. Freeze the vial.
b. Keep the large sample bottle (from the 500 mL initial sample) frozen for future use.
23. Third freeze-thaw cycle:
oe
3
Q
U
c. Thaw the sample vial contents to room temperature. §
QC
CL
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25. Verify that each kit contains all of the required contents:
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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 (either distilled or deionized water)
oe
3
Q
LU
u
o
QC
CL
U
O
QC
u
X
o
<
26. 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.
27. Adjust the microtiter plate, samples, standards, and the reagents to room temperature.
28. 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).
29. Prepare a negative control (NC) using distilled water
30. The standards, controls, antibody solution, enzyme conjugate, color solution, and stop
solutions are ready to use and do not require any further dilutions.
31. Dilute the wash solution with distilled or deionized water. (The wash solution is a 5X
concentrated solution.) In a 1L container, dilute the 5X solution 1:5 (i.e., 100 mL of the 5X wash
solution plus 400 mL of distilled or deionized water). Mix thoroughly. Set aside the diluted
solution to wash the microtiter wells later.
32. Handle the stop solution containing diluted H2S04 with care.
4.4.3 Insertion of Contents into Wells
This section describes the steps for placing the different solutions into the 96 wells. Because of the
potential for cross contamination using a shaker table, the following steps specify manual shaking of
the kits instead mechanized shaking.
33. 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.
34. Turn on the computer so that it can control and access the plate reader.
35. Print the template (Figure 4.1) to use as reference when loading the standards, controls, and
samples as described in the next step. Templates contain rows, labeled with a marking pen, of
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strips of 8 wells that snap into the blank frame. (If the laboratory wishes to use a different
template, provide a copy to the USEPA HQ Laboratory Review Coordinator for approval prior to
first use. (See Section 1.2 of the manual for contact information.)
36. 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 Figure 4.1. Verify that the software displays the same
template or make any necessary corrections.
10 11 12
A
SO
S4
NC
U4
U8
U12
U16
U20
U24
U28
U32
U36
B
SO
S4
NC
U4
U8
U12
U16
U20
U24
U28
U32
U36
C
SI
S5
U1
U5
U9
U13
U17
U21
U25
U29
U33
U37
D
SI
S5
U1
U5
U9
U13
U17
U21
U25
U29
U33
U37
E
S2
KC
U2
U6
U10
U14
U18
U22
U26
U30
U34
U38
F
S2
KC
U2
U6
U10
U14
U18
U22
U26
U30
U34
U38
G
S3
KC
U3
U7
Ull
U15
U19
U23
U27
U31
U35
U39
H
S3
NC
U3
U7
Ull
U15
U19
U23
U27
U31
U35
U39
Figure 4.1 Microcystin: sample template.
Key:
S0-S5 = Standards;
KC = Control supplied with Kit (i.e., Kit Control);
NC = Negative Control;
U = Unknown (sample collected by the field crew);
37. 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.
38. Place the sealing Parafilm over the wells.
39. 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.
40. Place the plate in an area away from light for 90 minutes.
41. After 90 minutes, carefully remove the Parafilm.
42. 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
multi-channel pipette. After adding the washing solution each time, empty the solution into the
sink and use the paper towels as before.
43. Add 100 piL of enzyme conjugate solution to all wells using the multi-channel pipettor.
44. Cover the wells with Parafilm.
45. 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.
46. Place the strip holder in an area away from light for 30 minutes.
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47. After 30 minutes, remove the Parafilm, decant, and rinse the wells three times again with 250
piL of washing solution as described in step 10.
48. Add 100 piL of color solution to the wells using the multi-channel pipette and reagent reservoir.
This color solution will make the contents have a blue hue.
49. Cover the wells with Parafilm.
50. 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.
51. Place the plate in an area away from light for 20 minutes.
52. 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.
53. 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.
54. Dispose of solution in plates in a laboratory sink. Rinse plates and sink with water to dilute the
weak acid present.
55. Perform QC evaluations of the data as follows:
a. If the following failures occur, then the laboratory must reanalyze all samples in the
analytical run:
i. Standard curve with a correlation coefficient of less than 0.99 (i.e., R<0.99)
ii. Standards S0-S5 must have decreasing absorbance values. First, calculate the average
values for each standard. That is, if A, is the absorbance average for S,, then the
absorbance averages must be:
£ iii. A0> Ai> A2> A3> A4>A5
2 iv. The average absorbance of the standard SO less than 0.8 (i.e., A0 < 0.8).
O v. Two or more negative control samples with detectable concentrations of microcystins
^ (i.e., values >0.1 ng/L). If this occurs, then evaluate possible causes (e.g., cross-
Si contamination between samples), and if appropriate, modify laboratory processes
l/l
^ before the next analytical run.
3 vi. Results for control samples of outside the acceptable range of 0.75 +/- 0.185 ppb. That
^ is, results must be between 0.565 and 0.935.
^ b. If either, or both, of the following failures occur, then the sample must be reanalyzed
fci (maximum of two analyses, consisting of the original analysis and, if necessary, one
^ reanalysis):
y i. The concentration value registers as HIGH (exceeds the calibration range). Dilute the
^ sample for the reanalysis per Section 4.4.4.
ii. The %CV > 15% between the duplicate absorbance values for a sample.
h 56. Record the results, even if the data failed the quality control requirements in #55b, for each
I
< well in the USEPA's data template. The required entries are for the following columns:
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a. TYPE should be one of the following codes: S0-S5 for standards; KC or NC for controls; U for
unknown sample.
b. CONC contains the numeric concentration value. Two special cases:
i. Non-detected concentrations: If the sample is non-detected, then provide the sample-
specific detection limit which is 0.1 ng/L if the sample is undiluted. See Section 4.4.4
for calculating the sample-specific detection limit for a diluted sample.
ii. If the result shows that it is "HI," this indicates that the sample value is outside of the
calibration range and must be diluted and re-run using another analytical run. Leave
the CONC column blank and record 'HI' in the DATA FLAG column.
c. DATA FLAGS have codes for the following special cases:
i. ND if the sample was non-detected;
ii. J if the value is detected but at a level below the reporting limit of 0.15 ng/L (for
undiluted samples);
iii. HI if the concentration value registers as HIGH (exceeds the calibration range).
d. QUALITY FLAGS have codes for the following special cases:
i. QCF if there is a QC failure per step 55 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).
4.4.4 Dilutions (if needed)
Dilutions if needed are prepared as follows (using clean glass tubes):
#1
LU
1:10 dilution 3
Q
LU
e. Add 900 piL of distilled or deionized water to a clean vial. (Note: Dilutions may also be made u
QC
CL
using the kit's diluent rather than distilled or deionized water.
f. Pipette 100 piL from the sample into the vial. (To provide more accurate dilutions and less
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f. Multiply the final concentration and Abraxis' detection limit of 0.1 ng/L by 100 to obtain
the sample-specific detection limit of 10 ng/L.
• Other dilutions can be calculated in the same manner as #1 and #2 if needed.
4.5 Pertinent QA/QC Procedures
This section describes the quality assurance and quality control measures used to ensure that the data
will meet the NLA's requirements.
4.5.1 QC Samples
The External QC Coordinator will instruct the QC contractor to provide one or two identical sets of
freshwater 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.
QC
3
Q
LU
U
o
QC
CL
u
o
QC
u
X
o
<
Each laboratory will run the QC samples following the same procedures used for the other samples.
The External QC Coordinator will compare the results and assess patterns in the data (e.g., one
laboratory being consistently higher or lower than all others). Based upon the evaluation, the External
QC Coordinator may request additional information from one or more laboratories about any
deviations from the method or unique laboratory practices that might account for differences between
the laboratory and others. With this additional information, the External QC Coordinator will determine
an appropriate course of action, including no action, flagging the data, or excluding some or all of the
laboratory's data.
4.5.2 Summary of QA/QC Requirements
Table 4-2 provides a summary of the quality control requirements described in Sections 4.4.2 and
4.4.3. For microcystin, 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 +/- 0.185).
Table 4-2 Microcystin: sample and analysis quality control activities and objectives.
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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Calibration
All of the following must be met:
o Standard curve must have a
correlation coefficient of >0.99;
o Average absorbance value, A0, for SO
must be >0.80; and
o Standards S0-S5 must have decreasing
average absorbance values. That is, if
A, is the average of the absorbance
values for S,, then the absorbance
average values must be: A0 > Ai > A2 >
A3 > A4 >As
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.
Negative Control
Sample
Evaluations
The values for the negative control replicates
must meet the following requirements:
o All concentration values must be <
0.15 ng/L (i.e., the reporting limit);
and
o One or more concentration results
must be nondetectable (i.e., <0.10
Hg/L)
All samples are run in duplicate. Each
duplicate pair must have %CV<15% between
its absorbance values.
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 USEPAfor
guidance on persistent
difficulties with calibration.
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.
If %CV of the absorbances for the
sample>15%, then:
• Record the results for both
duplicates using different start
dates and/or start times to
distinguish between the runs.
• Report the data for both
duplicate results using Quality
Control Failure flag "QCF"; and
• Re-analyze the sample in a
new analytical run. No
samples are to be run more
than twice.
If the second run passes, then the
data analyst will exclude the data
from the first run (which will have
been flagged with "QCF"). If both
runs fail, the data analyst will
determine if either value should
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be used in the analysis (e.g., it
might be acceptable to use data if
the CV is just slightly over 15%).
Results Within
Calibration
Range
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. The laboratory 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.
