United States Environmental Protection Agency
Office of Water
Washington, DC
EPA841-B-12-010
National Rivers and Streams Assessment
2013-2014
Laboratory Operations
Manual
Version 2.0 May 2014
-------
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page ii of 224
NOTICE
The intention of the National Rivers and Streams Assessment 2013-2014 is to provide a comprehensive
"State of Flowing Waters" assessment for rivers and streams across the United States. The complete
documentation of overall project management, design, methods, quality assurance, and standards is
contained in five companion documents:
National Rivers and Streams Assessment 2013-14: Quality Assurance Project Plan EPA-841-B-12-007
National Rivers and Streams Assessment 2013-14: Site Evaluation Guidelines EPA-841-B-12-008
National Rivers and Streams Assessment 2013-14: Non-Wadeable Field Operations Manual EPA-841-B-
12-009a
National Rivers and Streams Assessment 2013-14: Wadeable Field Operations Manual EPA-841-B-12-
009b
National Rivers and Streams Assessment 2013-14: Laboratory Operations Manual EPA 841-B-12-010
Addendum to the National Rivers and Streams Assessment 2013-14: Wadeable & Non-Wadeable Field
Operations Manuals
This document (Laboratory Operations Manual) contains information on the methods for analyses of the
samples to be collected during the project, quality assurance objectives, sample handling, and data
reporting. These methods are based on the guidelines developed and followed in the Western
Environmental Monitoring and Assessment Program (Peck et al. 2003). Methods described in this
document are to be used specifically in work relating to the NRSA 2013-2014. All Project Cooperator
laboratories should follow these guidelines. Mention of trade names or commercial products in this
document does not constitute endorsement or recommendation for use. More details on specific
methods for site evaluation, sampling, and sample processing can be found in the appropriate
companion document.
The suggested citation for this document is:
USEPA. 2012. National Rivers and Streams Assessment 2013-2014: Laboratory Operations Manual. EPA-
841-B-12-010. U.S. Environmental Protection Agency, Office of Water, Washington, DC.
y
O
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page iii of 224
TABLE OF CONTENTS
NOTICE ii
TABLE OF CONTENTS iii
LIST OF TABLES vi
LIST OF FIGURES vii
LIST OF ACRONYMS vii
1.0 INTRODUCTION 10
2.0 GENERAL LABORATORY GUIDELINES 11
2.1 RESPONSIBILITY AND PERSONNEL QUALIFICATIONS 11
2.2 ROLES AND CONTACT INFORMATION 11
2.3 SAMPLE TRACKING 11
2.4 REPORTING 12
3.0 ALGAL TOXIN (MICROCYSTIN) IMMUNOASSAY PROCEDURE 13
3.1 SUMMARY OF THE PROCEDURE 13
3.2 HEALTH AND SAFETY WARNINGS 14
3.3 DEFINITIONS AND REQUIRED RESOURCES (PERSONNEL, LABORATORIES, AND EQUIPMENT) 14
3.3.1 Definitions 14
3.4 GENERAL REQUIREMENTS FOR LABORATORIES 15
3.4.1 Expertise 15
3.4.2 Quality assurance and quality control requirements 15
3.4.3 Personnel 16
3.4.4 Equipment/Materials 16
3.5 SAMPLE RECEIPT 16
3.6 PROCEDURE 17
3.6.1 Sample Preparation 18
3.6.2 Kit Preparation 18
3.6.3 Insertion of Contents into Wells 19
3.6.4 Dilutions (if needed) 23
3.7 QUALITY MEASURES 24
3.7.1 Assistance Visits 24
3.7.2 QC Samples 24
3.7.3 Summary of QA/QC Requirements 24
3.8 SAMPLE AND RECORD RETENTION 26
3.9 REFERENCES 26
4.0 BENTHICMACROINVERTEBRATES 27
4.1 INTRODUCTION 27
4.2 SUMMARY OF METHOD 28
4.3 HEALTH AND SAFETY WARNINGS 28
4.4 DEFINITIONS AND REQUIRED RESOURCES (LABORATORY, PERSONNEL, AND EQUIPMENT) 28
4.4.1 Definitions 28 H
4.4.2 Laboratory 32 LU
4.4.3 Personnel 33 z
4.4.4 Equipment/Materials 34 u
4.4.4.1 Sample Preparation (Subsampling) and Sorting Equipment/Materials 34 Q
4.4.4.2 Taxonomy Identification Equipment/Materials 35 LU
4.5 SAMPLE RECEIPT 35 co
<ฃ
4.6 SAMPLE PREPARATION (SUBSAMPLING) 36 i-
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page iv of 224
4.7 SORTING 40
4.8 TAXONOMY IDENTIFICATION 44
4.9 DATA ENTRY 51
4.10 SAMPLE AND RECORD RETENTION 51
4.11 EXTERNAL TAXONOMIC QUALITY CONTROL 51
4.12 QUALITY ASSURANCE/QUALITY CONTROL (QA/QC) 53
4.13 REFERENCES 56
5.0 FECAL INDICATOR: ENTEROCOCCI 57
5.1 SCOPE & APPLICATION 57
5.2 SUMMARY OF METHOD 57
5.3 DEFINITIONS OF METHOD 57
5.4 INTERFERENCES 58
5.5 HEALTH & SAFETY WARNINGS 58
5.6 PERSONNEL QUALIFICATIONS 59
5.7 EQUIPMENT AND SUPPLIES 59
5.8 REAGENTS & STANDARDS 59
5.9 PREPARATIONS PRIOR TO DNA EXTRACTION & ANALYSIS 60
5.10 PROCEDURES FOR PROCESSING &QPCR ANALYSIS OF SAMPLE CONCENTRATES 61
5.10.1 Sample Processing (DNA Extraction) 61
5.10.2 Sample Analysis by Enterococcus qPCR 62
5.10.2.1 Preparation of qPCR assay mix 62
5.10.3 Sample analysis sequence for SmartCycler 64
5.11 STORAGE & TIMING OF PROCESSING/ANALYSIS OF FILTER CONCENTRATES 64
5.12 CHAIN OF CUSTODY 64
5.13 QUALITY ASSURANCE/QUALITY CONTROL (QA/QC) PROCEDURES 64
5.14 METHOD PERFORMANCE 65
5.15 RECORD KEEPING & DATA MANAGEMENT 65
5.16 WASTE MANAGEMENTS POLLUTION PREVENTION 65
5.17 LITERATURE CITED 66
5.18 TABLES, DIAGRAMS, FLOWCHARTS, CHECKLISTS, AND VALIDATION DATA 66
5.18.1 Enterococcus qPCR Analysis Decision Tree (ADT) 69
5.18.2 "Modified" MagNA Pure LC DNA Purification Kit III Protocol 69
6.0 FISH VOUCHER SPECIMENS 72
6.1 SUMMARY OF PROCEDURES 72
6.2 HEALTH AND SAFETY WARNINGS 72
6.3 DEFINITIONS AND REQUIRED RESOURCES (PERSONNEL, TAXONOMY LABORATORIES, AND EQUIPMENT) 73
6.3.1 Definitions 73
6.3.2 General Requirements for Taxonomists and Taxonomy Laboratories 74
6.3.3 Personnel 74
6.3.4 Equipment/Materials 74
6.4 SAMPLE RECEIPT 75
6.5 QC IDENTIFICATION 76
6.6 ASSISTANCE VISITS 81
6.7 SAMPLE AND RECORD RETENTION 81 ฃ
6.8 SUMMARY OF QC REQUIREMENTS FOR FISH VOUCHER SPECIMENS 81 g
6.9 REFERENCES 82 ^
7.0 FISH TISSUE FILLET (Whole Fish Collection) 84 8
LJ_
8.0 FISH TISSUE PLUG 85 ฐ
i
9.0 PERIPHYTON 87
IV
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page v of 224
9.1 SUMMARY OF PROCEDURE 87
9.2 HEALTH AND SAFETY WARNINGS 88
9.3 DEFINITIONS AND REQUIRED RESOURCES (LABORATORY, PERSONNEL, AND EQUIPMENT) 89
9.3.1 Definitions 89
9.3.2 Laboratory 90
9.3.3 Lab Personnel 91
9.3.4 Equipment/Materials 91
9.3.4.1 Subsampling Equipment/Materials (Section 9.5) 91
9.3.4.2 Diatoms 92
9.3.4.2.1 Cleaning Equipment/Materials (Section 9.6 Diatom Cleaning and Mounting) 92
9.3.4.2.2 Preparation of Diatom Slides (Section 9.6) 92
9.3.4.2.3 Analysis of Diatoms (Section 9.7Analysis of Diatoms and Soft Algae) 92
9.3.4.3 Soft algae (Section 9.7) 93
9.4 SAMPLE RECEIPT 93
9.5 SAMPLE PREPARATION (SUBSAMPLING) 94
9.6 DIATOM CLEANING AND MOUNTING 97
9.7 ANALYSIS OF DIATOMS AND SOFT ALGAE 102
9.7.1 Taxonomic Nomenclature and Photographic Specifications 103
9.7.2 Analysis of Diatoms 104
9.7.3 Analysis of Soft Algae 107
9.7.4 Internal Quality Control Ill
9.7.5 Required Data Elements for Diatom and Soft Algae Analyses Ill
9.8 DATA ENTRY 114
9.9 SAMPLE AND RECORD RETENTION 119
9.10 EXTERNAL TAXONOMIC QUALITY CONTROL EVALUATION 120
9.11 REFERENCES 124
10.0 PERIPHYTON META-GENOMICS (Pilot Research Effort) 128
11.0 WATER CHEMISTRY and CHLOROPHYLL A 129
11.1 ANALYTICAL PARAMETERS 129
11.2 SAMPLE PROCESSING AND PRESERVATION 129
11.2.1 Water Chemistry Samples 130
11.2.2 Chlorophyll-a Samples 131
11.3 PERFORMANCE-BASED METHODS 131
11.4 PERTINENT QA/QC PROCEDURES 133
11.4.1 Laboratory Performance Requirements 133
11.4.2 Laboratory Quality Control Samples 133
11.4.3 Data Reporting, Review, and Management 138
11.5 LITERATURE CITED 140
APPENDIX A: CONTACT INFORMATION 141
APPENDIX B: LABORATORY REMOTE EVALUATION FORMS 143
APPENDIX C: SAMPLE LABORATORY FORMS 150
BENTHIC MACROINVERTEBRATE: SORTING BENCH SHEET 151
BENTHIC MACROINVERTEBRATES: TAXONOMY BENCH SHEET (OPTIONAL) 152
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page vi of 224
LIST OF TABLES
TABLE 2.1 CONTACT INFORMATION 11
TABLE 3.1 MICROCYSTIN: REQUIRED DATA ELEMENTS-LOGIN 17
TABLE 3.2 MICROCYSTIN: REQUIRED DATA ELEMENTS-DATA SUBMISSION 22
TABLE 3.3 MICROCYSTIN: QUALITY CONTROL-SAMPLE ANALYSIS 24
TABLE 4.1 BENTHICMACROINVERTEBRATE: REQUIRED DATA ELEMENTS-LOGIN 36
TABLE 4.2 BENTHICMACROINVERTEBRATE: LIST OF TAXA THAT ARE NOT TO BE SORTED/COUNTED 41
TABLE 4.3 BENTHICMACROINVERTEBRATE: REQUIRED DATA ELEMENTS-SORTING 43
TABLE 4.4 BENTHIC MACROINVERTEBRATE: TARGET LEVEL OF TAXONOMIC IDENTIFICATION - BENTHICS COMMONLY FOUND IN
FRESHWATER 47
TABLE 4.5 BENTHIC MACROINVERTEBRATE: TARGET LEVEL OF TAXONOMIC IDENTIFICATION-CHIRONOMIDAE 48
TABLE 4.6 BENTHICMACROINVERTEBRATE: REQUIRED DATA ELEMENTS-TAXONOMIC IDENTIFICATION 50
TABLE 4.7 BENTHICMACROINVERTEBRATE: MEASUREMENT DATA QUALITY OBJECTIVES 54
TABLE 4.8 BENTHICMACROINVERTEBRATE: QUALITY CONTROL-LABORATORY 55
TABLE 5.1 ENTEROCOCCI: PCR ASSAY MIX COMPOSITION (ACCORDING TO DRAFT EPA METHOD 1606) 66
TABLE 5.2 ENTEROCOCCI: BATCH CALIBRATOR & ENTEROCOCCUS STANDARDS PCR RUN - 7 SAMPLES 66
TABLE 5.3 ENTEROCOCCI: SUB BATCH TEST SAMPLE PCR RUN -26 SAMPLES & 1 METHOD BLANK 66
TABLE 5.4 ENTEROCOCCI: LABORATORY METHODS 67
TABLE 5.5 ENTEROCOCCI: PARAMETER MEASUREMENT DATA QUALITY OBJECTIVES 68
TABLE 5.6 ENTEROCOCCI: LABORATORY QC PROCEDURES-ENTEROCOCCI DNA SEQUENCES 68
TABLE 6.1 FISH VOUCHER: REQUIRED DATA ELEMENTS-LOGIN 76
TABLE 6.2 FISH VOUCHER: REQUIRED DATA ELEMENTS-DATA SUBMISSION WORKSHEET 80
TABLE 6.3 FISH VOUCHER: MEASUREMENT DATA QUALITY OBJECTIVES 81
TABLE 6.4 FISH VOUCHER: QUALITY CONTROL-TAXONOMIC IDENTIFICATION 82
TABLE 6.5 FISH VOUCHER: DATA VALIDATION 82
TABLE 8.1 FISH TISSUE PLUG: MEASUREMENT DATA QUALITY OBJECTIVES 85
TABLE 8.2 FISH TISSUE PLUG: QUALITY CONTROL 85
TABLE 9.2 PERIPHYTON: REQUIRED DATA ELEMENTS-LOGIN 94
TABLE 9.3 PERIPHYTON: REQUIRED DATA ELEMENTS-SUBSAMPLING 97
TABLE 9.4 PERIPHYTON: REQUIRED DATA ELEMENTS-DIATOM CLEANING & MOUNTING 102
TABLE 9.5 PERIPHYTON: REQUIRED DATA ELEMENTS-ANALYSIS 112
TABLE 9.6 PERIPHYTON: REQUIRED DATA ELEMENTS-ALL 116
TABLE 9.7 PERIPHYTON: MEASUREMENT DATA QUALITY OBJECTIVES-DIATOMS 126
TABLE 9.8 PERIPHYTON: MEASUREMENT DATA QUALITY OBJECTIVES-SOFT BODIED ALGAE 126
TABLE 9.9 PERIPHYTON: QUALITY CONTROL-ALL ACTIVITIES 126
TABLE 9.10 PERIPHYTON: DATA VALIDATION 127
TABLE 10.1 WATER CHEMISTRY: NRSA 2013/14 PARAMETERS 129
TABLE 10.2 WATER CHEMISTRY: ACID PRESERVATIVES ADDED FOR VARIOUS ANALYTES 131
TABLE 10.3 WATER CHEMISTRY: NRSA 2013/14 ANALYTICAL METHODS (CENTRAL LABORATORY, EPAORD-CoRVALLis) 132
TABLE 10.4 WATER CHEMISTRY & CHLOROPHYLL-A: LABORATORY METHOD PERFORMANCE REQUIREMENTS 134
TABLE 10.5 WATER CHEMISTRY: QUALITY CONTROL-LABORATORY SAMPLES 136
TABLE 10.6 WATER CHEMISTRY: QUALITY CONTROL-DATA VALIDATION 138
TABLE 10.7 WATER CHEMISTRY: DATA REPORTING CRITERIA 138
TABLE 10.8 WATER CHEMISTRY: CONSTANTS FOR CONVERTING MAJOR ION CONCENTRATION FROM MG/LTO^EQ/L 139
TABLE 10.9 WATER CHEMISTRY: FACTORS TO CALCULATE EQUIVALENT CONDUCTIVITIES OF MAJOR IONS 140
TABLE D.O.I FISH IDENTIFICATION: STANDARD COMMON AND SCIENTIFIC NAMES 156
VI
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
LIST OF FIGURES
Laboratory Operations Manual
Page vii of 224
FIGURE 3.1 MICROCYSTIN: ABRAXISMICROCYSTIN TEST KIT 13
FIGURE 3.2 MICROCYSTIN: SAMPLE TEMPLATE 20
FIGURE 4.1 BENTHICMACROINVERTEBRATE: SUMMARY OF SORTING 37
FIGURE4.2 BENTHICMACROINVERTEBRATE:SORTING METHOD EXAMPLE 40
FIGURE 5.1 ENTEROCOCCI: oPCR ANALYSIS DECISION TREE (ADT) 69
FIGURE 6.1 FISH VOUCHER: EXAMPLE QC FISH TAXONOMY BENCH SHEET (OPTIONAL) 83
FIGURE 9.1 PERIPHYTON: SUMMARY OF PROCEDURE FOR IDENTIFYING SOFT ALGAE AND DIATOMS 88
FIGURE 10.1 WATER CHEMISTRY: SAMPLE PROCESSING PROCEDURES 130
LIST OF ACRONYMS
A absorbance HCI
ADT analysis decision tree HOPE
AFDM ash-free dry mass HNO3
ANC acid neutralizing capacity HQ
ANS Academy of Natural Sciences HRP
AQM absolute quantitation method H2S
ASTM American Society for Testing and Materials I-^SCU