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5.0 BACTERIA [E. COLI) METHOD
This method describes the analysis of water samples for the enumeration of Escherichia coli (E. coli)
using E. coli using the IDEXX Quanti-Tray/2000 System with Colilert reagent (Standard Method, 9223 B)
(Table 5-1) Laboratories also record information on Total Coiiforms and Presence/Absence of E.coli.
While the USEPA's recommended holding time is 8 hours, it is not possible for crews to submit samples
to laboratories in that timeframe. For the purposes of the NLA, crews will ship samples to the analysis
laboratory as soon as is practicable (either the same day as sample collection or the following day) via
overnight courier and the laboratories will begin processing the samples within 6 hours of receipt to
minimize exceeding the holding time as much as possible. The minimum detection limit for this analysis
is one Most Probable Number (MPN) per lOOmL of sample, and the maximum detection limits is up to
2419 MPN per lOOmL of sample.
Table 5-1 Bacteria: analytical methods.
Storage Requirements
Type
Method
Maintain at 4 C°
Bacteria
Standard Method 9223 B using IDEXX
Colilert and Quanti-Tray/2000
Crews will collect 200 ml of surface water in a 290 mL shrink-banded sterile IDEXX bottle (plastic) that
will later be separated into two 100 ml aliquots during test preparation to allow for duplicate testing, if
necessary. Crews will ship the samples overnight to the laboratory on wet ice maintaining a temperature
of 4 °C, The laboratories will analyze an undiluted water sample from the sampled The laboratory
technician will add the Colilert® reagent directly to one 100 mL aliquot of undiluted sample and mix it
thoroughly to dissolve the reagent. If a laboratory's duplicate is run, the laboratory will follow the same
procedure for the second 100 mL aliquot. The technician transfers the sample to QuantiTrays®/2000
(Figure 5.1) and seals the trays using the Quanti-Tray sealer. Samples are incubated at 35.0 ± 0.52 c for
24 hours. Results are reported as MPN/100 mL.
Figure 5.1 Bacteria [E.coli): disposable 97-well tray for use with the Quanti-Tray sealer
Image from the IDEXX website: https://www.idexx.com/water/products/Quanti-tray.html
d Because NLA participants will not know which sites need to be diluted, and with holding time constraints, it will not be
possible to run dilutions on the NLA 2017 samples. Analyses that show greater than the 2419 count will be reported as having a
result of 2419 with a flag that indicates the value has exceeded the upper limit of quantitation.
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5.1 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.
5.2 Precautions
The analysis involves handling of freshwater samples that may contain live microorganisms and
therefore pose some threat of infection. Laboratory personnel who are routinely exposed to such water
samples are encouraged to protect themselves from water borne illnesses by wearing clean disposable
gloves and washing their hands frequently. The Colilert® reagent is not hazardous although the
manufacturer does recommend wearing gloves and safety glasses while using this reagent and washing
hands after use.
Interferences - water samples containing humic or other material may be colored. If there is background
color, compare inoculated trays to a control tray containing only water (SM, 9223 A).
5.2.1 Storage and Stability
Colilert 100 mL format can be stored up to 12 months at 2-30°C.
5.3 Equipment/Reagents/Standards
Sample from field crew
Sterile bottles with cap for duplicates
Quanti-Tray Sealer® and rubber inserts
Incubator
Colilert® reagent: Snap packs for 100 ml samples, IDEXX
Quanti-Tray®/2000: Containing 97 wells each, IDEXX.
Squeeze bottles for blank
Sterile pipet for removing excess volume in sample container if needed
UV light
5.4 Sample Receipt
Q
o
o
u
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
£5 form
u b. Record the information in Table 3.3-1 for the NARS IM Team, including the Condition Code
cq 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. W: Sample is warm (>4°), 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 5-2 Bacteria 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 (>4°)
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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5.5 Procedure
1. Record all site identification numbers and sample identification numbers being analyzed in the
data spreadsheet.
2. Record initial temperature of samples in the data spreadsheet.
3. The laboratory technician then implements the following steps for test preparation and test
procedure outlined in Sections 5.5 and 5.5.2.
1. Turn on Quanti-tray Sealer and warm it to 35.0 ± 0.52C. Warm up time is approximately 10
minutes.
2. Shake the water sample bottle 25 times within 7 seconds making sure that the interval between
shaking and measuring the test portion is not greater than three minutes.
a. If there is at least 1" of headspace, the sample is shaken in the field sample container. Then
proceed to step 3.
b. If there is insufficient headspace (<1") for proper mixing, do not pour off the excess and
discard. Instead, pour the entire sample into a larger sterile container, mix properly and
proceed to step 3. Record this on the data spreadsheet.
3. Transfer 100 mL of sample into a sterile bottle with cap and sufficient volume to allow for at
least 1" of headspace. For duplicates, the laboratory transfers the second 100 mL into another
sterile bottle.
1. Open a Colilert ampule (reagent) and pour contents into one of the undiluted samples.
2. Re-cap the bottle and shake until reagent is dissolved.
3. Label back of tray with sample ID. If this is a laboratory duplicate or a laboratory reagent blank,
make sure the label clearly indicates this.
4. Use one hand to hold open the Quanti-Tray/2000 with the well side of the tray facing your palm.
5. Squeeze upper part of the tray so it bends toward the palm.
6. Gently pull open the tray. Avoid touching the inside of tray or the foil tab.
7. Pour 100 ml of the sample into the tray.
8. Tap the small wells 2-3 times to release bubbles. Failure to release the bubbles may result in the
wells filling or sealing improperly.
9. Place the tray with the sample into the rubber insert so that the wells sit within the cutouts.
10. Pace the rubber insert on the input shelf of the sealer.
11. Slide the rubber insert with the tray into the sealer.
12. Place the sealed tray/trays into the incubator and incubate at 35.0 ± 0.52C for 24 hours.
13. Record the lot number of the reagents and the wells used in the data spreadsheet.
1. From the back of the tray, record the sample ID in the data spreadsheet corresponding to the
correct NLA site ID. Count the number of small and large positive (yellow) wells and refer to the
MPN table to find the most probable number for Total Coliform. Use the color comparator to
confirm positive results. Document this number in the data spreadsheet.
2. Use an Ultra Violet lamp to check for fluorescence.
a. If no wells fluoresce, the test is negative for E. coli. If wells do fluoresce, the test is positive
for E. coli. Record Presence or Absence in the data spreadsheet.
5.5.1 Test preparation
5.5.2 Test Procedure (see also manufacturer's instructions for preparation
of Quanti-Tray/2000 and use of the Quanti-Tray Sealer)
5.5.3 Results
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b. Count the small and large fluorescing wells. Refer to table for most probable number (MPN).
Record the results in the data spreadsheet.
3. Calculate precision for laboratory duplicates. The desired precision objective is ± 20% (RPD).
Equation 5.1 Precision
Precision (as RPD) = (A - B) x 100%
(A + B)/2
Where: A = MPN from aliquot A and
B = MPN from aliquot B
5.5.4 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.
5.6 Pertinent QA/QC Procedures
Table 5-3 provides a summary of the quality control activities.
5.6.1 Internal QC
1. Initial laboratory demonstration of capability and for each new lot of Quanti-Tray/2000:
a. Use an Ultra Violet lamp to check for fluorescence of the media snap packs. Discard the lot if
fluoresces.
b. Dissolve one packet in 100 ml distilled water. Do not incubate. Check for fluorescence. If it
fluoresces, discard the lot.
2. Quanti-Cult Procedure (Validation of performance of Colilert) run once per new lot of Quanti-
Tray/2000. See Quanti-Cult instructions from manufacturer.
a. Pre-heat incubator to bring temperature up to 35^ ± 0.52C.
b. Pre-warm rehydration fluid vials at 35^C for 10 minutes.
c. Transfer colorless cap from desiccant vial onto pre-warmed rehydration vial. Discard the
blue cap and desiccant vial.
d. Place the vial into the foam rack/vial holder (provided with Quanti-Cult kit).
e. Invert the foam rack and place in the incubator for 10 minutes.
f. Fill four sterile IDEXX 100 ml (or other appropriate, sterilized bottles) bottles with distilled
water to the fill line.
g. Label three bottles with each bacteria name and one bottle "control".
h. Place in incubator until a temperature of 355 ± 0.55C is reached. Q
i. Remove the rehydration vial from the holder (one at a time). Hold the vial upside down and §
tap cap gently to mix. Remove the cap and look at the inside surface to ensure that no tj
undissolved black particles are present. Inoculate an additional 10 minutes if present. ^
j. Add entire contents of each appropriate bacteria vial to pre-warmed 100 ml labeled bottles. q
k. Add the Colilert reagent to sample bottles including the control. ^
LU
I. Place sample bottles in the incubator for 24 hours at 35^ ± 0.5^C. Do not place in Quanti- ^
Trays. E
LU
b
The following organism results should be observed: ^
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Escherichia coli- Yellow wells, fluorescence
Klebsiella pneumonia - Yellow wells, no fluorescence
Pseudomonas aeruginosa - Clear wells, no fluorescence
Method Blank - Clear wells, no fluorescence
3. Sample Batch QC: The analyst runs -
a. One laboratory duplicate for every ten samples (laboratory duplicate - two replicates taken
from the same collection bottle)
b. One (LRB) per sample batch to verify that there is a negative result from 24-28 hours.
5.6.2 External QC
1. Analyze 10 provided spiked samples (blind sample) provided by the USEPA. After processing the
samples, the laboratory will send the results to the USEPA HQ Laboratory Review Coordinator.
The results will be compared to the known concentrations and a determination made. Expected
success is correct analysis of 9 of 10 samples with no false negatives.
Table 5-3 Bacteria: quality control activities for samples.
Activity Evaluation Corrective Action
Demonstrate competency for
analyzing E. coli samples to meet
the performance measures
Demonstration of competency with
E. coli samples in achieving the
method detection limits, accuracy,
and precision targets.
The USEPA will not approve any
laboratory for NLA sample
processing if the laboratory cannot
demonstrate competency. In other
words, the USEPA will select
another laboratory that can
demonstrate competency for its
NLA samples.
Check condition of sample when it
arrives.
Sample issues such as cracked
container; missing label; sufficient
volume for test.
Assign appropriate condition code
identified in Table 5-2
Store sample appropriately. While
stored at the laboratory, the sample
must be kept at a temperature -
refrigerated at 4°C.
Check the temperature of the
refrigerator per laboratory's
standard operating procedures.
Record temperature of sample upon
arrival at the laboratory. If at any
other time, samples are warmer
than required, note temperature
and duration of deviation in
comment field in Table 5-2
Data analyst will consider
temperature deviations in
evaluating the data. The analyst will
flag the deviations and determine
whether the data appear to be
affected and/or the data should be
excluded from the analyses.
Analyze sample within holding time
(8 hour holding time will not be
The 24-hour test must be completed
within 30 hours of receipt (e.g.
analysis of this sample must begin
Perform test, but note reason for
performing test outside of a 36 hour
holding time (assume sample
collected by crew at 12:00 PM). The
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met, so a 36 hour holding time is
the objective for samples)
Perform one laboratory reagent
blank once at the start of each batch
upon receipt because of holding
times.)
Control limits cannot exceed the
method detection limit of 1
MP N/100 mL).
Compare results of one laboratory
duplicate sample
Results must be within the target
precision goal of +/-20%.
Maintain the required MDL
identified 1 MPN/100 ml
Participate in External Quality
Control
Evaluate for each sample
Evaluate QC samples provided by
the External QC Coordinator
USEPA expects that the laboratory
will exercise every effort to perform
tests before the holding time
expires. There will not be sufficient
sample for a re-test.
First, prepare and analyze one
additional blank. If the second blank
meets the requirement, then no
further action is required. If the
second blank fails, then determine
and correct the problem (e.g.,
contamination, instrument
calibration) before proceeding with
any sample analyses keeping in
mind that samples must be
processed within holding times.
Consider alternative options for
analyzing samples such as using
equipment/laboratory space not
involved with the failed blanks.
Reestablish statistical control by
analyzing three blank samples.
Report values of all blanks analyzed.
If both results are below LRL, then
conclude that the test has passed.
Otherwise, prepare and analyze a
split from a different sample in the
batch. If the second result is within
the target precision goal (see
Section 5.5.3) of the original
sample, then report the data and
findings for both QC samples.
However, if the two results differ by
more than the target precision goal,
check preparation of split sample;
etc. and report evaluation and
findings in the case narrative and
assign appropriate data code.
Consult with the USEPA HQ NLA
Laboratory Review Coordinator to
determine what if any changes in
laboratory protocols are needed.
If MDL could not be achieved, then
provide QC code and explanation in
the comment field.
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
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might account for incorrect or
unusual results. 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.
Contact the USEPA HQ NLA
Laboratory Review Coordinator
immediately if issues affect
laboratory's ability to meet
completeness objective.
*Section 1.2 provides contact information for the USEPA HQ NLA Laboratory Review Coordinator. Laboratories under contract
to the USEPA must contact the Task Order's Contracting Officer's Representative (TOCOR) instead of the Laboratory Review
Coordinator.
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Maintain completeness Completeness objective is 95% for
all parameters.
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6.0 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.
6.1 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.
6.2 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.
6.2.1 Sorting and Subsampling Precautions
6.2.2 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).
6.3 Equipment/Materials
6.3.1 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)
6.3.2 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)
Specimen vials with caps or stoppers
Sample labels for specimen vials
'Some laboratories may choose not to use the gridded screen in a plastic holding tray.
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70 - 80% denatured ethanol in plastic wash bottle
Benthic Macroinvertebrate Taxonomic Bench Sheet
Hand tally counter
6.4 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 6-1 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 6-1 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
CONDITION CODE
text
Condition codes describing the condition of the
sample upon arrival at the laboratory.
Flag
Definition
l/l
Q
O
<
QC
CO
LU
I—
QC
LU
>
o
QC
u
<
u
CO
41
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CONDITION
COMMENT
text
Laboratory Operations Manual
Page 42 of 124
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
Comments about the condition of the sample.
u
6.5 Procedure
6.5.1 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
b. Station Location
c. Station Number
d. Date Sorted
l/l
O e. Sorter's Initials
h f. "1 of x" or "2 of x", etc. if the sample is sorted into >1 vial (where x is the total number of
LU
^ vials for the sorted sample)
LU
< 6.5.2 Subsampling
QC
E2 1. Remove the lid from the sample container and remove the internal sample label (save the
g label—it will need to be returned to the sample container with the archived portion of the
^ sample that does not get processed). Record the sample collection information on a Benthic
O Macroinvertebrate Laboratory Bench Sheet. Header information required includes both project
u 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.
42
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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
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.
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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 6.5.3, #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.
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.
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:
6.5.3 Sorting
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a. Empty snail or bivalve shells
b. Specimens of surface-dwelling or strict water column2 arthropod taxa (e.g., Collembola,
Veliidae, Gerridae, Notonectidae, Corixidae, Culicidae, Cladocera, orCopepoda)
c. 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
6.5.2 #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."
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.
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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6.5.4 Taxonomy Procedures
1. The taxonomic target for benthic invertebrates is identified in Section 6.5.1 #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 6-2). 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
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.
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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.
6.5.4.1 Taxonomic Level of Effort
This is the Standard Taxonomic Effort list for benthic macroinvertebrates (Table 6-2). 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).
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Table 6-2 Required level of taxonomic identification for benthic macroinvertebrates.
Phylum Class
Required
Taxonomic
Identification Notes
CO
O
o
<
cc
CQ
LU
I—
cc
o
C£L
U
<
u
CQ
48
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
Mysidacea
Genus
COELENTERATA
MOLLUSCA
Bivalvia
Genus
Gastropoda
Except in the
following case:
Genus
Hydrobiidae
Family
NEMERTEA
Genus
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6.6 Pertinent QA/QC Procedures
6.6.1 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 6.1 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.
6.6.2 Taxonomic QC
6.6.2.1 Internal Taxonomic QC
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%. §
JZ
If the individual fails to maintain a > 90% identification as determined by QC checks, previous samples tj
will be re-counted and identified. ^
LU
5
6.6.2.2 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 2j
send those samples to a QC taxonomist (another experienced taxonomist who did not z
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 z
LU
each bench sheet is completed, fax it to the Project Facilitator. co
u
49
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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 6.2 Percent difference in enumeration (PDE).
PDE =
¦ x 100
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 6.3 Percent taxonomic disagreement (PTD).
PTD =
1-
COmPPos
N
x 100
where compp0s 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.
6.6.2.3 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 6-3 Laboratory quality control: benthic indicator.
Check or Sample
Description
Frequency
Acceptance Criteria
Corrective Action
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
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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 > 85%
If PDE > 5%, implement
recommended corrective actions.
If PTD < 85%, 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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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.
l/l
Q
O
o
I—
<
I
CL
O
£
53
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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
to calculate the total biovolume contributed by the taxon to its representative sample (Burkholder
and Wetzel, 1989).
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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:
^ count x chamber xlOOOmL ^
Equation 7.1 Phytoplankton abundance.
cells/ - \numfields x field x mlsettled
7mL
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 zooplankton. 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
q Duplicate processing (duplicate the processing steps presented in Section 7.5.1 - 7.5.5).
O
The remaining concentrated sample will be sent to the QC taxonomist.
Lu
^ 1. Using the same volume as the original Utermohl chamber, prepare a duplicate Utermohl
O chamber cell and enumerate 400 natural algal units. Complete another copy of the Taxonomic
t Bench Sheet for each sample. Label each bench sheet with the term "QC Dup-ID." As each bench
< sheet is completed, the laboratory sends it (through email or fax) to the Indicator QC
q Coordinator.
> 2. The Indicator QC Coordinator compares the taxonomic results generated by the primary and QC
S taxonomists for each sample and calculate percent difference using:
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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
External QC
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 SEDIMENT CONTAMINANTS, GRAIN SIZE, AND TOC
This method describes the analysis requirements for sediment samples. The purpose is to determine
concentrations of contaminants, grain size, and total organic carbon (TOC) in sediment samples
collected in the National Lakes Assessment (NLA) 2017 and related studies. The laboratory shall perform
analysis to determine the moisture content, concentrations of metals, mercury, pesticides, PAHs and
PCBs found in lake sediments.
At each sampling site, the FOM instructs the crews to collect sediment samples. The field crew then
ships the samples on wet ice to either its own state laboratory or the USEPA's batching laboratory. Once
the samples arrive, the laboratory will freeze the samples for the contaminant analyses and TOC, and
will refrigerate the grain size samples.
This chapter describes the contaminant, grain size, and TOC determination of sediment samples
collected for the USEPA's NLA 2017. As described in Section 8.5, unless otherwise contractually bound
by other requirements, the laboratory may choose to use any method that meets the USEPA's
specifications for contaminant and grain size measurements.
8.1 Definitions and Required Resources (Personnel, Laboratories, and
Equipment)
This section provides definitions and required resources for using the analytical procedures.