Avg Average IBD
BHI brain heart infusion ID
BV biovolume IM
Ca Calcium IPC
CCE calibrator cell equivalents ISBN
CEQ cell equivalent ISO
Chl-a chlorophyll-a
CO2 carbon dioxide IT IS
Ct threshold cycle
CPR cardiopulmonary resuscitation K
cv curriculum vitae KC
CV coefficent of variation LFB
DCF dilution/concentration factor LFM
Dl de-ionized LIMS
DIG differential interference contrast
DL detection limit LOM
DNA Deoxyribo-nucleic Acid LRL
DO dissolved oxygen Mg
DOC dissolved organic carbon MDL
DTH depositional targeted habitat MPCA
DW distilled water MSDS
ELISA enzyme-linked Immunosorbent assay N
EMAP Environmental Monitoring and Assessment Na
Program NABS
ENT enterococci NALMS
EPA Environmental Protection Agency NARS
ETON ethyl alcohol NAWQA
FOM Field Operations Manual
GEQ genomic equivalent NC
GIS geographic information system ND
GPS global positioning device
hydrogen chloride
high density polyethylene
nitric acid
headquarters
antibody-Horseradish Peroxidase
hydrogen sulfide
sulphuric acid
ionic balance difference
Identification
information management
internal positive control
International Standard Book Number
International Organization for
Standardization
Integrated Taxonomic Information System
(IT IS)
potassium
kit control
lab fortified blanks
lab fortified matrices
Laboratory Information Management
System
Lab Operations Manual
lower reporting limit
magnesium
method detection limit
Minnesota Pollution Control Agency
Materials Safety Data Sheet
nitrogen
sodium
North American Benthological Society
North American Lakes Management Society
National Aquatic Resource Surveys
National Water Quality Assessment
Program
negative control
nondetect
52
LI-
LI-
VII
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
NELAC National Environmental Laboratory
Accreditation Conference
NELAP National Environmental Laboratory
Accreditation Program
NH4 ammonium
NIST National Institute of Standards
NO2 nitrite
NO3 nitrate
NRSA National Rivers and Streams Assessment
NIL no template control
NTU Nephelometric Turbidity Units
OD optical density
OSHA Occupational Safety and Health
Administration
PBS phosphate buffered saline
PCB polychlorinated biphenyl
PctDIFF percent difference
PDE percent disagreement in enumeration
PCR polymerase chain reaction
PE performance evaluation
PES performance evaluation samples
PHab physical habitat
P-M Palmer-Maloney (P-M) count
PDE percent difference in enumeration
PSE percent sorting efficiency
PT performance testing
PTD percent taxonomic disagreement
QA quality assurance
QAPP Quality Assurance Project Plan
QA/QC quality assurance/quality control
QC quality control
QCCS quality control check solution
QMP Quality Management Plan
Laboratory Operations Manual
Page iii of 224
qPCR quantitative polymerase chain reaction
QRG Quick Reference Guide
RL reporting limit
RMSE root mean square error
RO reverse-osmosis
RPD relative percent difference
RQM relative quantitation method
RSD relative standard deviation
RTH richest targeted habitat
S standard deviation
SO-S5 standards provided with microcystin kit
SFS Society of Freshwater Science
SEG Site Evaluation Guidelines
SiO2 silica
SO4 sulphate
SOPs Standard Operating Procedures
SPC sample processing control
S-R Sedgewick-Rafter count
SRM standard reference material
SS salmon sperm
1MB tetramethylbenzidine
TN total nitrogen
TOC total organic carbon
TP total phosphorus
TRANS transect
TSN taxonomic serial number
TSS total suspended solids
TVS total volatile solids
LINK unknown
USGS United States Geological Survey
WSA Wadeable Streams Assessment
WQX Water Quality Exchange
52
LI-
LI-
-------
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 10 of 224
1.0 INTRODUCTION
This manual describes methods for analyses of the samples to be collected during the National Rivers
and Streams Assessment (NRSA), including quality assurance objectives, sample handling, and data
reporting. The NRSA is a probabilistic assessment of the condition of our Nation's rivers and streams and
is designed to:
Assess the condition of the Nation's rivers and streams
Establish a baseline to compare future rivers and streams surveys for trends assessments
Evaluate changes in condition from the 2004 Wadeable Streams Assessment
Help build State and Tribal capacity for monitoring and assessment and promote collaboration
across jurisdictional boundaries
This is one of a series of water assessments being conducted by states, tribes, the U.S. Environmental
Protection Agency (EPA), and other partners. In addition to rivers and streams, the water assessments
will also focus on coastal waters, lakes, and wetlands in a revolving sequence. The purpose of these
assessments is to generate statistically-valid reports on the condition of our Nation's water resources
and identify key stressors to these systems.
The goal of the NRSA is to address two key questions about the quality of the Nation's rivers and
streams:
What percent of the Nation's rivers and streams are in good, fair, and poor condition for key
indicators of water quality, ecological health, and recreation?
What is the relative importance of key stressors such as nutrients and pathogens?
EPA selected sampling locations using a probability based survey design. Sample surveys have been used
in a variety of fields (e.g., election polls, monthly labor estimates, forest inventory analysis) to determine
the status of populations or resources of interest using a representative sample of a relatively few
members or sites. Using this survey design allows data from the subset of sampled sites to be applied to
the larger target population, and assessments with known confidence bounds to be made.
With input from the states and other partners, EPA used an unequal probability design to select 900
wadeable streams and 900 non-wadeable rivers. To estimate change from the 2004 Wadeable Streams
Assessment (WSA), 450 of the 900 wadeable sites were selected using an unequal probability design
from the WSA original sites. Field crews will collect a variety of measurements and samples from
randomly predetermined sampling reaches (located with an assigned set of coordinates), and from
randomized stations along the sampling reach.
g
b
Q
o
10
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 11 of 224
2.0 GENERAL LABORATORY GUIDELINES
2.1 Responsibility and Personnel Qualifications
All laboratory personnel shall be trained in advance in the use of equipment and procedures used for
the standard operating procedure (SOP) in which they are responsible. All personnel shall be responsible
for complying with all of the QA/QC requirements that pertain to the samples to be analyzed. Each lab
should follow its institutional or organizational requirements for instrument maintenance. Specific lab
qualification documentation required for analysis is contained in the Quality Assurance Project Plan
(QAPP).
2.2 Roles and Contact Information
The EPA Headquarters Project Management Team consists of the Project Leader, Alternate Project
Leaders, Project QA Lead, and Laboratory Review Manager. The Team is responsible for overseeing all
aspects of the project and ensuring technical and quality assurance requirements are properly carried
out. The Team is the final authority on all decisions regarding laboratory analysis.
The NARS Information Management (IM) Coordinator tracks the location of each NRSA 2013-2014
sample that involves post-processing. The coordinator will be the labs main point of contact in regards
to sample tracking and data submission.
Table 2.1 Contact information
Title
EPA HQ NRSA Project Lead
EPA HQ NRSA Project QA
Lead
EPA HQ NRSA Laboratory
Review Manager
Information Management
Center Coordinator
Name
Ellen Tarquinio, OW
Sarah Lehmann, OW
Kendra Forde, OW
Marlys Cappaert, SRA
International Inc.
Contact Information
tarquinio.ellen@epa.gov
202-566-2267
lehmann.sarah@epa.gov
202-566-1379
kendra.forde@epa.gov
202-564-0417
cappaert.marlys@epa.gov
541-754-4467
541-754-4799 (fax)
2.3 Sample Tracking
Samples are collected by a large number of different field crews during the index period (May through
September). The actual number of rivers and streams sampled on a given day will vary widely during this
time. Field crews will submit electronic forms when they have shipped samples and the NARS IM Center
will input each sample into the NARS IM database. Laboratories can track sample shipment from field
crews by accessing the NARS IM database. Participating laboratories will be given access to the NARS IM
system, where they can acquire tracking numbers and information on samples that have been shipped
to them by field crews (either by overnight shipment for perishable samples or batch shipments for
preserved samples). Upon sample receipt, the laboratory must immediately log in to the database and
confirm that samples have arrived. Overnight samples may not be loaded into the database prior to
sample arrival, but should be tracked by the laboratory and receipt information inputted into the
database when sample information is loaded. Each lab will make arrangements with the NARS IM
Coordinator, listed above, to ensure access is granted.
>
o
QC
O
en
5
(D
11
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 12 of 224
When the samples arrive from the field crews, laboratories should also receive tracking forms in the
shipment (refer to the NRSA 2013-2014 FOM). These forms will list the samples that should be included
in the shipment. Laboratory personnel should cross check the forms with the samples received to verify
that there are not any inconsistencies. If any sample is missing or damaged, contact the NARS IM
Coordinator immediately.
2.4 Reporting
All labs must provide data analysis information to the HQ Project Management Team and the NARS IM
Center by March 30, 2015 or as stipulated in contractual agreements. These reports must include the
following information:
Sample Type (indicator)
Site ID (ex: CAS9-0918)
Sample ID (ex: 999000)
Pertinent information to the indicator
Metadata for all fields
See Appendix C for reporting templates that labs will submit electronically.
The submitted file name must state the following:
Indicator name (ex: microcystin)
Date of files submission to NARS IM Center by year, month, and day (ex: 2013_11_01)
Lab name (ex: MyLab)
Combined, the file name would look as follows: WaterChemistry_2013_ll_01_MyLab.xlsx
As specified in the QAPP, remaining sample material and specimens must be maintained by the EPA's
designated laboratory or facilities as directed by the NRSA 2013-2014 Project Lead. All samples and raw
data files (including logbooks, bench sheets, and instrument tracings) are to be retained by the
laboratory for 3 years or until authorized for disposal, in writing, by the EPA Project Lead. Deliverables
from contractors and cooperators, including raw data, are permanent as per EPA Record Schedule 258.
EPA's project records are scheduled 501 and are also permanent.
>
o
QC
O
CD
5
(D
12
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 13 of 224
3.0 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-ADDATest Kits
(Figure 3.1; "kits"). Each kit is an enzyme-linked immunosorbent assay (ELISA) for the determination of
microcystins and nodularins in water samples. Microcystins refers to the entire group of toxins, all of the
different congeners, rather than just one congener. Algae can produce one or many different congeners
at any one time, including Microcystin-LR (used in the kit's calibration standards), Microcystin-LA, and
Microcystin-RR. The different letters on the end signify the chemical structure (each one is slightly
different) which makes each congener different.