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8.1.1 Definitions
The analytical procedures use the following terms:
Detection Limit is the minimum concentration at which the analyte can be detected with
confidence. In other words, the outcome can be reported with confidence that it is greater than
zero (i.e., present in the sample). Also see "Sample-Specific Detection Limit."
Duplicates are defined as two aliquots of the same sample which are analyzed separately using
identical procedures. The results are used to evaluate the precision of the laboratory analyses.
h NARS Information Management System (NARS IM): The IM system established to support all
¦z. surveys, including NLA, in the NARS program. The IM system is used to track the samples from field
- collection to the laboratory.
M
1/1 Percent Recovery: Recovery is measured by comparing the concentrations of a sample split into two
^ parts; where one part is spiked with a known concentration value. Cs is the concentration measured
^ in the spiked part; C is the concentration measured in the unspiked part; and s is the known
of concentration amount for the spike. The following equation is used to calculate the percent
^ recovery:
Equation 8.1 Percent recovery
cs- C
%Rs = — x 100
^ Relative Percent Difference (RPD): Relative percent difference compares the matrix spike (S) and
q the matrix spike duplicate (D) using the following equation:
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Equation 8.2 Relative percent difference
RPD
Reporting Limit: A reporting limit is the point at which the measured value of the analyte can be
reported with confidence.
Sample-Specific Detection Limit: Most samples will have a sample-specific detection equal to the
method's detection limit. For diluted samples, the sample-specific detection limit will be the product
of the method's detection limit and the dilution factor. Typical values for the dilution factors will be
10 or 100.
Spiked Sample: See Percent Recovery definition for purpose of spiked samples.
TOC: Total Organic Carbon
TOCOR: Task Order Contracting Officer's Representative is the USEPA's contact person for
laboratories under contract to the USEPA.
\S-D\
(S + D)jr
¦x 100
8.1.2 Personnel
The analytical procedures refer to the following personnel:
Laboratory Technician: These procedures may be used by any laboratory technician who is
familiar with the NLA Quality Assurance Project Plan, and this procedure in the NLA Laboratory
Operations Manual.
External QC Coordinator is an USEPA 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 the NLA. The QC contractor is responsible for complying with instructions from
the External QC Coordinator; coordinating and paying for shipments of the PT samples to ^
participating laboratories; and preparing brief summary reports. Q
<
8.2 Precautions
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The laboratory must require its staff to abide by appropriate health and safety precautions. In addition
to the laboratory's usual requirements such as a Chemical Hygiene Plan, the laboratory must adhere to ^
the following health and safety procedures: ^
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8.3 Equipment/Materials
The analytical methods, selected by the laboratory, specify the required equipment.
8.4 Sample Receipt
Because the USEPA initiates tracking procedures designed to recover any missing shipments, the
laboratory personnel responsible for tracking samples must start the following login steps within 24
clock hours of receiving a delivery. The laboratory must inspect the samples promptly upon receipt. As
samples arrive, the laboratory must:
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 for
contact information).
2. Check that each shipping container has arrived undamaged. Check the temperature of one of
the samples in the cooler using either a thermometer that reads from 21 (i.e., room
temperature) down to -20 ^C or lower (i.e., the expected temperature of frozen samples), or an
infra-red (IR) temperature "gun" and record the reading.
3. Record the condition and temperature of the sample on the NARS IM sample tracking
spreadsheet using the codes in Table 8-1.
4. Verify that all required data elements, per Table 8-1, have been recorded.
5. Transfer the samples to the freezer or refrigerator for long-term storage. Except during the
processing and analysis stages, the contaminant samples and TOC must be stored frozen to less
than or equal -20 °C and the grain size samples must be stored in the refrigerator.
6. Notify the USEPA immediately about any problems involving sample integrity, conformity, or
inconsistencies as soon as possible following sample receipt and inspection.
7. Maintain the sample tracking forms with the samples.
Table 8-1 Sediment chemistry, grain size, and TOC login: required data elements.
Variable
Type
Description
SITEJD
Character
Site identification code
VISIT_NO
Numeric
Sequential visits to site (1 or 2)
SAMPLE_ ID
Character
Sample number
DATE_COLLECT
MMDDYY
Date that the field crew collected the sample
ANALYSIS_TYPE
Character
Contaminant, TOC, or GRAIN SIZE
ARRIVAL_TEMP
Numeric
Temperature of sample upon arrival at the laboratory
CONDITION_CODE
Character
Condition codes describing the condition of the sample upon
arrival at the laboratory; leave blank for control
Flag
Definition
OK
Sample is in good condition
C
Sample container is cracked
L
Sample or container is leaking
ML
Sample label is missing
VT
Volume not sufficient for testing
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Variable
Type
Description
VR
Volume not sufficient for a retest, if required
Q
Other quality concerns, not identified above
COND_COMMENT
Character
Explanation for Q FLAG (if needed)
8.5 Laboratory Analysis: Requirements
The laboratory shall perform analyses of the sediment samples to determine the moisture content, grain
size, and concentrations of TOC, metals, mercury, pesticides, PAHs, and PCBs.
Table 8-2 identifies the storage requirements. Laboratories may choose to use any analysis method,
including those in Table 8-2, which measures the parameters to the levels of the method detection
limits identified in Table 8-3. In addition, the contaminant analysis method must meet the precision and
accuracy targets of 30% and 20%, respectively. For each batch of contaminant samples, precision is
assessed using the RPD between the matrix spike (MS) and the matrix spike duplicate (MSD); and
accuracy by the average percent recovery (%Rs) between the matrix spike and matrix spike duplicate.
Section 8.1.1 provides the equations used to calculate the RPD and %Rs. The precision and accuracy
targets for each batch of TOC are both 10% and determined by the RPD of one sample and its duplicate
(for precision) and the analysis of Certified Reference Material (CRM; for accuracy). The grain size target
precision is 10% as determined using a Laboratory Control Sample (LCS) (accuracy is not applicable).
Table 8-2 Sediment chemistry, grain size, and TOC: storage requirements and analytical methods.
Storage Requirements
Type
Methods that Meet the QA/QC
Requirements (any method that meets
the QA/QC requirements is
acceptable)
Freeze samples with
maximum of-20° C
Metals (except Mercury)
Extraction: USEPA Method 3051A
Analysis: USEPA Method 6020A6
Mercury
USEPA Method 245.7f
PCBs, Pesticides, PAHs
Extraction: USEPA Method 3540C
Analysis: USEPA Method 8270DS
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e For example, see:
• Method 3051A "Microwave Assisted Acid Digestion of Sediments, Sludges, Soils, And Oils" retrieved April
28, 2017 fromhttps://www.epa.gov/hw-sw846/sw-846-test-method-3051a-microwave-assisted-acid-
digestion-sediments-sludges-soils-and-oils; and
• Method 6020A "Inductively Coupled Plasma-Mass Spectrometry" retrieved April 28, 2017 from
https://www.epa.gov/sites/production/files/2015-07/documents/epa-6Q20a.pdf
f For example, see Method 245.7 "Mercury in Water by Cold Vapor Atomic Fluorescence Spectrometry, Revision
2.0" (EPA-821-R-05-001, February 2005), retrieved June 27, 2014 from
http://water.epa.gov/scitech/methods/cwa/bioindicators/upload/2007 07 10 methods method 245 7.pdf.
g For example, see:
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TOC
US EPA Method 9060 e
Refrigerate at 4° C
(do not freeze)
Grain Size
Any method that reports the
determination as %silt and meets
QA/QC requirements
Table 8-3 Sediment chemistry, grain size, and TOC: required parameters.
Type
UNITS
Parameter
CAS Number
PCB
MDL
Number
Target
(where
applicable)
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% sand
Grain Size
not applicable
0.05%
% silt
% clay
mg/kg and %
Total Organic Carbon (TOC)
not applicable
0.01%
METAL
dry weight ng/g
Aluminum
7429-90-5
1500
(ppm)
Antimony
7440-36-0
0.2
Arsenic
7440-38-2
1.5
Cadmium
7440-43-9
0.05
Chromium
7440-47-3
5.0
Copper
7440-50-8
5.0
Iron
7439-89-6
500
Lead
7439-92-1
1.0
Manganese
7439-96-5
1.0
Mercury
7439-97-6
0.01
Nickel
7440-02-0
1.0
Selenium
7782-49-2
0.1
Silver
7440-22-4
0.3
Tin
7440-31-5
0.1
Vanadium
7440-62-2
1.0
Zinc
7440-66-6
2.0
PCB
dry weight
2,2',3,3',4,4l,5,5l,6,6l-Decachlorobiphenyl
2051-24-3
209
1.0
ng/g
2,4'-Dichlorobiphenyl
34883-43-7
8
1.0
(ppb)
2,2',3,3l,4,4',5-Heptachlorobiphenyl
35065-30-6
170
1.0
• Method 3540C "Soxhlet Extraction" retrieved April 28, 2017 from
https://www.epa.gov/sites/production/files/2015-12/documents/3540c.pdf; and
• Method 8270D "Semivolatile Organic Compounds by Gas Chromatography/Mass Spectrometry (GC/MS)
retrieved April 28, 2017 fromhttps://www.epa.gov/sites/production/files/2015-07/documents/epa-
8270d.pdf .