Figure 3.1 Microcystin: Abraxis microcystin test kit
(from James, page 3, 2010)
3.1 Summary of the Procedure
The procedure is an adaption of the instructions provided by Abraxis for determining total microcystins
concentrations using its ELISA-ADDA kits.3 For freshwater samples, the procedure's reporting range is
0.15 u.g/L to 5.0 u.g/L, although, theoretically, the procedure can detect, not quantify, microcystins
concentrations as low as 0.10 u.g/L. For samples with higher concentrations of microcystins, the
procedure includes the necessary dilution steps.
u
O
CL.
>
LO
LO
O
Z
=>
LO
b
O
u
a 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.
X
O
<
<
13
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 14 of 224
3.2 Health and Safety Warnings
The laboratory must require its staff to abide by appropriate health and safety precautions, because the
kit substrate solution contains tetramethylbenzidine (1MB) and the stop solution contains diluted
sulfuric acid. In addition to the laboratory's usual requirements such as a Chemical Hygiene Plan, the
laboratory must adhere to the following health and safety procedures:
1. Laboratory facilities must properly store and dispose of solutions of weak acid.
2. Laboratory personnel must wear proper personal protection clothing and equipment (e.g. lab
coat, protective eyeware, gloves).
3. When working with potential hazardous chemicals (e.g., weak acid), laboratory personnel must
avoid inhalation, skin contact, eye contact, or ingestion. Laboratory personnel must avoid
contacting skin and mucous membranes with the TMB and stopping solution. If skin contact
occurs, remove clothing immediately. Wash and rinse the affected skin areas thoroughly with
large amounts of water.
3.3 Definitions and Required Resources (Personnel, Laboratories, and
Equipment)
This section provides definitions and required resources for using the procedure.
3.3.1 Definitions
The following terms are used throughout the procedure:
Absorbance (A) is a measure of the amount of light in a sample. A standard statistical curve is used to
convert the absorbance value to the concentration value of microcystins.
Calibration Range is the assay range for which analysis results can be reported with confidence. For
undiluted samples, it ranges from the reporting limit of 0.15 u.g/L to a maximum value of 5.0 u.g/L.
Values outside the range are handled as follows. If the value is:
< 0.10 u.g/L, then the laboratory reports the result as being non-detected ("<0.10 u.g/L"). ^
Between 0.10 u.g/L and the reporting limit of 0.15 u.g/L (i.e., >0.10 u.g/L and <0.15 u.g/L), the ฃ
laboratory should record the value, but assign a QC code to the value (i.e., DATA_FLAG=J). ง
5.0 u.g/L, the laboratory must dilute and reanalyze the sample. >
CO
CO
Coefficient of Variation (CV): The precision for a sample is reported in terms of the percent CV of its <
absorbance values. To calculate the %CV, first calculate S (standard deviation) as follows: ^
lV2 i
L i = l
where n is the number of replicate samples, A/, is the absorbance measured for the /th replicate. Per >
Section 3.5.3, samples are evaluated in duplicate (i=l or 2); controls are either evaluated in duplicate or o
triplicate (i=l, 2, 3). A is the average absorbance of the replicates. Then, calculate %CV as: y
%CV =
S
xlOO
Dark or Dimly Lit: Away from sunlight, but under incandescent lighting is acceptable.
14
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 15 of 224
Detection Limit is the minimum concentration at which the analyte can be detected with confidence. In
other words, the outcome can be reported with confidence that it is greater than zero (i.e., present in
the sample). The detection limit is less than the reporting limit of 0.15 u.g/L at which the measured value
of the analyte can be reported with confidence. Also see "Sample-Specific Detection Limit."
Duplicates are defined as two aliquots of the same sample which are analyzed separately using identical
procedures. The results are used to evaluate the precision of the laboratory analyses. Per Section 3.5.3,
controls are evaluated in duplicate or triplicate (i.e., three aliquots).
NARS: National Aquatic Resource Surveys. The National Rivers and Streams Assessment (NRSA) is part of
the NARS program.
NARS Information Management System (NARS IM): The IM system established to support all surveys,
including NRSA, in the NARS program. The IM system is used to track the samples from field collection to
the laboratory.
NRSA: National Rivers and Streams Assessment. Freshwater samples will be collected during the field
stage of NRSA.
Relative Standard Deviation (RSD) is the same as the coefficient of variation (%CV). Because many of
the plate reader software programs provides the CV in their outputs, the procedure presents the quality
control requirement in terms of %CV instead of RSD.
Reporting Limit: For undiluted freshwater sample, the reporting limit is 0.15 u.g/L. A reporting limit is
the point at which the measured value of the analyte can be reported with confidence.
Standard Deviation (S) shows variation from the average
Sample-Specific Detection Limit: Most samples will have a sample-specific detection equal to the
method's detection limit of 0.1 u.g/L. For diluted samples, the sample-specific detection limit will be the
product of the method's detection limit of 0.1 u.g/L and the dilution factor. Typical values for the dilution
factor will be 10 or 100.
3.4 General Requirements for Laboratories ฃ
3.4.1 Expertise ฃ
To demonstrate its expertise, the laboratory shall provide EPA with one or more of the following: cz
Q_
Memorandum that identifies the relevant services that the laboratory provided for the National <
CO
Aquatic Resource Surveys in the past five years. <
O
Documentation detailing the expertise of the organization, including professional certifications "z.
for water-related analyses, membership in professional societies, and experience with analyses ^
that are the same or similar to the requirements of this method. ^
^
3.4.2 Quality assurance and quality control requirements ฃ
u
To demonstrate its expertise in quality assurance and quality control procedures, the organization shall O
provide EPA with copies of the quality-related documents relevant to the procedure. Examples include ^
Quality Management Plans (QMP), QAPPs, and applicable Standard Operating Procedures (SOPs). -
^_
To demonstrate its ongoing commitment, the person in charge of quality issues for the organization g
shall sign the NRSA QAPP Certification Page. ^j
15
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 16 of 224
3.4.3 Personnel
Laboratory Technician: This procedure may be used by any laboratory technician who is familiar with
the NRSA 2013-14 QAPP, and this procedure in the NRSA 2013-14 LOM (which differs from the Abraxis
instructions). The laboratory technician also must be familiar with the use of a multichannel pipette and
plate readers.
External QC Coordinator is an EPA staff person who is responsible for selecting and managing the "QC
contractor." To eliminate the appearance of any inherent bias, the QC contractor must be dedicated to
QA/QC functions, and thus, must not be a primary laboratory or a field sampling contractor for NRSA.
The QC contractor is responsible for complying with instructions from the External QC Coordinator;
coordinating and paying for shipments of the performance samples to participating laboratories;
comparing immunoassay results from the laboratories; and preparing brief summary reports.
3.4.4 Equipment/Materials
The procedures require the following equipment and information:
Abraxis ADDA Test Kit, Product #520011 (see items in Section 3.6.2)
Adhesive Sealing Film (Parafilm) for Micro Plates (such as Rainin, non-sterile, Cat. No. 96-SP-
100): Used to cover plates during incubation.
Data Template-See Figure 3.2
Distilled or Deionized Water: For diluting samples when necessary.
ELISA evaluation software
Glass scintillation, LC, vials (two vials of 2 mL each)
Glass vials with Teflon-lined caps of size:
o 20 mL
o 4 mL (for dilutions)
Multichannel Pipette & Tips: A single-channel and an 8-channel pipette are used for this
method. ^
Norm-ject syringes (or equivalent) o
LJJ
Paper Towels: For blotting the microtiter plates dry after washing. ^
Permanent Marker (Sharpie Fine Point): For labeling samples, bottles, plates and covers. a.
* >
Plate Reader (e.g., Metertech Model M965 AccuReader; ChroMate ; or equivalent readers with <
CO
software to read the microtiter plates and measure absorbances). <
Reagent Reservoirs (e.g., Costar Cat Number 4870): Plain plastic reservoir for reagents that ^
accommodate the use of a multi-channel pipette. ^
Test tubes: For dilutions, if needed. ^
Timer: For measuring incubation times. P
Vortex Genie: For mixing dilutions. u
Whatman Glass fiber syringe filter (25mm, GF 0.45 u.m filter) ^
ง
3.5 Sample Receipt -^
x
Field crews hold the microcystins samples on ice while in the field and then pack the samples in ice for O
delivery to a central facility ("batching laboratory") or the State's laboratory. The batching and State ^
laboratories freeze the samples upon receipt. Periodically, the batching laboratory ships samples to the 53
<
16
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 17 of 224
microcystins laboratory. The batching and microcystins laboratory may retain the frozen samples for
several months before analysis.
Because EPA initiates tracking procedures designed to recover any missing shipment, the laboratory
personnel responsible for tracking samples must start the following login steps within 24 clock hours of
receiving a delivery.
1. Report receipt of samples in the NARS IM sample tracking system (within 24 clock hours).
2. Inspect each sample THE SAME DAY THEY ARE RECEIVED:
a. Verify that the sample IDs in the shipment match those recorded on the:
i. Chain of custody forms when the batching laboratory sends the samples to the
microcystins laboratory; or
ii. Sample tracking form if the field crew sends the shipment directly to the State
laboratory.
b. Record the information in Table 3.1 into NARS IM, including the Condition Code for each
sample:
i. OK: Sample is in good condition
ii. C: Sample container was cracked
iii. L: Sample container is leaking
iv. ML: Sample label is missing
v. NF: Sample not frozen
c. If any sample is damaged or missing, contact the EPA HQ Laboratory Review Manager to
discuss whether the sample can be analyzed. (See contact information in Table 2.1).
3. Store samples in the freezer until sample preparation begins.
4. Maintain the chain of custody or sample tracking forms with the samples.
Table 3.1 Microcystin: required data elements- login
FIELD FORMAT DESCRIPTION
LAB ID
DATE RECEIVED
SITE ID
VISIT NUMBER
SAMPLE ID
DATE COLLECTED
CONDITION CODE
text
MMDDYY
text
numeric
numeric
MMDDYY
text
Name or abbreviation for QC laboratory
Date sample was received by lab
NRSA site id as used on sample label
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
OK
C
L
ML
NF
0
Definition
Sample is in good condition
Sample container is cracked
Sample or container is leaking
Sample label is missing
Sample is not frozen
Other quality concerns, not identified above
U
O
QC
CL
<
CO
l/l
<
O
O
QC
U
X
O
<
I
<
3.6 Procedure
The following sections describe the sample and kit preparation and analysis.
17
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 18 of 224
3.6.1 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 25ฐ C). Swirl the sample to check for
ice crystals. At this temperature, no ice crystals should be present in the sample.
c. Shake well to homogenize the sample, then transfer 10 ml to an appropriately labeled clean
20 ml glass vial.
3. Second freeze-thaw cycle:
a. Freeze the vial.
b. Keep the large sample bottle (from the 500 ml initial sample) frozen for future use.
c. Thaw the sample vial contents to room temperature.
4. Third freeze-thaw cycle:
a. Freeze the vial.
b. Thaw the vial contents to room temperature.
c. Filter the vial contents through a new, syringe filter (0.45 u.m) into a new, labeled 20 ml
glass scintillation vial. Norm-ject syringes and Whatman Glass fiber syringe filters (25mm, GF
0.45 u.m filter) or other similar alternative are acceptable. One new syringe and filter should
be used per sample.
3.6.2 Kit Preparation
The technician prepares the kits using the following instructions: LU
Cฃ.
1. Check the expiration date on the kit box and verify that it has not expired. If the kit has expired, Q
LJJ
discard and select a kit that is still within its marked shelf life. (Instead of discarding the kit, <=>
consider keeping it for training activities.) CL
2. Verify that each kit contains all of the required contents: <
i/i
<
Microtiter plate O
Standards (6) referenced in this procedure as follows with the associated concentration: ^
o SO: 0 u.g/L I
o SI: 0.15 u.g/L ^
o S2: 0.40 u.g/L, ฃ
o S3: 1.0 u.g/L O
o S4: 2.0 u.g/L -
o S5: 5.0 ug/L ~
Kit Control (KC): 0.75 ug/L x
Antibody solution [3
<
Anti-Sheep-HRP Conjugate (J
18
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Wash Solution 5X Concentrate
Color Solution
Stop Solution
Diluent
Laboratory Operations Manual
Page 19 of 224
3. If any bottles are missing or damaged, discard the kit. This step is important because Abraxis has
calibrated the standards and reagents separately for each kit.
4. Adjust the microtiter plate, samples, standards, and the reagents to room temperature.
5. Remove 12 microtiter plate strips (each for 8 wells) from the foil bag for each kit. The plates
contain 12 strips of 8 wells. If running less than a whole plate, remove unneeded strips from the
strip holder and store in the foil bag, ziplocked closed, and place in the refrigerator.
6. Store the remaining strips in the refrigerator (4-8ฐ C).
7. Prepare a negative control (NC) using distilled water
8. The standards, controls, antibody solution, enzyme conjugate, color solution, and stop solutions
are ready to use and do not require any further dilutions.
9. Dilute the wash solution with deionized water. (The wash solution is a 5X concentrated
solution.) In a 1L container, dilute the 5X solution 1:5 (i.e., 100 ml of the 5X wash solution plus
400 ml of deionized water). Mix thoroughly. Set aside the diluted solution to wash the
microtiter wells later.
10. Handle the stop solution containing diluted H2SO4 with care.
3.6.3 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 3.2) to use as reference when loading the standards, controls, and
samples as described in the next step. Templates contain rows, labeled with a marking pen, of
strips of 8 wells that snap into the blank frame. (If the laboratory wishes to use a different
template, provide a copy to the EPA HQ Laboratory Review Manager for approval prior to first
use. (See Section 2 of the manual for contact information.)
4. Using the 100-u.L pipette, add 50 ul, 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 u.g/L),
the kit control (0.75 ul), and negative control, in pairs, starting in the well in the upper left-hand
corner of the kit as shown in Figure 3.2. Verify that the software displays the same template or
make any necessary corrections.
cc.
u
O
CL
>
CO
CO
O
Z
=)
O
QC
U
X
O
<
i
<
19
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 20 of 224
c
D
E
F
G
H
SI
SI
S2
S2
S3
S3
S5
S5
KC
KC
KC
NC
Ul
Ul
U2
U2
U3
U3
U5
U5
U6
U6
U7
U7
U9
U9
U10
U10
Ull
Ull
U13
U13
U14
U14
U15
U15
U17
U17
U18
U18
U19
U19
U21
U21
U22
U22
U23
U23
U25
U25
U26
U26
U27
U27
U29
U29
U30
U30
U31
U31
U33
U33
U34
U34
U35
U35
U37
U37
U38
U38
U39
U39
Figure 3.2 Microcystin: sample template
Key:
SO-S5 = Standards;
KC = Control supplied with Kit (i.e., Kit Control);
NC = Negative Control;
U = Unknown (sample collected by the field crew);
5. Add 50 u.L of the pink antibody solution to each well using the multi-channel pipettor and a
reagent reservoir. Use dedicated reagent reservoirs for each reagent to avoid contamination
from one reagent to another.
6. Place the sealing Parafilm over the wells.
7. Manually mix the contents by moving the strip holder in a rapid circular motion on the benchtop
for 30 seconds. Be careful not to spill the contents.
8. Place the plate in an area away from light for 90 minutes.
9. After 90 minutes, carefully remove the Parafilm.
10. Empty the contents of the plate into the sink, pat inverted plate dry on a stack of paper towels,
and then wash the wells of the plate three times with 250 u.L 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 enzyme conjugate solution to all wells using the multi-channel pipettor.
12. Cover the wells with Parafilm.
13. Manually mix the contents by moving the strip holder in a rapid circular motion on the benchtop
for 30 seconds. Be careful not to spill the contents.
14. Place the strip holder in an area away from light for 30 minutes.
15. 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.
16. Add 100 piL of color solution to the wells using the multi-channel pipette and reagent reservoir.
This color solution will make the contents have a blue hue.
17. Cover the wells with Parafilm.
18. Manually mix the contents by moving the strip holder in a rapid circular motion on the benchtop
for 30 seconds. Be careful not to spill the contents.
19. Place the plate in an area away from light for 20 minutes.
20. After 20 minutes, remove the Parafilm and add 50 piL of stopping solution to the wells in the
same sequence as for the color solution. This will turn the contents a bright yellow color. After
adding the stopping solution, read the plate within 15 minutes.
cc.
U
O
CL
>
CO
CO
O
Z
=)
O
cc
U
X
O
<
i
<
20
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 21 of 224
21. Within 15 minutes of adding the stopping solution, use the microplate ELISA photometer (plate
reader) to determine the absorbance at 450 nm. The software (i.e., commercial ELISA evaluation
program) calculates the absorbance and concentration values of the samples from the
calibration curve and the average values for each pair. Use a 4-parameter standard curve fit to
determine the concentrations.
22. Dispose of solution in plates in a lab sink. Rinse plates and sink with water to dilute the weak
acid present.
23. Perform QC evaluations of the data as follows:
a. If the following failures occur, then the laboratory must reanalyze all samples in the
analytical run:
Standard curve with a correlation coefficient of less than 0.99 (i.e., R<0.99)
Standards SO-S5 must have decreasing absorbance values. First, calculate the average
values for each standard. That is, if A, is the absorbance average for S,, then the
absorbance averages must be:
iii. A0> Ai> A2> A3> A4>A5
iv. The average absorbance of the standard SO less than 0.8 (i.e., A0 < 0.8).
v. Two or more negative control samples with detectable concentrations of microcystins
(i.e., values > 0.1 u.g/L). If this occurs, then evaluate possible causes (e.g., cross-
contamination between samples), and if appropriate, modify laboratory processes
before the next analytical run.
vi. Results for control samples of outside the acceptable range of 0.75 +/- 0.185 ppb. That
is, results must be between 0.565 and 0.935.
b. If either, or both, of the following failures occur, then the sample must be reanalyzed
(maximum of two analyses, consisting of the original analysis and, if necessary, one
reanalysis):
i. The concentration value registers as HIGH (exceeds the calibration range). Dilute the ^
sample for the reanalysis per Section 3.6.4. LU
ii. The %CV > 15% between the duplicate absorbance values for a sample. ง
n
24. Record the results, even if the data failed the quality control requirements in #23b, for each well >
for unknown sample. ^
b. CONC contains the numeric concentration value. Two special cases: ^
\. Non-detected concentrations: If the sample is non-detected, then provide the sample- ti
specific detection limit which is 0.1 u.g/L if the sample is undiluted. See Section 3.6.4 for Q
calculating the sample-specific detection limit for a diluted sample. y
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. H
i
c. DATA FLAGS have codes for the following special cases: <
i. ND if the sample was non-detected; <
21
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 22 of 224
ii. J if the value is detected but at a level below the reporting limit of 0.15 u.g/L (for
undiluted samples);
iii. HI if the concentration value registers as HIGH (exceeds the calibration range).
d. QUALITY FLAGS have codes for the following special cases:
i. QCF if there is a QC failure per step 23 above. The QCF code must be used for all failures
to facilitate data analysis.
ii. Qfor any other quality issue (describe in COMMENTS)
e. DILUTION FACTOR is only required if the sample was diluted.
f. DUP AVG and DUP CV are required for duplicate samples and control samples (use all three
values if the controls are used in triplicate).
Table 3.2 Microcystin: required data elements - data submission
STAGE FIELD FORMAT DESCRIPTION
LOGIN
ANALYSIS
LAB ID
DATE RECEIVED
SITE ID
VISIT NUMBER
SAMPLE ID
DATE COLLECTED
CONDITION CODE
TECHNICIAN
KIT EXPIRE DATE
KIT ID
R2
TYPE
LOCATION
SALINITY
text
text
text
numeric
numeric
MMDDYY
text
text
MMDDYY
text
numeric
text
text
numeric
Name or abbreviation for QC laboratory
Date sample was received by lab
NRSA site ID code as recorded on sample label or tracking form
(blank if standard or control)
Sequential visits to site (1 or 2) (blank if standard or control)
6-digit Sample ID number as recorded on sample jar or tracking
form (blank if standard or control)
Date sample was collected (blank if standard or control)
Sample condition upon arrival at the laboratory (blank if standard
or control)
Flag Definition
Blank or N Not a sample (blank, standard, or control)
OK Sample
C Sample
L Sample
ML Sample
NF Sample
is in good condition
container is cracked
or container is leaking
label is missing
is not frozen
Name or initials of technician performing the procedure
Expiration date on kit box
Kit identification code. If one does not exist, assign a unique code
to each kit.
R2 from curve fit to the average absorbance values for the
standards. Value is between 0 and 1.
Type of solution being tested in the well
Code
KC
NC
SO,S1, S2,S3, S4, S5
U
Definition
Kit Control
Negative Control
Standard
Sample of unknown concentration
Location of well in the kit (e.g., B5 would be the fifth well from the
left in the second row B)
If the sample vial has the salinity marked on the vial, record the
value in units of parts per thousand. Otherwise, leave blank.
QC
U
o
QC
CL
<
CO
l/l
<
O
O
QC
U
X
o
<
I
<
22
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 23 of 224
CONC
ABSORBANCE
DILUTION FACTOR
CV_ABSORB
AVG_ABSORB
AVG_CONC
DATA FLAG (if
appropriate)
QUAL FLAG
COMMENTS
numeric
numeric
numeric
numeric
numeric
numeric
text
QCF/Q
text
Concentration or sample-specific detection limit of contents of
well in u.g/L. Sample-specific detection limit should be 0.1 u.g/L if
the sample hasn't been diluted.
Absorbance value
10, 100, etc for number of times the sample was diluted. If not
diluted, leave blank or record 1
Calculated %CV of duplicate values of absorbance for a sample.
Only calculated forTYPE=U, KC, or NC. Enter %CV. Value is
between 0 and 100%.
Calculated average of absorbance values for a sample. Only
provided for TYPE=U, KC, NC, or SC. Average value of the original
sample and its duplicate (or replicates for KC and NC).
Calculated average of concentration values for a sample.
Substitute 0.15 u.g/Lfor any result recorded as <0.15 u.g/L
Data qualifier codes associated with specific identifications of
voucher samples. These codes provide more information that
those used when reporting receipt of samples. A technician may
use alternative or additional qualifiers if definitions are provided
as part of the submitted data package (e.g., as a separate
worksheet page of the data submission file).
Flag
ND
HI
J
QCF
0
Definition
Concentration below detection. Unless the sample was
diluted, the concentration will be 0.1 u.g/L
Result indicated that a high concentration (i.e., outside
calibration range)
Concentration above detection but below reporting limit.
Without dilution, these values are between 0.1 and 0.15
Hg/L
QC failure
Other quality concerns, not identified above
Explanation for data flag(s) (if needed) or other comments.
cc.
u
o
CL
>
CO
CO
O
Z
=)
3.6.4 Dilutions (if needed)
Dilutions if needed are prepared as follows (using clean glass tubes):
1:10 dilution
a. Add 900 ul of distilled water to a clean vial. (Note: Dilutions may also be made using the
kit's diluent rather than distilled water.)
b. Pipette 100 ul from the sample into the vial. (To provide more accurate dilutions and less
chance of contaminating the diluent, the diluent should be added to the vial before the
sample.)
c. Mix by vortexing.
d. Multiply final concentration and Abraxis' detection limit of 0.1 u.g/L by 10 to obtain the
sample-specific detection limit of 1.0 u.g/L.
1:100 dilution
O
QC
U
X
o
<
i
<
23
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 24 of 224
a. Add 3.96 ml of distilled water to a clean, appropriately labeled glass vial. (Note: Dilutions
may also be made using the kit's diluent rather than distilled water.)
b. Vortex the sample to mix thoroughly, then pipette 40 ul from the sample and add to the
water (or diluent) in the appropriate labeled vial. Vortex.
c. Multiply the final concentration and Abraxis' detection limit of 0.1 u.g/L by 100 to obtain the
sample-specific detection limit of 10 u.g/L.
Other dilutions can be calculated in the same manner as #1 and #2 if needed.
3.7 Quality Measures
This section describes the quality assurance and quality control measures used to ensure that the data
will meet NRSA's requirements.
3.7.1 Assistance Visits
Assistance visits are intended to familiarize EPA with actual procedures being implemented by different
laboratories; and to ensure a clear and consistent understanding of procedures and activities by both
EPA and the laboratories. If EPA decides to conduct an assistance visit, a qualified EPA scientist or
contractor will administer a checklist based upon the steps described in this chapter.
3.7.2 QC Samples
During the course of the survey, the External QC Coordinator will instruct the QC contractor to 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 NRSA reference 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 QC contractor 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.
3.7.3 Summary of QA/QC Requirements
Table 3.3 provides a summary of the quality control requirements described in Sections 3.5 and 3.6.
Table 3.3 Microcystin: quality control - sample analysis
Quality Control
Activity
Kit - Shelf Life
Description and Requirements
Is within its expiration date listed on kit box.
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.
Corrective Action
If kit has expired, then discard or set
aside for training activities.
If any bottles are missing or damaged,
discard the kit.
QC
u
o
QC
CL
<
CO
l/l
<
O
O
QC
U
X
o
<
I
<
Calibration
All of the following must be met:
If any requirement fails:
24
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 25 of 224
Standard curve must have a correlation
coefficient of >0.99;
Average absorbance value, A0, for SO must
be >0.80; and
Standards SO-S5 must have decreasing
average absorbance values. That is, if A, is
the average of the absorbance values for
Si, then the absorbance average values
must be: A0> AI> A2> A3> A4>A5
Results from the analytical run
are not reported.
All samples in the analytical run
are reanalyzed until calibtration
provides acceptable results.
Kit Control
The average concentration value of the duplicates
(or triplicate) must be within the range of 0.75 +/-
0.185 u.g/L That is, results must be between 0.565
and 0.935.
Negative Control
The values for the negative control replicates must
meet the following requirements:
o All concentration values must be < 0.15
u.g/L (i.e., the reporting limit); and
o One or more concentration results must
be nondetectable (i.e., <0.10 u.g/L)
If either requirement fails:
Results from the analytical run
are not reported
The lab evaluates its processes,
and if appropriate, modifies its
processes to correct possible
contamination or other problems.
The lab reanalyzes all samples in
the analytical run until the
controls meet the requirements.
Sample
Evaluations
All samples are run in duplicate. Each duplicate pair
must have %CV<15% between its absorbance
values.
If %CV of the absorbances for the
sample>15%, then:
Record the results for both
duplicates.
Report the data for both duplicate
results as Quality Control Failure
"QCF"; and
Re-analyze the sample in a new
analytical run. No samples are to
be run more than twice.
If the second run passes, then the
data analyst will exclude the data
from the first run. If both runs fail, the
data analyst will determine if either
value should be used in the analysis
(e.g., it might be acceptable to use
data if the CV is just slightly over
15%).
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 u.g/Lfor undiluted samples), then the
requirement is met.
If one or both duplicates register as
'HIGH,' then the sample must be
diluted and re-run until both results
are within the calibration range. No
samples are to be run more than
twice.
External Quality External QC Coordinator, supported by QC
Control Sample 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
25
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 26 of 224
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.
3.8 Sample and Record Retention
The laboratory shall retain:
1. The sample materials, including vials, for a minimum of 3 years from the date the EPA publishes
the final report. During this time, the laboratory shall freeze the materials. The laboratory shall
periodically check the sample materials for degradation.
2. Original records, including laboratory notebooks and the reference library, for a minimum of 10
years from the date that EPA publishes the final report.
After the stated time periods, the laboratory shall follow its internal protocols for disposal.
3.9 References
Abraxis, "Microcystins-ADDA ELISA (Microtiter Plate)," Product 520011, R021412, Undated. Retrieved
January 2014 from
http://www.abraxiskits.com/uploads/products/docfiles/278 Microcystin%20PL%20ADDA%20users%20R120214.p
df.