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2,2',3,4',5,5',6-Heptachlorobiphenyl
52663-68-0
187
1.0
2,2',3,4l,5,5',6-Heptachlorobiphenyl
35065-29-3
180
1.0
2,2',3,3l,4,4'-Hexachlorobiphenyl
38380-07-3
128
1.0
2,2',3,4,4',5'-Hexachlorobiphenyl
35065-28-2
138
1.0
2,2',4,4l,5,5'-Hexachlorobiphenyl
35065-27-1
153
1.0
2,2',3,3',4,4l,5,5l,6-Nonachlorobiphenyl
40186-72-9
206
1.0
2,2',3,3',4,4',5,6-Octachlorobiphenyl
52663-78-2
195
1.0
2,3,3',4,4'-Pentachlorobiphenyl
32598-14-4
105
1.0
2,2',4,5,5'-Pentachlorobiphenyl
37680-73-2
101
1.0
2,3',4,4',5-Pentachlorobiphenyl
31508-00-6
118
1.0
2,3,3',4,6'-Pentachlorobiphenyl
38380-03-9
110
1.0
3,3',4,4',5-Pentachlorobiphenyl
57465-28-8
126
1.0
2,2',3,5'-Tetrachlorobiphenyl
41464-39-5
44
1.0
3,3',4,4'-Tetrachlorobiphenyl
32598-13-3
77
1.0
2,2',5,5'-Tetrachlorobiphenyl
35693-99-3
52
1.0
2,3',4,4'-Tetrachlorobiphenyl
32598-10-0
66
1.0
2,2',5-Trichlorobiphenyl
37680-65-2
18
1.0
2,4,4'-Trichlorobiphenyl
7012-37-5
28
1.0
PEST
dry weight
2,4'-DDD
53-19-0
1.0
ng/g
2,4'-DDE
3424-82-6
1.0
(ppb)
2,4'-DDT
789-02-6
1.0
4,4'-DDD
72-54-8
1.0
4,4'-DDE
72-55-9
1.0
4,4'-DDT
50-29-3
1.0
Aldrin
309-00-2
1.0
Alpha-BHC
319-84-6
1.0
Beta-BHC
319-85-7
1.0
Delta-BHC
319-86-8
1.0
Alpha-Chlordane
5103-71-9
1.0
Gamma-Chlordane
5566-34-7
1.0
Dieldrin
60-57-1
1.0
Endosulfan 1
959-98-8
1.0
Endosulfan II
33213-65-9
1.0
Endosulfan Sulfate
1031-07-8
1.0
Endrin
72-20-8
1.0
Endrin Aldehyde
7421-93-4
1.0
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Endrin Ketone
53494-70-5
1.0
Heptachlor
76-44-8
1.0
Heptachlor Epoxide
1024-57-3
1.0
Hexachlorobenzene
118-74-1
1.0
Lindane
58-89-9
1.0
Mi rex
2385-85-5
1.0
Cis-Nonachlor
5103-73-1
1.0
Oxychlordane
26880-48-8
1.0
Trans-Nonachlor
39765-80-5
1.0
PAHs
dry weight
Acenaphthene
83-32-9
1
ng/g
Acenaphthylene
208-96-8
1
(ppb)
Anthracene
120-12-7
1
Benz(a)anthracene
200-280-6
1
Benzo(b)fluoranthene
205-99-2
1
Benzo(k)fluoranthene
207-08-9
1
Benzo(g,h,i)perylene
191-24-27-2
1
Benzo(a)pyrene
50-32-8
1
Benzo(e)pyrene
192-9
1
Biphenyl
92-54-4
1
Chrysene
218-01-9
1
Dibenz(a,h)anthracene
53-70-3
1
Dibenzothiophene
132-65-0
1
2,6-Dimethylnaphthalene
581-42-0
1
Fluoranthene
205-99-2
1
Fluorene
86-73-7
1
lndeno(l,2,3-c,d)pyrene
193-39-5
1
1-Methylnaphthalene
90-12-0
1
2-Methylnaphthalene
91-57-6
1
1-Methylphenanthrene
832-69-9
1
Naphthalene
91-20-3
1
Perylene
198-55-0
1
Phenanthrene
85-01-8
1
Pyrene
129-00-0
1
2,3,5-Trimethylnaphthalene
2245-38-7
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8.5.1 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
This section describes the quality assurance and quality control measures used to ensure that the data
will meet the NLA's requirements.
8.6.1 QC Samples
Once or twice during the performance period (preferably once at the beginning and once at the end),
the External QC Coordinator will provide one or two identical sets of QC samples to all participating
laboratories. Each set will contain up to five QC samples. As determined by the External QC Coordinator,
the QC samples may be synthetic; aliquots of additional samples collected at NLA sites; or reference
samples obtained from an organization such as the National Institute of Standards. Each laboratory will
run the QC samples following the same procedures used for the other samples. The External QC
Coordinator will compare the results to the expected value and determine consistency between
laboratories (e.g., determine if one laboratory is 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 unique laboratory practices that might account for differences between the
laboratory and others. The contractor shall analyze the external QC samples using the same procedures
as those for the field samples.
8.6.2 Summary of QA/QC Requirements
QC protocols are an integral part of all analytical procedures to ensure that the results are reliable and
the analytical stage of the measurement system is maintained in a state of statistical control. The
laboratory must conduct QC analyses for each batch of samples. Each batch shall consist of no more
than 20 samples. Unique laboratory quality control lot numbers must be assigned to each batch of
samples. The lot number must associate each batch of field samples to the appropriate measures such
as laboratory control sample, matrix spike, matrix spike duplicate, laboratory duplicate, and method
blank samples. Also, each laboratory QC samples (i.e., preparation and instrument blanks, laboratory
control sample (LCS), spike/duplicate, etc.) must be given a unique sample identification. Table 8-4
provides a summary of the quality control requirements.
Table 8-4 Sediment chemistry, grain size, and TOC: quality control activities for samples.
Activity
Evaluation
Corrective Action
Demonstrate competency for
analyzing sediment samples to meet
the performance measures
Demonstration of competency with
sediment samples in achieving the
method detection limits, accuracy,
and precision targets.
The USEPA will not approve any
laboratory for the NLA sample
processing if the laboratory cannot
demonstrate competency. In other
words, the USEPA will select another
laboratory that can demonstrate
competency for its NLA samples.
Check condition of sample when it
arrives.
Sample issues such as cracked
containers; missing labels;
insufficient volume for testing.
Assign appropriate condition code
identified in Table 8-1.Error!
Reference source not found..
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Activity Evaluation Corrective Action
Store sample appropriately. While
stored at the laboratory, the sample
must be kept at a temperature
<-20° C except jars for grain size
analyses are refrigerated at 4°C.
Check the temperature of the
refrigerator/freezer and refrigerator
per the laboratory's SOPs.
Record temperature of sample upon
arrival at the laboratory. If at any
other time, samples are warmer
than required, note temperature
and duration of deviation in
comment field.
Data analyst will consider
temperature deviations in
evaluating the data. He/she will flag
the deviations and determine
whether the data appear to be
affected and/or the data should be
excluded from the analyses.
Analyze sample within holding time
The test must be completed within
the holding time of 1 year. If the
original test fails, then the retest
also must be conducted within the
holding time.
Perform test, but note reason for
performing test outside holding
time. The USEPA expects that the
laboratory will exercise every effort
to perform tests before the holding
time expires.
Perform once at the start of each
batch to evaluate the labeled
compound recovery (LCR) in a
Laboratory Control Sample (LCS).
This tests the performance of the
equipment.
Control limits for recovery cannot
exceed 100±20%.
First, prepare and analyze one
additional LCS. If the second blank
meets the requirement, then no
further action is required. If the
second LCS fails, then determine
and correct the problem before
proceeding with any sample
analyses.
Perform once at the start of each
batch to evaluate the entire
extraction and analysis process
using a Method Blank
Control limits cannot exceed the
laboratory reporting level (LRL).
First, prepare and analyze one
additional blank. If the second blank
meets the requirement, then no
further action is required. If the
second blank fails, then determine
and correct the problem (e.g.,
contamination, instrument
calibration) before proceeding with
any sample analyses. Reestablish
statistical control by analyzing three
blank samples. Report values of all
blanks analyzed.
Check calibration immediately
before and immediately after the
sample batch (abbreviated as QCCS
for quality control check sample)
Results must be ±10% of each other
or as specified in method criteria
If calibration fails before analysis,
recalibrate and reanalyze QCCS until
it passes. If check fails after all
samples in the batch have been
analyzed, verify the QCCS reading. If
the QCCS reading fails a second
time, then reanalyze all samples in
the batch and report only the set of
results associated with the
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Activity Evaluation Corrective Action
acceptable QCCS reading. Also
report all QCCS readings for the
batch.
Compare results of one laboratory
duplicate sample (forTOC) or matrix
spike duplicate sample (for
contaminants) for each batch (not
required for grain size)
Results must be within the target
precision goal in Section 8.5.
If both results are below LRL, then
conclude that the test has passed.
Otherwise, prepare and analyze a
split from different sample in the
batch. If the second result is within
the target precision goal (see
Section 8.5) of the original sample,
then report the data and findings
for both QC samples. However, if
the two results differ by more than
the target precision goal, review
precision of QCCS measurements
for batch; check preparation of split
sample; etc. and report evaluation
and findings in the case narrative.
Consult with the USEPA HQ NLA
Laboratory Review Coordinator to
determine if reanalysis of the entire
batch (at the laboratory's expense)
is necessary. If no reanalysis is
necessary, report and quantify all
samples in batch. If reanalysis is
necessary, then report all QC
samples and the 2nd analysis of the
batch. If the second set also is
unacceptable, then assign a QC code
to each sample in the batch.
Compare results of one matrix spike
sample per batch to evaluate
performance in matrix (not required
for TOC and grain size)
Evaluate performance after the first
3 batches; and then every
subsequent batch. Ideally, control
limits for recovery will not exceed
the target accuracy goal, but this
may not be realistic for all
parameters with this matrix.