Abraxis, "Microcystin-ADDA ELISA Kit, Detailed Procedure," Undated. Retrieved January 2014 from
http://www.abraxiskits.com/uploads/products/docfiles/253 PN520011FLOW.pdf.
James, R., et al., "Environmental Technology Verification Report: Abraxis Microcystin Test Kits: ADDA
ELISA Test Kit; DM ELISA Test Kit; Strip Test Kit," in Environmental Technology Verification System Center
2010. Retrieved March 2013 from http://nepis.epa.gov/Adobe/PDF/P100EL6B.pdf
QC
u
o
QC
CL
<
CO
l/l
<
O
O
QC
U
X
o
<
I
<
26
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 27 of 224
4.0 BENTHIC MACROINVERTEBRATES
4.1 Introduction
This chapter describes the steps for identifying benthic macroinvertebrate organisms in samples
collected in the National Rivers and Streams Assessment (NRSA). The procedure is adapted from
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 rivers and streams.
Field crews preserve samples in the field with ethanol and ship them to a central holding facility or
directly to the laboratory. Samples are sent to the laboratory on a regular basis to avoid delays in
processing and sample identification.
In the following discussion, Sections 4.1, 4.2, 4.3, and 4.4 summarize the procedure; health and safety
concerns; and required resources. Section 4.5 provides the steps for acknowledging sample receipt.
Section 4.6 and Section 4.7 provide the steps for preparing and sorting the sample. Sections 4.8 - 4.10
provide the steps for the taxonomy identification, data entry, and sample and record retention. Section
4.11 describes EPA's external review of laboratory operations. Section 4.12 identifies references used in
developing the procedure.
CC
CD
o
cc
u
u
27
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 28 of 224
Table 4.8 summarizes the quality control measures.
4.2 Summary of Method
The procedure is designed to pick and identify organisms from sediment samples. This section provides
a summary of the procedure and quality control measures.
The sorter evenly distributes each sample across a sorting tray(s), and then places an evenly divided grid
over the sample, usually 30 grids per tray ("Caton-style tray"). The sorter randomly selects a minimum of
three grids for sorting and picking organisms. The sorter continues until all grids are sorted or the sorter
has picked the 500th organism from a subsample. If a sorter reaches 500 organisms in the middle of a
subsample, then the sorter continues until that the subsample is completed. Figure 4.1 summarizes the
sorting process.
During the identification step, a taxonomist identifies the picked organisms to the target taxonomic
levels for the survey and discards materials that do not meet the identification criteria. If necessary, the
sorter repeats the sorting and identification process to ensure that the target of 500 organisms is
reached. The taxonomist also creates a reference collection with at least one organism from each genus
or lowest taxonomic level identified.
As part of the quality control measures, a second taxonomist will re-identify a subset of the samples to
quantify enumeration and taxonomic precision, or consistency, as percent difference in enumeration
(PDE) and percent taxonomic disagreement (PTD), to help target corrective actions, and ultimately to
help minimize problems during data analysis.
4.3 Health and Safety Warnings
In addition to the laboratory's usual requirements, the following health and safety procedures must be
followed for this procedure:
1. All proper personal protection clothing and equipment (e.g. lab coat, protective eyewear /
goggles) must be worn or applied.
2. When working with potential hazardous chemicals (e.g. 95% ethanol) or biological agents
(benthic organisms and sediments) avoid inhalation, skin contact, eye contact, or ingestion. If
skin contact occurs remove clothing immediately and wash / rinse thoroughly. Wash the
affected skin areas thoroughly with large amounts of soap and water.
4.4 Definitions and Required Resources (Laboratory, Personnel, and Equipment)
This section provides definitions and required resources for using this procedure. Section 4.4.1 defines
the terms used throughout the procedure. Section 4.4.2 describes the expertise required for each (s>
LU
laboratory using the procedure. Section 4.4.3 describes the roles and responsibilities of the personnel b
involved in the procedure. Section 4.4.4 identifies the equipment necessary to apply the procedure in ^
preparing, sorting, and identifying benthic macroinvertebrate organisms in samples. H
4.4.1 Definitions >
The following terms are used throughout the procedure: ง
u
Caton-style grid: Subsampling grid that consists of a solid outer tray, a mesh-bottomed inner tray, ^
evenly spaced squares (e.g., 30 or 36), a square "cookie cutter" and a scoop. y
i
Dissecting microscope: Microscope that is configured to allow low magnification of three-dimensional ^
objects that are larger or thicker than the compound microscope can accommodate. co
28
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 29 of 224
Distinct taxa: Data analysts use the number of distinct (i.e., unique) taxa within a given sample to
evaluate the richness associated with the sample location. The distinctness attribute is assessed sample
by sample, and not across all samples. To facilitate the data analyses, the database includes an
additional variable ("flag") that is used for the first identification of a particular taxon in a sample.
Section 4.8 provides the steps used to identify which taxa are flagged.
Good quality digital photograph: Good quality means that other taxonomists can readily identify the
taxon from one or multiple photographs and the library can readily locate the photographs. To ensure
that the photographs meet these objectives, the image must be:
Taken through the microscope at a high enough resolution so that the key diagnostic features
are distinguishable and clear. Include all features that would be necessary for an experienced
taxonomist to identify the specimen, this may require multiple photographs and at different
magnifications.
Positioned so that it includes:
o Only one taxon in the photo. If necessary, the laboratory may edit (e.g., crop) the digital
photograph and save the file with a new filename. Both the original and edited files
must be included in the digital library.
o A scale bar or measurements in an appropriate location to indicate the size of the
specimen.
o One specimen that lies flat on the surface instead of tilted (to the extent practicable).
Saved using a format that preserves the image in the highest resolution possible.
Saved with a filename that is consistent within the digital library and shall include the following
elements in the order listed below:
o NRSA2 (for the second NRSA conducted in 2013-2014)
o Laboratory name (or abbreviation)
o Sample number
o Taxa name
o Magnification (if applicable, otherwise indicate no magnification as "Ix")
o Date (format YYYYMMDD) that the photograph was taken.
o Appendage of "e" if the photograph was edited (e.g., cropped).
For example, on September 8, 2013, laboratory ABC identified the specimen in sample 1234 to
be a Homoeothrixjanthina and took a digital photograph at a resolution of 40x and then
cropped the photograph to eliminate extraneous material. The filenames of the original and
edited photographs would be: NRSA2_ABC_1234_ homoeothrixjanthina_40x_20130908.gif and
NRSA2_ABC_1234_ homoeothrixjanthina_40x_20130908e.gif.
^
Grid: Each individual square within the Caton tray ง
u
Inorganic material: Material that is not part of the animal or vegetable kingdom (e.g., gravel, sand, silt) ^
Integrated Taxonomic Information System (ITIS): Database with standardized, reliable information on ^
species nomenclature and their hierarchical taxonomic classification. EPA has incorporated the ITIS ^
information into classifications used by WQX. m
29
ft
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 30 of 224
NARS: National Aquatic Resource Surveys. The National Rivers and Streams Assessment (NRSA) is part of
the NARS program.
NARS Information Management (IM) System: The IM system established to support all surveys,
including NRSA, in the NARS program. The IM system is used to track the samples from field collection to
the laboratory.
NRSA: National Rivers and Streams Assessment. The samples were collected during the field stage of
NRSA.
Organic material: Material that is capable of decay or the product of decay (e.g., leaves, sticks, algae).
Percent sorting efficiency (PSE): Number of organisms recovered by sorter (A) compared to the
combined (total) number of recoveries by the sorter (A) and QC Officer (B) for a sample.
PSE = A xlOO
A + B (i)
Percent disagreement in enumeration (PDE): measure of taxonomic precision comparing the number of
organisms, ni, counted in a sample by the primary taxonomist with the number of organisms, n2,
counted by the internal or external QC taxonomist.
n, -n7
PDE = -xlOO
Percent taxonomic disagreement (PTD): measure of taxonomic precision comparing the number of
agreements (positive comparisons, compp0s) of the primary taxonomist and internal or external QC
taxnomists. In the following equation, N is the total number of organisms in the larger of the two counts.
PTD =
1-
comPpos
xlOO
(3)
Pickate: Sort residue from all grids originally sorted. This is the remaining material left from the grid,
after the sorter has removed all benthic macroinvertebrates. This could include small stones, sticks or
leaves, etc.
Quarter: A subsection of a grid. If a sorter finds many organisms within a grid, they will evenly divide the
grid into four "quarters".
Sorting Bench Sheet: Form used by the laboratory to record information about the sample during the
sample preparation and sorting procedure.
Primary laboratory: The laboratory that 1) sorts the sample; and 2) provides the first identification of
benthic macroinvertebrates in the sample. ฃ!
<
Secondary laboratory: The laboratory selected by the External QC Coordinator. It provides an co
independent identification of the benthic macroinvertebrates in the sample. The secondary laboratory ^
must provide QC taxonomists who did not participate in the original identifications for the sample. >
z
Subsample: Portion of the sample obtained by random selection and division. O
u
Target number of organisms: 500 per sample. If the 500th organism is counted in a grid (or quarter), <
then the macroinvertebrates in the rest of the grid (quarter) are picked and counted until that grid (or
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 31 of 224
Table 4.4 and Table 4.5 provide the target taxonomic levels for typical benthic macroinvertebrates
found in freshwater. EPA developed these tables considering its data analysis objectives, and thus, does
not require more specific identifications. In other words, taxonomists need only identify benthic
macroinvertebrates to the level specified in
CO
CC
LJJ
O
cc
u
u
31
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 32 of 224
Table 4.4 and Table 4.5.
Taxonomic Bench Sheet: Form used by the laboratory to record information about the sample during
the identification procedure.
Taxonomic Serial Number (TSN): stable and unique identifier that the Integrated Taxonomic
Information System (ITIS), Encyclopedia of Life, and/or Catalogue of Life couples with each scientific
name to serve as the "common denominator" for accessing information. ITIS numbers are preferred,
but when they are not available secondary sources are acceptable. WQX incorporates this identifier into
its "ExternallD" variable in the database. For entries that are not present in the other databases, WQX
assigns an identification number that should be used in the same manner as theTSNs. (This is notthe
identifier that should be used to identify organisms with this procedure. See WQX below for appropriate
identifier.)
Water Quality Exchange (WQX): a database framework that allows for data submissions and sharing of
water quality monitoring data, including data about benthic macroinvertebrates. It has assigned
identification numbers using information from ITIS, Encyclopedia of Life, and Catalogue of Life. The
following steps will retrieve the taxon information:
1. Access the WQX database at
http://www.epa.gov/storet/wqx/wqx getdomainvalueswebservice.html
2. Click on the link for "Taxon" listed near the bottom of the webpage.
3. Select "Save" to the file download popup box that asks "Do you want to open or save this file?"
4. Extract the saved file using software such as WinZip.
5. To open the extracted file in Excel:
a. When prompted, select "Open the file with the following stylesheet applied (select one):"
The selection should show the default of
"http://www.epa.gov/storet/download/domain_value_use.xsl"
b. Click "yes" to the warning about format (i.e., "The file you are trying to open...is in a
different format...Do you want to open the file now?")
6. Search for the taxon in the "Name" column. The "Description" column identifies a citation to a
webpage with more information about the taxon. The "ExternallD" column provides an
identifier commonly used by other references, and is left blank if none exists. Because the
"Uniqueldentifier" column has a value for every taxon listed, its identifier is the one that should
be used in recording the findings from this procedure.
4.4.2 Laboratory
i/i
LU
The procedure may be used by any laboratory that has expertise in each of the following areas: b
Cฃ
1. Analytical work: To demonstrate its expertise, the laboratory shall provide EPA with one or more ฃ
of the following: ^
a. Memorandum that identifies the relevant services that the laboratory provided for the -z.
National Aquatic Resource Surveys in the past five years. ง
u
b. Dated copy of relevant Accreditation or Certification (NELAC, ISO, state, etc.) for the <
laboratory and/or its experts who will perform and/or oversee the analyses. The u
accreditation must be for the entirety of analysis that the laboratory will be performing. H
LJJ
CO
32
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 33 of 224
c. Memorandum that describes the laboratory's participation in round robin studies and/or
performance studies.
d. Report of findings from an on-site technical assessment or audit.
2. Quality procedures:
a. To demonstrate its expertise in quality assurance and quality control procedures, the
laboratory shall provide EPA with copies of the quality-related documents relevant to the
procedure. Examples include Quality Management Plans (QMP), QAPPs, and applicable
Standard Operating Procedures (SOPs).
b. To demonstrate its ongoing commitment, the person in charge of quality issues for the
laboratory shall sign the NRSA 13/14 QAPP Certification Page.
3. Reporting standardized data. To demonstrate its expertise, the laboratory shall provide EPA with
a memorandum that confirms that the laboratory has a computerized Laboratory Information
Management System (LIMS) that is routinely used to track samples and record laboratory
results. The memorandum also shall confirm that the laboratory will use LIMS to record and
report results from the procedure.
4.4.3 Personnel
The procedure may be used by any person who has received training in processing and identification of
benthic macroinvertebrates; however, within a given laboratory, the work is generally parsed out to
several personnel:
Quality Control (QC) Officers provide oversight of daily operations, sample processing, monitors QC
activities to determine conformance, and conducts performance and systems audits of the procedures.
Documentation for the QC Officer meeting these requirements must be kept at the lab, and made
available to the EPA QC officer if requested. The following types of QC Officers are used by this
procedure:
Sorting QC Officer is an experienced sorter who:
Demonstrated an initial sorting proficiency (measured by PSE) of >90% in 5 consecutive
samples evaluated by an experienced sorter; and
Maintains a sorting proficiency of >90% in periodic QC checks (i.e., 1 in 10 samples).
Verifies the completeness of every Preparation/Sorting Bench Sheet to ensure header
information is correctly entered.