If both the original and duplicate
results are below LRL, then conclude
that the test has passed for the
batch. Otherwise, if any results are
not within the target accuracy goal
for the first 3 batches, within 2
working days, contact the USEPA
HQ NLA Laboratory Review
Coordinator to discuss method
performance and potential
improvements. After achieving
acceptable results or the USEPA's
permission to continue, perform the
test for every subsequent batch. For
each batch, report the results from
the original analysis and its
duplicate and their RPD for TOC; the
matrix spike, matrix spike duplicate,
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67
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Activity
Evaluation
Corrective Action
RPD and %recovery for
contaminants.
Compare results of TOC Certified
Reference Material once per each
batch
Value must be within 10% of the
certified value.
If value is outside the acceptable
range, analyze a second CRM. If the
second CRM also is measured
outside the acceptable range, then
determine and correct the problem
(e.g., contamination, instrument
calibration) before reanalyzing all
samples in the batch.
Maintain the required MDL targets
identified in Section 8.5 and Table
8-3
Evaluate for each sample
If MDL could not be achieved, then
provide dilution factor or QC code
and explanation in the comment
field.
Participate in External Quality
Control
Evaluate QC samples provided by
the External QC Coordinator
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.
Maintain completeness
Completeness objective is 95% for
all parameters.
Contact the USEPA HQ NLA
Laboratory Review Coordinator
immediately if issues affect
laboratory's ability to meet
completeness objective.
*Section 1.2 provides contact information for the USEPA HQ NLA Laboratory Review Coordinator. Laboratories
under contract to the USEPA must contact the Task Order's Contracting Officer's Representative (TOCOR) instead
of the Laboratory Review Coordinator.
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9.0 ATRAZINE PESTICIDE SCREEN
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 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.
Cold atrazine pesticide screen samples will be shipped on ice from the field crews to the contract
batching laboratory. The contract batching laboratory will store samples in the refrigerator and send the
batched samples to the analysis laboratory in coolers on 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 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.
9.1 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.
9.2 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.
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.
9.2.1 Storage and Stability
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9.3 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
9.4 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
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 9-1 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
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 9-1 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
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VISIT NUMBER numeric
SAMPLE ID numeric
DATE COLLECTED MMDDYY
CONDITION CODE text
CONDITION
COMMENT
text
Laboratory Operations Manual
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Sequential visits to site (1 or 2)
Sample id as used on field sheet (on sample label)
Date sample was collected
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 (>4°)
Q
Other quality concerns, not identified
above
Comments about the condition of the sample. If
the condition code='W' then provide the
temperature
9.5 Procedure
9.5.1 Test preparation
1. Filter all lake water samples with a 0.2 pim 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.
9.5.2 Procedural notes and precautions
• A consistent technique is important for optimal performance. For the greatest precision, treat
each tube in an identical manner.
• Add reagents directly to the bottom of the tube while avoiding contact between the reagents
already added to the tube and the pipet tip. This will help assure consistent quantities of
reagent in the test mixture.
• 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.
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9.5.3 Assay procedure
1. Label test tubes for standards, controls, and samples (Table 9-2).
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.
9.5.4 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.
Table 9-2 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
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9.5.5 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.
9.6 Pertinent QA/QC Procedures
9.6.1 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 9-3 provides a summary of the quality control requirements.
Table 9-3 Atrazine: quality control requirements.
Quality
Description and Requirements
Corrective Action
Control
Activity
Kit-Shelf
Life
Is within its expiration date listed on
kit box.
If kit has expired, then discard or 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
A, is the average of the
absorbance values for S,, then
the absorbance average
values must be: A0> 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.
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.
Negative
Control
The values for the negative control
replicates must meet the following
requirements:
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o All concentration values must
be < 0.05 ng/L (i.e., the
reporting limit)
• The lab reanalyzes all samples in the
analytical run until the controls meet the
requirements.
Sample : Samples are run in duplicate: requires
Evaluations : 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.
Results
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.
9.6.2 External QC
1. Analyze 10 provided spiked samples (blind sample) provided by the USEPA HQ Laboratory
Review Coordinator. After processing the samples, the laboratory will send the results to the
USEPA HQ Laboratory Review Coordinator. The results will be compared to the known
concentrations and a determination made.
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10.0 WATER CHEMISTRY and CHLOROPHYLL A
10.1 Analytical Parameters
A total of 19 parameters are determined from each bulk water chemistry sample collected (Table 10-1).
In addition, chlorophyll-o 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 2017.
Analyte Units Comments
Conductivity
|j.S/cm at 25°C
PH
Standard (Std) Units
Turbidity
NTU
Acid Neutralizing Capacity
(ANC)
|aeq/L
(20 neq/L=l mg as CaC03)
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 (N03-N02)
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
Sulfate (S04)
mg /L
Chloride (CI)
mg/L
Nitrate (N03)
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)
Aluminum (Al)
mg/L
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-o
|ag/L (in extract)
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10.2 Sample Receipt
Laboratory Operations Manual
Page 76 of 124
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
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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
Q_
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CONDITION
COMMENT
text
Laboratory Operations Manual
Page 77 of 124
L Sample or container is leaking
ML Sample label is missing
W Sample is warm (>7°)
Q Other quality concerns, not identified
above
Comments about the condition of the sample. If
the condition code='W' then provide the
temperature
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.
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10.3.1 Water Chemistry Samples
Sample Receipt
4 L Bulk Sample
Inspect samples and complete
tracking form
Store at 4°C in darkness
r
Process Sample
Within 24 hours
y
Filtration (0.4um)
Not Filtered
N
<
HDPE
HDPE
HDPE
bottle
bottle
bottle
Acid
Not acid
Acid
washed
washed
washed
Preserv
Store at
Preserv
e with
4 °C in
e with
HNO„
V-- |
h2§o4
J
Analyses
Calcium (180
days)
Magnesium
(180 days)
Sodium (180
days)
Potassium
(180 days)
Aluminum (180
days)
Analyses
Chloride (28
days)
Nitrate (7 days)
Sulfate (28
days)
Silica (28 days)
Nitrate-Nitrite
(with IC) (7
days)
Nitrate (with IC)
(7 days)
True Color (3
days)
Analyses
Ammonia-N (28 days)
Dissolved Organic
Carbon (28 days)
Nit rate-Nit rite (with FIA)
(28 days)
Nitrate (with FIA) (28
days)
HDPE
bottle
Acid
washed
Preserve
with
H2SOf
-P—''
HDPE
bottle
Not acid
washed
Store at
4 °C in
darknes
/ N
Analyses
Analyses
Total Phosphorus
pH (3 days)
(28 days)
ANC (7 days)
Total Nitrogen (28
Conductivity
days)
(28 days)
Turbidity (3
days)
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x
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Figure 10.1 Water chemistry sample processing procedures.
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
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C£
78
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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.
Table 10-3 Acid preservatives added for various analytes.
Preservatives
h2so4
HNO3
DOC
Al
nh3-n
Ca
Total N
Mg
Total P
Na
NO2-NO3
K
10.3.2 Chlorophyll-a Samples
Chlorophyll-o 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 represents the value of the lowest non-zero calibration
standard used. 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 z!
Wilson (1986). The transition value is the value at which performance objectives for precision and bias i
switch from absolute (< transition value) to relative (> transition value). For pH, the objectives are O
established for samples with lower H+ (or OH") concentrations (pH between 5.75 and 8.25) and higher H+ O
(or OH") concentrations (pH < 5.75 or > 8.25).
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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 value and the target value of a performance t
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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.
Analytical methods used at the central laboratory (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 determine whether the laboratories meet the necessary requirements. The information
from this process is maintained in the NLA 2017 QA files by the USEPA HQ Laboratory Review
Coordinator.
Table 10-4 Summary of analytical methods used by NLA 2017 (Central Laboratory, USEPA ORD-Corvallis).
Analyte Summary of Method*1 References' WRSSOPj
pH (laboratory)
Automated, using ManSci PC-Titrate w/ Titra-Sip
autotitrator and Ross combination pH electrode. Initial pH
determination for ANC titration
USEPA 150.6 (modified)
WRS 16A.0 (April
2011)
Specific conductance
<5> 25°C
Electrolytic, Man-Tech TitraSip automated analysis
OR manual analysis, electrolytic
USEPA 120.6
WRS 16A.0 (April
2011)
WRS 11A.4 (April
2011)
Acid neutralizing
capacity (ANC)
Automated acidimetric titration to pH<3.5, with modified
Gran plot analysis
U.S. USEPA (1987)
WRS 16A.0 (April
2011)
Turbidity
Nephelometric; Man-Tech TitraSip automated analysis,
OR
Manual analysis using Hach turbidimeter (high turbidity
samples)
APHA 214 A, USEPA 180.1 U.S.
EPA (1987)
WRS 16A.0 (April
2011)
WRS 13A.3 (April
2011)
True color (Hach Kit)
Visual comparison to calibrated glass color disk.
APHA 204 A (modified), USEPA
110.2 (modified), U.S. EPA
(1987)
WRS 15A.3 (April
2011)
Dissolved Organic
Carbon (DOC)k
UV promoted persulfate oxidation to CO2 with infrared
detection
APHA 5310-C
U.S. EPA (1987)
WRS 21A.4 (May
2011)
Nitrate+Nitrite, as N
(fresh waters)
Ion Chromatography
OR
FIA automated colorimetric (cadmium reduction)
USEPA 300.6; SW-846 9056A;
APHA 4110B
USEPA 353.2
APHA 4500-N03-N-E
Lachat 10-107-04-1-C
WRS 36A.0 (April
2011
WRS 40A.5 (May
2011)
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.4 (April
2011)
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FIA=Flow injection analysis. AAS=Atomic Absorption Spectrometry
1 U.S. EPA, 1987. Handbook of Methods for Acid Deposition Studies: Laboratory Analyses for Surface Water Chemistry.
EPA/600/4-87/026. U.S. Environmental Protection Agency, Office of Research and Development, Washington D.C. APHA=
American Public Health Association (Standard Methods). ASTM=American Society of Testing and Materials.
J WRS= Willamette Research Station. References are to laboratory SOP being used at central laboratory. Available upon request,
(contact the Project Lead)
k For DOC, "dissolved" is defined as that portion passing through a 0.45 [am nominal pore size filter. For other analytes,
"dissolved" is defined as that portion passing through a 0.4 [am pore size filter (Nucleopore or equivalent).
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Silica, dissolved
(Si02)
Fresh waters
FIA automated colorimetric (molybdate, stannous chloride)
EPA366.0,APHA 425 C
Lachat 10-114-27-1-B
WRS 32A.5
(February 2010)
Total nitrogen (TN)
Persulfate Digestion; FIA Automated Colorimetric Analysis
(Cadmium Reduction, sulfanilamide)
EPA353.2 (modified)
APHA 4500-N-C (modified)
ASTM WK31786
U.S. EPA (1987)
Lachat 10-107-04-1-C
(modified)
WRS 34A.5 (April
2011)
Total phosphorus
(TP)
Persulfate Digestion; Automated Colorimetric Analysis
(molybdate, ascorbic acid)
APHA 4500-P-E
USGS 1-4650-03
U.S. EPA (1987)
Lachat 115-01-1-B (modified)
WRS 34A.5 (April
2011)
Major anions,
dissolved
chloride, nitrate,
nitrite, sulfate
Ion Chromatography
USEPA 300.6; SW-846 9056A;
APHA 4110B
WRS 40A.5 (May
2011)
Major cations,
dissolved
calcium, sodium,
potassium,
magnesium,
aluminum
Inductively-coupled Plasma Atomic Emission Spectroscopy
(ICP-AES)
OR
Flame AAS
USEPA 200.7; USEPA 6010B
U.S. EPA (1987), USEPA 215.1
USEPA 273.1, USEPA 258.1
USEPA 242.1
WRS SOP 3.04 v3
(October 2011)
WRS 50A.4
(March 2007)
Chlorophyll-a
(Chl-a)
Extraction 90% acetone analysis by fluorometry
USEPA 445.0, USEPA 446.0
WRS 71A.3 (April
2011)
10.5 Pertinent QA/QC Procedures
A single central 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 central laboratory demonstrated in previous studies that it can meet the required LRL (USEPA 2004).
QA/QC procedures outlined in this manual and the NLA 2017 QAPP will be followed to ensure these LRLs
are met for the NLA 2017.
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 Lower Transition Precision Bias
Range of Reporting Value" Objective0 Objective13
Samples' Limit"1
Conductivity
|j.S/cm at
25°C
1 to 15,000
2.0
20
± 2 or±10%
± 2 or 5%
pH (laboratory)
Std Units
3.5 to 10
N/A
5.75, 8.25
±0.07 or
±0.15
>5.75 and <
8.25: ±0.15
±0.05 or
±0.10
>5.75 and <
8.25: ±0.15
Turbidity
NTU
0 to 44,000
2.0
20
± 2 or±10%
± 2 or±10%
Dissolved Organic
Carbon (DOC)
mg/L
0.1 to 109
0.20
< 1
> 1
±0.10 or
±10%
±0.10 or
±10%
Ammonia as
N(NH3-N)
mg/L
Oto 17
0.02 (1.4
Heq/L)
0.10
±0.01 or
±10%
±0.01 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
0.1 to 90
0.02
0.10
±0.01 or
±10%
±0.01 or
±10%
Total Phosphorus
(TP)
Hg/L
0 to 22,000
4
20
± 2 or±10%
± 2 or±10%
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1 Estimated from samples analyzed at the WED-Corvallis laboratory between 1999 and 2005 for TIME, EMAP-West,
and WSA streams from across the U.S.
m The lower reporting limit is the lowest value that needs to be quantified (as opposed to just detected), and
represents the value of the lowest nonzero calibration standard used. 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.
" 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: General Principles and Techniques. 2nd ed. Royal Society of Chemistry, London, England.
° For duplicate samples, precision is estimated as the pooled standard deviation (calculated as the root-mean
square) of all samples at the lower concentration range, and as the pooled percent relative standard deviation of
all samples at the higher concentration range. For standard samples, precision is estimated as the standard
deviation of repeated measurements across batches at the lower concentration range, and as percent relative
standard deviation of repeated measurements across batches at the higher concentration range.
p Bias (systematic error) is estimated as the difference between the mean measured value and the target value of a
performance evaluation and/or internal reference samples at the lower concentration range measured across
sample batches, and as the percent difference at the higher concentration range.
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Sulfate (S04
Chloride (CI)
Nitrate (N03
Calcium (Ca)
Sodium (Na)
Silica (Si02)
True Color
Chlorophyll a
mg/L
mg/L
mg/L
mg /L
Magnesium (Mg) mg/L
mg/L
Potassium (K) mg/L
PCU
0 to 5,000
0 to 5,000
0 to 360
0.04 to 5,000
0.1 to 350
0.08 to 3,500
0.01 to 120
mg/L 0.01 to 100
0 to 350
0.50 (10
Heq/L)
0.20 (6
Heq/L)
0.02 (4
Heq/L)
0.10 (5
M-eq/L)
0.10 (8
M-eq/L)
0.10 (4
Heq/L)
0.10 (2
Heq/L)
0.10
j^ig/L (in 0.7 to 11,000 0.5
extract)
2.5
1
0.1
0.5
0.5
0.5
0.5
0.5
50
15
±0.25 or
±10%
±0.10 or
±10%
±0.01 or
±10%
±0.05 or
±10%
±0.05 or
±10%
±0.05 or
±10%
±0.05 or
±10%
±0.05 or
±10%
±5 or ±10%
± 1.5 or ±10%
±0.25 or
±10%
±0.10 or
±10%
± 0.01 ±10%
± 0.05 or
±10%
± 0.05 or
±10%
± 0.05 or
±10%
± 0.05 or
±10%
± 0.05 or
±10%
±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
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Laboratory/
All
Once per day
Control limits
Prepare and analyze new
Reagent
prior to
< LRL
blank. Determine and
Blank
sample
correct problem (e.g.,
analysis
reagent contamination,
instrument calibration, or
contamination introduced
during filtration) before
proceeding with any
sample analyses.
Reestablish statistical
control by analyzing three
blank samples.
Filtration
All dissolved
ASTM Type II
Prepare once
Measured
Measure archived
Blank
analytes
reagent
per week
concentrations
samples if review of other
water
and archive
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Laboratory
Duplicate
Sample
All analyses
One per
batch
Control limits
< precision
objective
If results are below LRL:
Prepare and analyze split
from different sample
(volume permitting).
Review precision of QCCS
measurements for batch.
Check preparation of split
sample. Qualify all
samples in batch for
possible reanalysis.
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
and QCCS solutions and
standards for
contamination and
preparation error. Correct
before any further
analyses of routine
samples are conducted.
Reestablish control by
three successive
reference standard
measurements that are
acceptable. Qualify all
sample batches analyzed
since the last acceptable
reference standard
measurement for possible
reanalysis.
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 2017 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)
Review holding times
Ion balance:
Calculate percent ion balance difference
(%IBD) using data from cations, anions,
pH, and ANC. See Equation 10.1.
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)
Review data from QA samples (laboratory
PE samples, and inter-laboratory
comparison samples)
Correct reporting errors or qualify as suspect or invalid.
Qualify value for additional review
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
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.
Indicator QC Coordinator determines impact and possible
limitations on overall usability of data based on the specific
issue.
Table 10-8 Data reporting criteria: water chemistry indicator.
Measu rement
Units
No. Significant
Figures
Maximum No.
Decimal Places
DO
mg/L
2
1
Temperature
°C
2
1
PH
pH units
3
2
Carbon, dissolved organic
mg/L
3
1
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ANC
|aeq/L
3
1
Conductivity
|j.S/cm at 25 °C
3
1
Aluminum, calcium, magnesium, sodium,
potassium, chloride, nitrate, and sulfate
|aeq/L
3
1
Silica
mg/L
3
2
Total phosphorus
M-g/L
3
0
Total 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- V anions) - ANC
%IBD = ^-= 1 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.
Table 10-9 Constants for converting major ion concentration from mg/L to |ieq/L
Analyte Conversion from mg/L to |aeq/Lq
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
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q Measured values are multiplied by the conversion factor. ;>
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Table 10-10 Factors to calculate equivalent conductivities of major ions/
Ion
Equivalent Conductance per mg/L Ion
Equivalent Conductance per
(|oS/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 105s
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.
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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 2017 method. Macrozooplankton are counted from a sample using a
150 pirn 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 50 pim mesh nets.
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, 1x3 inch
Cover slips
Tubes for concentrating plankton samples (see below)
Small sieves with 45 and 140-nm 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).
l/l
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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:
OK: Sample is in good condition
C: Sample container was cracked
in. 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).
l/l
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3. Store samples until sample preparation begins.