Checks sorted grids of all inexperienced laboratory personnel (those who have not achieved
CO
a >90% sorting efficiency) for missed organisms and records the number of missed ^
organisms in the appropriate field of the Bench Sheet. cz
Checks 1 in 10 of an experienced individual's samples, with a minimum of 1 NRSA sample. If H
the Sorting QC Officer performs the QC check more frequently, then the additional QC >
results must be submitted with the data for the required QC checks.
Determines the sorting efficiency for ea
is recorded on the Sorter Bench Sheet.
Determines the sorting efficiency for each sample and sorter. The sorter's sorting efficiency u
y
Internal Taxonomy QC Officer is an experienced taxonomist who: !=
33
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 34 of 224
Demonstrated an initial enumeration and identification proficiency (as measured by
PDE<5%and PTD<15%.
Maintains enumeration and identification proficiency in periodic QC checks (i.e., 1 in 10
samples with a minimum of one sample checked).
External QC Coordinator is an EPA staff person. Alternatively, the External QC Coordinator may
be a contractor that has not previously been involved with field sampling and/or benthic
macroinvertebrate analyses. Because the assigned duties are primarily logistical in nature, the
QC Coordinator is not required to have laboratory experience, although such experience would
be preferable.
External QC Taxonomists, are selected by the External QC Coordinator, and have demonstrated
expertise and experience to be used as a quasi "gold standard" for taxonomic evaluations.
Taxonomists are trained, and have considerable experience, in identifying benthic macroinvertebrates,
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. EPA prefers, but does not require, that the
taxonomists are certified by the Society of Freshwater Science (SFS). Each laboratory must submit the
resume or curriculum vitae for the taxonomists who identify benthic macroinvertebrates for the NRSA
samples to the EPA Project QC Officer.
Sorters are laboratory technicians who have basic training in laboratory procedures; and training in the
use of the Caton-style tray. An "experienced" sorter is one that has achieved >90% sorting efficiency in 5
consecutive samples. An experienced sorter can serve as the Sorting QC Officer.
4.4.4 Equipment/Materials
The procedure requires the following equipment and materials for sample preparation (subsampling),
sorting, and taxonomic identifications.
4.4.4.1 Sample Preparation (Subsampling) and Sorting Equipment/Materials
U.S. 35 sieve (500 u.m)
Round buckets
Standardized gridded screen (40 Mesh (380-u.m openings, T304 stainless steel wire, 34GA
(0.010"))
Mesh screen, square grids (e.g., 30 squares of 6 cm2 each) with white plastic holding tray (e.g.,
Caton tray, 30cm x 36cm, 4cm deep)
6-cm scoop [ฃ
6-cm2 metal dividing frame ("cookie cutter") <
White plastic or enamel pan (6" x 9") for sorting ฃ
Scissors 2j
>
Teaspoon ^
O
Permanent ink pen (e.g Pigma Micronฎ pen) g
Dropper ^
Fine-tipped forceps (watchmaker type, straight and curved)
Vials with caps or stoppers ^
LJJ
CO
34
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 35 of 224
Sample labels for vials
70-80% ethanol ( denatured or non-denatured)
Sorting Bench Sheet or other Sheet or database approved by EPA's External QC Coordinator
Stereo zoom microscope (6-10X magnification)
4.4.4.2 Taxonomy Identification Equipment/Materials
Stereo dissecting microscope with fiber optics light source (50-60X magnification)
Compound microscope (10, 40, and 100X objectives, with phase-contrast capability)
Digital camera with high resolution capability mounted on a microscope
Petri dishes
Microscope slides (1" x 3" flat, precleaned)
Cover slips (appropriately sized)
CMCP-10 (or other appropriate mounting medium)
Permanent ink pen (e.g Pigma Micronฎ pen)
Dropper
Fine-tipped forceps (watchmaker type, straight and curved)
Vials with caps or stoppers
Sample labels for vials
70 - 80% non-denatured ethanol in plastic wash bottle
Taxonomic Bench Sheet ( provided to each participating laboratory by EPA)
Hand tally counter
4.5 Sample Receipt
Under U.S. regulations, samples preserved in ethanol are classified as "Dangerous Goods" and must be
shipped according to hazardous material shipping requirements and regulations. Laboratory personnel
receiving the shipment must be certified to handle hazardous material. Because EPA initiates tracking
procedures designed to recover any missing shipment, the laboratory personnel should start the
following login steps within 24 clock hours of receiving a delivery.
1. Record receipt of samples in the NARS IM system (within 24 clock hours) and the laboratory's
Information Management System (LIMS). Assign the appropriate chronological bench number to
each sample.
2. Inspect each jar THE SAME DAY THEY ARE RECEIVED:
CO
a. Refill them with 70-80% ethanol if necessary. ฃ
^
b. Verify that the site identification and sample number on the label also appear on the chain <*
of custody form in the shipment. H
c. Notify the NARS IM team if any jars were broken and/or there are discrepancies between >
the custody form and jars. Q
3. After refilling the sample containers, store them at room temperature until sorting begins. u
<
4. Maintain the chain-of-custody form with the samples; it will be needed if the samples are ^
transported to any other location (e.g., for taxonomic identification, external QC evaluation). ^
i-
LJJ
CO
35
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 36 of 224
5. Verify that the login information includes the required data elements in Table 4.1. After
completing all required elements, provide the information to the data entry personnel.
Table 4.1 Benthic macroinvertebrate: required data elements - login
FIELD FORMAT DESCRIPTION
LAB NAME
LAB ID (optional)
DATE RECEIVED
SITE ID
VISIT NUMBER
SAMPLE ID
DATE COLLECTED
QA FLAG (if
appropriate)
QA.COMMENTS
LAB COMMENTS
text
text
MMDDYY
text
numeric
numeric
MMDDYY
text
text
text
name of lab
lab sample
id
date sample was received by lab
NRSA site identification code as used on sample label
sequential
visits to site (1 or 2, if specified on label)
sample number as used on field sheet (on sample label)
date sample was taken
QA/QC flag (lab may use its own flags, if defined in QA_COMMENTS
field or provided to NARS IM team)
Flag
NP
S
Q
Definition
Not enough preservative used
Sample shipping problem (explain in QA_COMMENTS
field)
Other quality concerns, not identified above
explanation for QA FLAG (if needed)
general laboratory analysis comments
4.6 Sample Preparation (Subsampling)
In preparation for sorting and picking organisms from the samples as described in Section 4.5, the
sorters first prepare the sample as described in the following steps.
CO
QC
LJJ
O
cc
u
u
36
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 37 of 224
Sampl
e received
sorting snoum resuu m ai
least:
1 jar of organisms
1 jar of pickate/unsorted
sample
V J
\
(
Sample logged
]
^ r
Removal of all inorganic and large organic
material and spread in a Caton-style tray
T
Randomly select three sample grids (may need more if
target of 500 organisms is not reached in first three)
Once a sorter begins a sample grid, that sample grid most
be completedly sorted even if 500 organisms are counted
before the sample grid is complete
The sorter should subdivided a sample grid into fourths if
it appears that sorting the entire subsample grid will
exceed 600 organisms; as with the larger subsample
grids, once sorting beings in a quarter grid, it must be
completed, even if 500 organisms are counted part way
v through the quarter grid /
Ensure all sorted organisms are place in
a single vial; bottle unsorted material
and pickate; label all vials and bottles
with proper labels
Figure 4.1 Benthic macroinvertebrate: summary of sorting
Figure 4.1 provides a simplified version that may be useful as a quick reference guide to the steps in
Sections 4.6 and 4.7. APPENDIX C: SAMPLE LABORATORY FORMS provides an example of the Benthic
Macroinvertebrate: Sorting Bench Sheet. If EPA's External QC Coordinator agrees, the laboratory may
substitute its own Sorting Bench Sheet or computer format instead of using the example. The alternative
must include the QC elements used for the evaluations described in Table 4.6.
1. Remove the lid from the sample container and remove the internal sample label (save the
labelit 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 Sorting
Bench Sheet. Header information required includes station name, station location, station
number, project name, bench number, sample type, date the sample was collected, and the
CD
o
cc
u
u
37
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 38 of 224
field team who collected the sample (e.g., Team 1). After the Sorting QC Officer verifies the
information, set the bench sheet aside.
2. Consider the contents of the sample:
3. If the sample has heavy amounts of inorganic substrate (e.g., sample that has 4 or 5 jars total
and 2 or 3 with gravel or sand), it is acceptable for the sorter to elutriate the sample, before
subsampling. An example of an acceptable elutriation method is as follows:
4. Pour ethanol off of sample containers through sieve (500 u.m or smaller). Also deposit leaf litter
and any other organic material (leaves, sticks, algae) onto sieve.
5. Depending on amount of inorganic material (gravel, sand, silt), pour all or a portion of this
material into a rectangular Tupperwareฎ/Rubbermaidฎ container and cover with water.
6. 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 a sieve (U.S. 35).
7. Repeat this until the water runs clear.
8. 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 with 3x magnification for any
remaining organisms. Have another sorter double check for organisms.
9. Once no organisms remain in the plastic container, wash the water through the sieve and dump
the inorganic material into a waste bucket.
10. Repeat this process until all of the inorganic material has been elutriated and checked for
heavier organisms, such as clams, mussels, or worms.
a. For all other samples:
i. Carefully decant the ethanol from the sample container by pouring the fluid through
a sieve (U.S. 35) into a separate container.
ii. Inspect the mesh of the sieve for any organisms and return any organisms found to
the sample container so they can be included in the sample sort process.
11. Spread the sample now in the circular sieve over the Caton-style tray. Use multiple trays if
necessary. If there is more than one jar for any particular sample, empty and wash each jar onto
the tray one at a time, making sure to spread each jar's contents evenly across the gridded
screen. If the amount of leaf litter or other detrital material fills the tray to the top of the wall
panels, divide it among two or more trays.
12. Mix the sample around in the tray(s) so that large and small particles are evenly distributed
throughout.
13. Add enough water to spread the sample evenly throughout the grid (the water level should be t2
relatively close to the top of the white tray). Spread the sample material over the bottom of the ^
CO
pan as evenly as possible. Move the sample into the corners of the pan using forceps, spoon, or LU
by hand. Vibrate or shake the pan gently to help spread the sample. 2j
14. Lift the screen out of the white tray to drain. Pour off or siphon excess water from the white tray ^.
O
and set the screen back into the tray. Leave just enough water in the bottom of the tray so that g
it barely covers the screen once it is returned to the tray to allow the sample to remain moist. ^
15. Label the grids by marking the side of the gridded tray with letters on one side and numbers on ^
the connecting side. For example, each grid could be labeled with one letter and one number, ^
e.g., A-5, E-l, to represent its position in the tray as shown in the example of the sorting method co
38
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 39 of 224
(Figure 4.2). If the sample is spread over more than one tray, label the grids identically in each
tray. See Example.
16. Use a random number generator to select at least 10% of the grids to ensure that the subsample
material is representative of the overall sample. For example, if the tray is subdivided into 30
squares (<6 cm2 each), then 3 squares ("grids") would be selected for sorting.
17. Make an initial assessment of the sample. If the number of organisms appears to exceed the
target number (500 organisms) in the collective three grids (or however many grids were
selected), quarter each grid, label the quarters (see example below) and randomly select a
quarter for initial sorting.
18. Assign unique identifiers on the Benthic Macroinvertebrate: Sorting Bench Sheet for each grid
and quarter processed. Provide a copy of the Sorting Bench Sheet, or its approved alternative, to
EPA's External QC Coordinator.
19. Remove all the material from the selected grid/quarter as follows:
20. 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.
21. 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 (or quarter'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:
22. An organism belongs to the grid containing its head.
23. 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.
24. 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.
25. Place the material from each selected grid(s) into a separate white plastic or enamel pan. Add
the necessary amount of water to the pan to facilitate sorting.
CD
o
cc
u
u
39
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Figure 4.2 Benthic Macroinvertebrate: sorting method example
Laboratory Operations Manual
Page 40 of 224
EXAMPLE: The sorter spreads the sample into a single Caton tray and labels the sides with letters and
numbers as shown below. The sorter randomly selects grids E-l, A-5, and B-4, in that order. The sorter
processes grid E-l and finds 50 organisms, and then continues to grid A-5 and finds 40 organisms. The
sorter then assesses grid B-4 and decides that it will produce more than 410 organisms needed to reach
the sample target of 500 organisms. The sorter then quarters grid B-4 and randomly selects the quarter
labeled a-1. The quarter has 350 organisms. The sorter then randomly selects a second quarter p-2. The
sorter processes the quarter and counts 75 organisms. The sorter now has picked a total of 515
organisms in the sample.
Place an X to indicate each grid sorted:
If more than one tray needed for the sample,
Tray #: 1 of 1
A
B
C
D
E
1 2
X
3456
X
X
If a grid has been quartered,
complete the following (see example
in Step 10):
Grid #: B-4
Place an X to indicate each quarter
sorted:
26. Set the subsampling device aside in case more grids need to be retrieved during the sorting
process. Cover the sample with aluminum foil to prevent desiccation of the sample and damage
to organisms (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 organisms, which could possibly change the probability of
selection.
4.7 Sorting
After dividing the sample into grids as described in Section 4.6, the sorter follows the following steps to
pick the organisms from the sample:
1. Remove the macroinvertebrates from the detritus with forceps.
a. In general, do not remove or count:
Empty snail or bivalve shells
Organisms of surface-dwelling or strict water column2 arthropod taxa (e.g., Collembola,
Veliidae, Gerridae, Notonectidae, Corixidae, Culicidae, Cladocera, or Copepoda, see
Table 4.2)
Incidentally-collected terrestrial taxa.
CO
CC
LJJ
O
cc
u
u
2Strict water column taxa are those that do not have at least one life stage that is benthic (i.e., bottom-dwelling).
40
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
iv. Fragments such as legs, antennae, gills, or wings.
Table 4.2 Benthic macroinvertebrate: list of taxa that are not to be sorted/counted
All terrestrial life history stages are to be excluded from the counts.