4. Maintain the sample tracking forms with the samples.
O
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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 log book 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 O
size. ti
2. Be sure to rinse the corresponding sample bottles thoroughly with reverse osmosis ^
(RO)/DI/distilled water into the 45-nm mesh and 145-nm mesh sieve to remove any residual o
organisms adhering to walls of the bottle. Rinse all containers from which zooplankton are g
transferred thoroughly, including the 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 q
time, to prevent organisms from sticking to the sides of the containers and from floating at the ^
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l/l
Q
O
O
I—
CL
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o
N
surface of the sample.
3. Stir the sample gently to break up algal clumps and then pour the entire sample into the Folsom
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 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 National
Lakes Assessment (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.
Species-level resolution will be the taxonomic requirement for macrozooplankton.
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).
Concentrate the sub-sample by using the small sieve or the condensing tube and place in
a circular (or other suitable) counting chamber.
Identify all macrozooplankton under a dissecting microscope and enumerate using a
mechanical or electronic tally counter.
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
1.0
2.0
3.0
4.0
5.0
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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.
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 Hydra carina. 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
dadocerans <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
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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
1. Place the sub-sample in an Utermohl counting chamber or Sedgwick-Rafter cell and cover with a
glass cover slip.
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 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 11.5.2.4.2 respectively.
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.
1/1 l/l
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<
i
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O
11.6.1 Volume of water filtered
Equation 11.1 Volume of water filtered.
Q
o 11.6 Calculating and Reporting o
Report zooplankton densities as number of organisms per cubic meter, which is calculated in the
following equations.
O
I—
<
I
Q_
2 V = LxA O
o
N N
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where:
V = Volume of water filtered (m3)
L = Length of vertical tow
A = Area of the mouth of the net (m2) = 0.1963 m2 for 0.5-m diameter net
11.6.2 Macrozooplankton Densities
Equation 11.2 Microcrustacean densities.
D
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.
(NxVsxS)
D =
NxV
where:
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)
11.6.4 Zooplankton Biomass Estimates
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).
11.6.5 Results of Laboratory Processing, Sample Archiving
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
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"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.
11.7 Pertinent QA/QC Procedures
Table 11-2 provides a summary of quality assurance/quality control procedures for the zooplankton
indicator.
11.7.1 Sorting and Subsampling QC
For each laboratory, approximately 10% of the samples are randomly-selected for evaluation of
subsampling precision (consistency of duplicate processing) by the laboratory. For these samples, the
unused fractions will be treated in an identical manner as the primary fractions (taxonomic identification
and enumeration). There are two precision calculations, one for tracking error for individual samples,
and the other for estimating error for the overall dataset. Differences between the two sample fractions
are an indication of subsampling consistency, quantified by relative percent difference (RPD) as follows:
Equation 11.4 Relative percent difference (RPD).
In, -nA
RPD=-±^ y—xlOO
(n2 + n2)/2
where rii is the metric or index value from the first subsample, and n2 is the metric or index value from
the second. The magnitude of error expected to be associated with splitting zooplankton samples is
unknown; thus a specific measurement quality objective is not proposed here. For estimating
subsampling error for the overall dataset, root mean square error (RMSE) is calculated. Also called
standard error of estimate, this statistic is an estimate of the standard deviation of a population of
observations and is calculated by:
Equation 11.5 Root mean square error (RMSE) or standard error of estimate.
RMSE =
k "i , ,
zzk-y,)2
i-i 2-i
2>/,
where yrj- is the ith individual observation in group j, j = l...k (Zar 1999). More simply put, the equation can
be described as the root of the sums of squared residuals across all subsample pairs, divided by the
number of sample pairs. For computational convenience, RMSE is often calculated by taking the root of
the mean square error (MSE), which can be output from an analysis of variance (ANOVA).
1/1 l/l
Q
O
11.7.2 Taxonomic QC ^
o
lu 11.7.2.1 Internal Taxonomic QC lu
¦> ¦>
^ As directed by the Indicator QC Coordinator, an in-house QC Analyst will randomly select 5 of the z
O samples counted and identified by individual taxonomists to ensure that each meets the acceptable O
criteria for percent identification efficiency which is 90%.
CL
O
o
N N
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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.
11.7.2.2 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 jars, vials, and slides for each of these samples are sent by the original
laboratory to a QC taxonomist for complete re-identification and re-enumeration. The
laboratory will complete and send with the samples a sample tracking form. Differences
between the two samples are an indication of taxonomic precision.
2. Precision of taxonomic identifications is determined by calculating percent taxonomic
disagreement (PTD) of taxonomic results from two independent taxonomists, using the formula:
Equation 11.6 Percent taxonomic disagreement (PTD).
PTD =
1-
fcomppos>
N
x 100
where compp0s is the number of agreements, and N is the total number of organisms in the
larger of the two counts (Stribling et al. 2003).
3. 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
are investigated and logged for indication of error patterns or trends.
4. The calculation of PTD is dependent on the number of organisms reported by the taxonomist.
Where there is potential for a large loss of organisms between the two sample fractions and the
PTD results prove to be too impractical for use, an additional proportional analysis will be used
to assist in eliminating the dependence on the difference of total numbers found with the
following formula:
Where: (ni/N)*100
ni = number of taxa 7 that the taxonomist counted
N= total count of all organisms in sample by the taxonomist
5. 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.
i/i
11.7.2.3 Taxonomic QC Review & Reconciliation §
The Indicator QC Coordinator prepares a report or technical memorandum to quantify aspects of jE
taxonomic precision, assess data acceptability, highlight taxonomic problem areas, and provide ^
recommendations for improving precision. This report is submitted to the HQ Project Management z
Team, with copies sent to the primary and QC taxonomists. Another copy is maintained in the project h
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 §
N
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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 (PTD) > 85%
If PTD < 85%, 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
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12.0 RESEARCH INDICATOR: DISSOLVED GASES
Carbon dioxide (C02), methane (CH4), and nitrous oxide (N20) concentrations in air and dissolved gases
will be measured using gas chromatography at the USEPA's Office of Research and Development
Laboratory in Cincinnati, Ohio. The relative abundance of the stable isotopes 12C and 13C in the dissolved
C02 and CH4 will be measured by colleagues at the University of Quebec. The laboratory procedures for
these analysis will not be included in this manual, but are available upon request.
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13.0 RESEARCH INDICATOR: FISH eDNA
Information on this indicator is contained in other research documents.
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14.0 LITERATURE CITED
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Havens, K.E. & J.R. Beaver. 2010. Composition, size and biomass of zooplankton in large productive Florida
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ADDA ELISA Test Kit; DM ELISATest Kit; Strip Test Kit," in Environmental Technology Verification
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James, R., et al., "Environmental Technology Verification Report: Abraxis Microcystin Test Kits: ADDA
ELISATest Kit; DM ELISATest Kit; Strip Test Kit," in Environmental Technology Verification System
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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.
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.
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
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.
44: 264-274.
Standard Method, "9223 A. 5.2 precautions.
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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. 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-
microcvstins-nodularins-drinking-water-ambient-water-adda-enzvme-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.
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
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
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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.
Website:
http://www.waterboards.ca.gov/water_issues/programs/tmdl/records/region_l/2013/ref4112.pdf
Website: http://www.ohiowea.org/docs/E_Coli_QuanitTray_.pdf
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) 2017, 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 2017.
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...)
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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 2017 Laboratory Operations Manual (or
equivalent). If using equivalent procedures, please provide procedures manual.
2.) Read and abide by the NLA 2017 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 May 1, 2018 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) 2017, 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 2017.
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 2017.
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...)
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> ~ Documentation of NABS certification for the taxonomists performing analyses (if available) >
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not covered in NLA 2017 Laboratory Manual) !<
~ Other documentation supporting your laboratory's ability to meet the required level of data 3
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This documentation may be submitted electronically via e-mail to forde.kendra@epa.gov. Questions h
concerning this request can be submitted to pollard.amina@epa.gov (202-566-2369) or ^
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Laboratory Signature Form - Biology Laboratories
i
certify that the
located in
_, will abide by the following standards in
laboratory,
performing biology data analysis and reporting for the National Lakes Assessment (NLA).
2.
8.)
Utilize procedures identified in the NLA 2017 Laboratory Operations Manual (or
equivalent). If using equivalent procedures, please provide procedures manual.
Read and abide by the NLA 2017 Quality Assurance Project Plan (QAPP) and
related Standard Operating Procedures (SOPs).
Have an organized IT system in place for recording sample tracking and analysis
data.
Use taxonomic standards outlined in the NLA 2017 Laboratory Manual.
Participate in taxonomic reconciliation exercises during the field and data
analysis season, which include conference calls and other laboratory reviews.
Provide data using the template provided in the Laboratory Operations Manual.
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.
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 Serial 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
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Phytoplankton Measurement Data Sheet
Sample # Lake
Laboratory Operations Manual
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Date Collected
Depth of tow_
Laboratory #_
Analyzed by_
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Zooplankton Sample Log In Form
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Zooplankton Enumeration Data Sheet
Sample # Lake
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Laboratory #
Date Collected
Depth of tow
Analyzed by
Working Volume (mL)
¦i-Taxa / Count
Milliliters in subsample (rotifers)
Split
Total Mature Copepoda
Total Immature Copepoda
Total Cladocera
Total Rotifera
Total Other Organisms
Note: For Rotifers only A and B counts are made.
117
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Sample # Lake
Date Collected Depth of tow
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Laboratory #
Analyzed by
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APPENDIX C: STATE SAMPLE TRACKING SPREADSHEET
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Provided on the NARS SharePoint site or from the Laboratory Review Coordinator.
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APPENDIX D: REPORTING TEMPLATES
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Templates will be provided on the NARS SharePoint Site.
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APPENDIX E: SUPPORTING METHODS
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As needed.
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