Laboratory Operations Manual
Page 41 of 224
Phylum
ANNELIDA
ARTHROPODA
BRYOZOA
NEMATODA
Class (or Subclass) Order
C//te//oto
Cladocera and other
Branchiopoda
Collembola
Insecta
Ostracoda
Branchiobdellida
Ephemeroptera (adults only)
Dipt era (larvae of listed families, plus all adults)
Coleoptera
Heteroptera
Neuroptera (adults)
Odonata (adults)
Plecoptera (adults)
Trichoptera (adults)
Family
Chaoboridae
Culicidae
Dytiscidae (adult)
Gyrinidae (adult)
Corixidae
Gerridae
Mesoveliidae
Notonectidae
Pleidae
b. 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.
c. In case of uncertainties, place the organism in the sort vial without counting it (the final
count is made by the taxonomist).
2. Place picked organisms for the selected grids and quarters into a single set of jars and vials for
the sample.
3. Sort all samples under a minimum of 6x (maximum of lOx) dissecting microscope. Once a grid,
or quarter, enters the sorting process, all organisms must be sorted to minimize bias. For the
survey, the target number of organisms is 500 per sample. If the 500th organism is counted in a
grid (or quarter), then the macroinvertebrates in the rest of the grid (quarter) are picked until
that grid (or quarter) is completed.
a. If the sample covers more than one tray, then sort the first selected grid in each tray, before
moving to the second grid, etc. For example, assume that a sample covers 3 trays Tl, T2,
and T3. Further assume that grids El, A3, and B5 were randomly selected in that order.
Then, in this example, the sorting order would be: T1E1, T2E1, T3E1, T1A3, T2A3, T3A3, and
T1B5,T2B5, andT3B5.
CC
CO
CC
LJJ
O
cc
u
u
41
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 42 of 224
b. If the grids have been quartered, then sort the first selected quarter in each grid. If the
target count has not been reached after sorting the selected quarter in the selected grids,
then randomly select another quarter for sorting. For example, if a sample covers a single
tray; grids El, A3, and B5 were randomly selected in that order; and quarter Q3 was
randomly selected; then the sorting order would be: E1Q3, A3Q3, B5Q3 before randomly
selecting another quarter.
4. Keep a rough count of the number of organisms removed and enter the number of organisms
found in each grid under that column on the Sorting Bench Sheet.
5. When all randomly selected grids have been sorted, verify that the cumulative count is greater
than 500 organisms.
6. This step is performed if: 1) the sorter has not reached 90% proficiency in 5 consecutive samples
("proficiency QC check", see Section 4.4.1 for definition and explanation); or 2) the Sorting QC
Officer has selected this sample to use as the 1 in 10 sample QC check for experienced sorters
("periodic QC check"). (The Sorting QC Officer must check a minimum of one NRSA sample if an
experienced sorter processes less than ten NRSA samples. If the laboratory performs QC checks
more frequently, then the all of the QC data must be submitted to EPA.) The Sorting QC Officer:
a. Performs QC checks using the same power microscope as the sorter;
b. Counts any missed organisms found and places them into the sample vial, or other suitable
sample vial;
c. Notes the number of organisms missed on the Sorting Bench Sheet; and
d. Adds that number to the final count of the sample.
e. Calculates the PSE for the sample (see Section 4.4.1 for definition; equation 1). If the PSE is:
i. <90% and the sample is the:
1) Proficiency QC check, the Sorting QC Officer must check the next 5 samples until the
sorter has PSE>90% for 5 consecutive samples.
2) Periodic QC check, then the sorter's samples since the last QC check are examined
for organisms missed in sorted grids and quarters. The sorter must again
demonstrate proficiency by achieving a PSE>90% in 5 consecutive samples, unless
he or she shows marked improvement in their sorting efficiency prior to completion
of the next five samples, the Sorting QC Officer may, at his/her discretion, consider
this individual to be "experienced" and check only 1 in the next 10 samples.
ii. >90% and the sample is the:
1) Proficiency QC check, the sample counts towards the 1 in 5 consecutive samples t2
used to establish proficiency. ^
CO
2) Periodic QC check, no corrective action is required. LU
7. Place organisms removed from grid to an internally-labeled vial (or larger container, if 2j
necessary) containing 70-80% ethanol. Record the following information on internal sample ^.
O
labels used for vials of sorted material with Pigma Micronฎ pen on cotton rag paper or an g
acceptable substitute. ^
8. Remove the remaining material from that particular grid that was just sorted left on the sorting ^
pan (i.e. material such as sticks, organic debris)and place it in a separate container with ^
preservative (70-80% ethanol). Label the container "Pickate," on both internal and external co
42
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 43 of 224
labels. This material is kept by the lab until notified by TOPO. Internal sample labels should be
made of cotton rag paper or an acceptable substitute. Include the following information on the
label:
a. Station Name
b. Station Location
c. Station Number
d. Date Sorted
e. Sorter's Initials
f. "1 of x" or "2 of x", etc. if the sample is sorted into more than one vial (where x is the total
number of vials for the sorted sample)
9. Search the entire caton 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."
10. Return all material not subsampled (remaining on the Caton tray) to the original container with
the preservative that was set aside. 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), and in accordance with survey and laboratory requirements.
11. Verify that the bench sheet includes the required data elements in Table 4.3. After completing
all required elements, provide the sorting bench sheets to the data entry personnel.
12. Thoroughly clean all sample preparation and sorting equipment and make sure all equipment is
free of organisms prior to sorting the next sample.
Table 4.3 Benthic macroinvertebrate: required data elements - sorting
LAB NAME
LAB ID (optional)
DATE RECEIVED
SITE ID
VISIT NUMBER
SAMPLE ID
DATE COLLECTED
DATE SORTED
PROPORTION
SORTED
CORRECTION
FACTOR
QA FLAG (if
appropriate)
FORMAT
Text
Text
MMDDYY
Text
numeric
numeric
MMDDYY
MMDDYY
numeric
numeric
text
DESCRIPTION
name of lab
lab sample id
date sample was received by lab
NRSA site identification code as used on sample label
sequential visits to site (1 or 2, if specified on label)
sample number as used on field sheet (on sample label)
date sample was taken
date that the sorter started working on the sample
proportion of sample sorted based upon number of grids and
quarters selected and available
subsampling correction factor
QA/QC flag (lab may use its own flags, if defined in QA_COMMENTS
field or provided to NARS IM team)
Flag Definition
C Organism in poor condition or fragments
DD Damaged Organism
CO
QC
LJJ
O
cc
u
u
43
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 44 of 224
QA.COMMENTS
LAB COMMENTS
text
text
IM
IN
NP
NT
S
UN
0
Immature
Indeterminate (explain in QA COMMENTS field)
Not enough preservative used
Not able to meet target level for identification (may be used
with other codes, or explain in QA_COMMENTS field)
Sample shipping problem (explain in QA_COMMENTS field)
Unknown. Identification is tentative. Organism has been sent
to expert taxonomist for definitive identification.
Other quality concerns, not identified above
explanation for QA FLAG (if needed)
general laboratory analysis comments
4.8 Taxonomy Identification
The taxonomist performs the following steps in identifying the benthic macroinvertebrate organisms:
1. Upon receipt of a set of sample vials from the sorter:
a. Compare all site identification codes and sample numbers on the form with those entered
on the labels of samples, and resolve any discrepancies with the sorter.
b. Determine if any vials are broken. For any broken vial, request that the sorting laboratory
subsample its jar of unsorted sample and use the new vials to replace the original vials
(broken and unbroken).
c. Maintain the chain-of-custody form with the sample vials; it will be needed to return/store
them.
2. Empty one sample vial at a time into a small Petri dish. Add 70-80% ethanol to keep the
organisms covered. Remove the internal sample label and complete the top portion of a
Taxonomic Bench Sheet (APPENDIX C: SAMPLE LABORATORY FORMS or comparable system),
using the information from the label. Depending on the type of organisms, select the
appropriate step:
a. For all Chironomidae and Oligochaeta organisms, extract the organisms from the Petri dish.
i. Prepare slide mounts using CMCP-10 (or CMC-9, CMC-10, or other media) and applying
a coverslip. All organisms should be visible, which generally means a maximum of 10-20
organisms per slide. Label the slides with the same sample identification code or log-in
number as the ethanol organisms.
ii. If the laboratory prefers to use another method than slide mounting, the EPA External
QC Coordinator will grant a waiver if the following applies:
1) The request is for a laboratory located at a single location. For example, EPA would
not consider the combined qualifications of a prime contract laboratory and its
subcontract laboratories. Instead, for whichever laboratories met the requirements,
EPA would evaluate and grant a waiver for the prime contract laboratory separate
from each of its subcontractor laboratories.
2) The request for a waiver must identify and describe a minimum of three studies. For
each study, the external QC evaluation must demonstrate that the laboratory met
or exceeded the NRSA QC requirements (i.e., PDE<5% and PTD<15%) for its
Chironomidae and/or Oligochaeta organisms.
QC
CO
QC
LJJ
O
QC
u
u
44
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 45 of 224
3) The laboratory agrees to mount the organisms on slides if it fails one of the periodic
(NRSA) external QC evaluations, as follows:
a) It must mount all Chironomidae and Oligochaeta organisms in samples
processed since the previous external QC evaluation (i.e., for which it met the
PDE and PTD requirements).
b) It must continue to mount all Chironomidae and Oligochaeta organisms for the
unprocessed samples.
b. For all other organisms, remove similar organisms to other dishes (keep these covered with
70-80% ethanol).
3. View the sample to ensure that all necessary diagnostic characters have been observed,
according to the taxonomic key or other literature using:
a. A stereo dissecting microscope for organisms in dishes.
b. A compound microscope for slides of Chironomidae and Oligochaeta organisms
4. To the extent possible, identify organisms to the target taxonomic level for the survey (usually
genus, see
QC
CO
QC
LJJ
O
QC
u
u
45
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 46 of 224
5. Table 4.4 for the typical benthic macroinvertebrates found in freshwater and their target
taxonomic levels). Add any necessary data qualifiers (see list provided with Required Data
Elements in Table 4.6).
a. Enter the Taxonomic Serial Number (TSN) as it appears in the column "Unique Identifier" of
EPA's WQX taxon database. Section 4.4.1 provides instructions in extracting the TSN values
from WQX.
b. Note whether the identification of a group of organisms is distinct (Distinct=Y/N) from other
organisms in the same sample as follows:
If the organisms can be identified to the target level, then Distinct="Y."
If an organism cannot be identified to the target level then assign values as follows:
1) If at least some of the organisms in the sample can be identified to the target level,
then:
a) Distinct="Y" for organisms identified at the target level; and
b) Distinct="N" for organisms that were identified at a higher taxonomic level (e.g.,
family) that may contain a target level taxa already identified in a given sample
(e.g., genus).
c) An example would be, if some organisms from a sample are identified to Baetis,
but other organisms in the sample could only be identified to Baetidae and/or
Ephemeroptera, then Baetis would be distinct, but Baetidae and/or
Ephemeroptera would not be Distinct.
2) If none of the organisms in the sample could be identified at the target level, then:
a) Distinct="Y" for organisms identified at the lowest taxonomic level (e.g., family);
and
b) Distinct="N" for organisms identified at a higher level (e.g., order).
c) For example, if a taxonomist can identify a number of Ephemeroptera (Order)
families, but a number of the organisms could not be taken past
Ephemeroptera, then the individual families would be distinct, but the order
would not be distinct.
CD
o
cc
u
u
46
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 47 of 224
Table 4.4 Benthic macroinvertebrate: target level of taxonomic identification-benthics commonly found in
freshwater
Target Level of
TAXON Identification
Phylum
ANNELIDA
ARTHROPODA
COELENTERATAf
Class (or
Subclass)
(any, except
Hirudinea,
Polychaeta)
Hirudinea
Oligochaeta
Polychaeta
Arachnoidea
Insecta
Malacostraca
Order
(any, including
Tubificinae, except as
noted below)
Enchytraeidae
Lumbriculidae
(any)
Acari
(any, except as noted)
Coleoptera
Diptera (any, except as
noted)
Ephemeroptera
Hemiptera (Heteroptera)
Lepidoptera
Megaloptera
Odonata
Plecoptera
Trichoptera
(any)
Amphipoda
Decapoda
Isopoda
Mysidacea
Family
Ceratogopogoninae
Chironomidae
Dolichopodidae
Empididae
Ephydiridae
Muscidae
Phohdae
Scathophagidae
Sciomyzidae
Stratiomyidae
Syrphidae
Tabanidae
Family
X
X
X
subfamily
see Table
4
X
X
X
X
X
X
X
X
X
X
Genu
s
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
CC
CO
O
cc
u
u
47
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 48 of 224
MOLLUSCA
NEMERTEA
(any, except
Hydrobiidae)
Bivalvia
Gastropoda
(any, except Hydrobiidae)
Hydrobiidae
X
X
X
X
X
^Colelenterata need only be identified to the phylum level.
Table 4.5 Benthic macro-invertebrate: target Level of taxonomic identification - chironomidae
Chironomidae (except as noted)
Cricotopus
Orthocladius
Conchapelopia
Hayesomyia
Helopelopia
Meropelopia
Rheopelopia
Telopelopia
Thienemannimyia
REQUIRED LEVEL OF IDENTIFICATION
Genus
Cricotopus/'Orthocladius if a clear distinction can not be
made
Cricotopus/Orthocladius if a clear distinction can not be
made
Thienemannimyia genus group
6. Record the identifications. For example, using the taxonomic bench sheet in Appendix C, the
identification would be recorded in the Column labeled "taxon." Enter the number of larvae,
pupae, and adults, or total count (e.g. mollusks), if appropriate life history column does not
apply, of each taxon under the appropriate columns.
a. If the target taxonomic level cannot be achieved due to immature or damaged organisms
this should be noted in the data file in the QA_FLAG field (e.g., QA_FLAG=IM). Appendix 2
provides other codes for the QA_FLAG field.
b. If damaged organisms can be identified, they are counted ONLY if the:
i. Fragment includes the head, and, in the case of arthropods, the thorax;
ii. Oligochaetes have a sufficient number of segments in the head;
iii. Mollusk shell (bivalve or gastropod) is occupied by a organism;
iv. Organism is the sole representative of a taxon in the sample.
c. If a unique taxon is determined for which the appropriate taxonomic level is not available in
the literature and there are other taxa in that taxonomic level:
i. Provide good quality digital photographs of the organism to outside experts for
identification; and
ii. Include the tentative identification in the database with a data qualifier code of
QA_FLAG='UN' so that these organisms can be distinguished from other organisms in
the data analysis.
iii. When the outside expert identifies the organism, update the database with the correct
identification.
7. Compare taxa names from WQX (see Section 4.4.1) to the names used for the identifications.
Check the non-matches for the following common problems and correct them.
QC
CO
QC
LJJ
O
cc
u
u
48
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 49 of 224
a. Abbreviations
b. Extra information identifiers (e.g., sp., spp.,, nr., cf., genus 1, w/ hair chaetae)
c. Extra character (e.g., "?", "Acentrella ?turbida", blank space)
d. The word "probably" or "prob" (e.g., "Microcylloepus prob. similis")
e. Identifying to a lower level than in WQX (e.g, to species rather than genus)
f. Double names (e.g., Callibaetis callibaetis)
g. Common misspellings
h. Tribes/subfamilies/subgenus sometimes do not appear in WQX
i. Species with incorrect genus (Hydatopsyche betteni)
j. Split level taxonomy (e.g., Cricotopus/Orthocladius)
k. Invalid name (e.g., taxonomic change, synonym; Sphaeriidae vs. Pisiidae)
8. Complete the identification by entering the totals for each developmental stage and the total
number of each taxon in the cells at the bottom of the sheet. Cross-check to be sure the totals
were summed correctly.
9. If the number of counted organisms is <500 and part of the sample is still unprocessed, return to
the sorting steps to obtain more organisms for identifying and counting.
10. Provide the data to the Internal Taxonomic Officer for another review to confirm that the
identifications use the same nomenclature as the WQX database and the laboratory's reference
collection.
11. Make two copies of the bench sheet or computer file used to record the identifications. They
are distributed as follows: 1) the project file; and 2) EPA's External QC Coordinator.
12. 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 Internal Taxonomic QC
Officer, who will periodically review the reference collection to ensure that it is complete.
13. Verify that the reference collection contains at least one organism that represents each genus
(or lowest taxonomic level) identified from all sample. For any missing references, choose an
appropriate organism(s) from the sample to represent a taxon name in the master taxa list:
a. Place the physical specimen in the reference library.
b. Place two labels in the sample container to identify: organisms placed in the reference
collection, and those in the non-reference organisms.
c. Obtain a good quality representative digital photographs of the specimen (see instructions t2
in Section 4.4.1). ^
CO
14. If the Internal Taxonomy QC Officer selects the sample for a QC check, the Internal Taxonomy LU
QC Officer re-counts and re-identifies the organisms in the sample following the same steps 2j
above for the original taxonomist. One in 10 of the taxonomist's samples must be checked. The ^.
O
Internal Taxonomy QC Officer records the independent verifications on a bench sheet or g
computer file. The Internal Taxonomy QC Officer will also supply a list of taxa that were found ^
to be problematic during their QC sorting check, which can be submitted in an Excel or Word ^
document format. (If the Internal Taxonomy QC Officer performs the QC check more frequently, ^
then all QC data must be submitted.) co
49
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 50 of 224
15. Carefully return the rest of the organisms to the original sample vial, fill with 70-80% ethanol,
and cap tightly.
16. Re-package the samples and slide-mounted organisms carefully, and sign and date the chain-of-
custody form. Return or store the samples according to laboratory protocols and any
contractual requirements.
17. Verify that all required data elements in Table 4.6 have been recorded by the taxonomist and
Internal Taxonomy QC Officer. If the results were recorded on paper, provide the Taxonomic
Bench Sheet to the data entry personnel.
Table 4.6 Benthic macroinvertebrate: required data elements-taxonomic identification
FIELD
LAB NAME
LAB ID (optional)
DATE RECEIVED
SITE ID
VISIT NUMBER
SAMPLE ID
DATE COLLECTED
DATE TAXON
ANALYST NAME
QC VERIFICATION
FAMILY
SUBFAMILY
TRIBE
GENUS GROUP
GENUS
SPECIES
WQX_TSN
LAB TIN (OPTIONAL)
TAXANAME
ABUNDANCE LARVAE
ABUNDANCE PUPAE
ABUNDANCE ADULT
ABUNDANCE TOTAL
DISTINCT
CITATION
QA FLAG (if appropriate)
FORMAT
text
text
MMDDYY
text
numeric
numeric
MMDDYY
MMDDYY
text
text
text
text
text
text
text
text
numeric
text
text
numeric
numeric
numeric
numeric
text
text
text
DESCRIPTION
name of lab
lab sample id
date sample was received by lab
NRSA site identification code as used on sample label
sequential visits to site (1 or 2, if specified on label)
sample number as used on field sheet (on sample label)
date sample was taken
date that the taxonomist started identifying organisms in the sample
name of taxonomist or Internal Taxonomy QC Officer (if record
provides results of QC check)
Y if the record provides the results from the QC check
taxonomic family
taxonomic subfamily
taxonomic tribe
taxonomic genus group (e.g., Thienemannimyia)
taxonomic genus
taxonomic species
Taxonomic Serial Number as defined by "Uniqueldentifier" in WQX. If
taxon is not in WQX, provide citation for reference used to identify
organism in CITATION field
lab taxa ID number
unique taxon name in WQX
number of individual larvae or immature bugs
number of individual pupae
number of individual adults
total number of individuals
distinct taxa in sample (y/n) (See description in Section 4.8)
citation for reference used to identify organism, if taxon not present
in WQX database
QA/QC flag (lab may use its own flags, if defined in QA_COMMENTS
field or provided to NARS IM team)
Flag Definition
DD Damaged Organism, poor condition or fragments
IM Immature
IN Indeterminate (explain in QA_COMMENTS field)
NP Not enough preservative used
CO
QC
LJJ
O
cc
u
u
50
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 51 of 224
QA.COMMENTS
LAB COMMENTS
text
text
NT
S
UN
Q
Not able to meet target level for identification (may be used
with other codes, or explain in QA_COMMENTS field)
Sample shipping problem (explain in QA_COMMENTS field)
Unknown. Identification is tentative. Organism has been sent
to expert taxonomist for definitive identification.
Other quality concerns, not identified above
explanation for QA FLAG (if needed)
general laboratory analysis comments
4.9 Data Entry
Table 4.1, Table 4.3 and Table 4.6 identify the required data elements that the sorting and taxonomic
laboratories must provide to EPA, preferably in EPA's data template provided to each of the laboratories
and found in Appendix C, available separately from EPA. In addition, the laboratory must provide the
resume or curriculum vitae for each taxonomist who identifies benthic macroinvertebrates for the NRSA
samples. The resume or cv for each taxonomist is submitted once to EPA's External QC Coordinator.
4.10 Sample and Record Retention
The laboratory shall retain:
1. The sample materials, including vials, slides, and sorting residuals, for a minimum of 3 years
from the date the EPA publishes the final report. During this time, the laboratory shall store the
materials in a cool location away from sunlight. The laboratory shall periodically check the
sample materials for degradation and refill jars and vials with 70-80% ethanol if necessary.
Original records, including laboratory notebooks and the reference library, for a minimum of 10
2.
years from the date that EPA publishes the final report.
After the stated time periods, the laboratory shall follow its internal protocols for disposal.
4.11 External Taxonomic Quality Control
Two laboratories and multiple experts together conduct the necessary work for the external taxonomic
quality control. They perform the following steps:
1. The External QC Coordinator randomly selects 10% of each laboratory's samples, subject to the
following constraints:
a. If the primary laboratory received fewer than 30 samples, then the External QC Coordinator
will randomly select three samples for the evaluation.
b. If the laboratory is responsible for processing 100 samples or more for the survey, the
External QC Coordinator will perform the evaluation in batches (e.g., each 50 to 100
samples) so that performance can be evaluated and corrected as necessary. The External QC
Coordinator will determine the size of the batches based upon the total number of samples
that will be shipped to the laboratory, the delivery schedule, processing schedule, and
availability of samples from other laboratories (e.g., the External QC Coordinator might
combine samples from three laboratories into one batch at a convenient time for the QC
taxonomist).
QC
CO
QC
LJJ
O
QC
u
u
51
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 52 of 224
c. The External QC Coordinator will arrange for a minimum of 4 QC evaluations in each year
(2013, 2014). To the extent practicable, the QC Coordinator will schedule batch evaluations
evenly throughout the project period.
2. The External QC Coordinator coordinates and pays for delivery of the selected samples, bench
sheets, and chain-of-custody form.
3. The primary laboratory packs the selected samples for shipping. The QC taxonomist's review will
be independent, and thus, will not have access to the original laboratory results. Although the
External QC Coordinator may tailor the directions for the independent taxonomist's
preferences, the directions are likely to include instructions such as:
a. Containers (e.g., jars, slides, vials) must include the site identification code and sample
number.
b. Containers must not contain the complete identification of the organisms. If reference
specimens were kept from a sample that is being QC'd, then these taxa must be noted and
this information must be given to the External QC Coordinator. In the event that the
reference specimens are the only organisms representing a given taxa for a given sample,
then these specimens must be made available to the QC taxonomist, through the External
QC coordinator.
c. Shipment must contain chain-of-custody documentation.
4. Upon receipt, the secondary laboratory follows the tracking procedures in Section 4.5.
5. The QC taxonomist:
a. Performs whole-sample re-identifications following the procedures in Section 4.8, taking
care to ensure inclusion of all slide-mounted organisms.
b. Completes a separate copy of the taxonomic bench sheet for each sample.
c. Enters the data using EPA's template (Template found in APPENDIX C: SAMPLE
LABORATORY FORMS, see Section 4.9 for data entry instructions). The QC taxonomist shall
label the bench sheet and database entries with the term "QC Re-ID."
6. The External QC Coordinator will compare the taxonomic results (counts AND identifications)
generated by the primary and secondary laboratories. If either laboratory identified the
organisms to a lower level than required by
CD
o
cc
u
u
52
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 53 of 224
7. Table 4.4 and Table 4.5 or by nomenclature different than used by WQX, the External QC
Coordinator will substitute the required level or name, respectively, in the comparisons. The
External QC Coordinator will calculate the percent difference in:
a. Enumeration as measured by PDE defined in Section 4.4.1; and
b. Taxonomy as measured by PTD defined in Section 4.4.1. Table 3 of Stribling (2003) provides
examples of what would be considered agreements.
8. If any sample has PDE>5% and/or PTD>15%, the External QC Coordinator will facilitate a
reconciliation call with EPA and the laboratories. The External QC Coordinator may decide that a
reconciliation call is unnecessary if there appears to be an obvious explanation for differences,
few samples are affected or other reasons.
9. After completing the reconciliation calls and/or documenting decisions to skip them, the
External QC Coordinator will provide EPA with a report or memorandum with recommendations
for resolving the differences. Significant differences may result in the re-identification of
samples by the primary taxonomist and a second QC check by the secondary taxonomist.
10. As a final QC check on the data, the External QC Coordinator will arrange for an independent
data validation of the database. For each sample, the data analyst will evaluate whether the
reported taxa are known to occur in geographic area in which the sample site resides. For any
discrepancy, the External QC Coordinator will arrange for another taxonomist to evaluate the
specimen or its digital photograph.
11. As an additional verification on the generation of the data, EPA may conduct assistance visits at
the laboratories. If EPA decides to conduct an assistance visit, a qualified EPA scientist or
contractor will administer a checklist based upon the steps described in this LOM. The objective
of the visit would be to:
Confirm the sorting and identification steps are properly implemented.
Assist with questions from laboratory personnel.
Suggest corrections if any errors are made.
4.12 Quality Assurance/Quality Control (QA/QC)
Equation 4.1 Percent sorting efficiency (PSE).
Number of organisms recovered by the sorter compared to the combined (total) number of recoveries
by the sorter (A) and QC Officer (B) for a sample. PSE should be >90%.
,
Measure of taxonomic precision comparing the number of organisms, nlf counted in a sample by the ^.
primary taxonomist with the number of organisms, n2, counted by the internal or external QC cc
taxonomist. PDE should be <5%. <
^
wi ~ni -
PDE= -xlOO E
+n2
53
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Equation 4.3 Percent taxonomic disagreement (PTD).
Laboratory Operations Manual
Page 54 of 224
Measure of taxonomic precision comparing the number of agreements (positive comparisons, comppos)
of the primary taxonomist and internal or external QC taxonomists. In the following equation, N is the
total number of organisms in the larger of the two counts. PTD should be <15%.
PTD =
1-
comp
pos
N
xlOO
Table 4.7 Benthic macroinvertebrate: measurement data quality objectives
Variable or Measurement Precision Accuracy
Sort and Pick
Identification
90% a
85% b
90% a
95% c
NA = not applicable;a As measured by PSE;b As measured by (100%-PTD);c As measured by (100%-PDE)
QC
CO
QC
LJJ
O
QC
u
u
54
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Table 4.8 Benthic macroinvertebrate: quality control - laboratory
Check or Sample
Description
Frequency
Acceptance Criteria
Laboratory Operations Manual
Page 55 of 224
Corrective Action
SAMPLE PROCESSING AND SORTING
Sample pickate
examined by
different analyst
within lab
10% of all samples
completed per
analyst
PSE > 90%
If < 90%, examine all residuals of
samples by that analyst and retrain
analyst
Sorting QC Officer
counts number of
organisms not
found in sorted
grids
All samples
Sorter achieves PSE >90% in
5 consecutive samples.
Sorter is now considered
"experienced"
Sorting QC Officer checks all
samples until acceptance criteria
met
Sorting QC Officer
counts number of
organisms not
found in sorted
grids for
experienced sorters
1 in 10 samples
completed per
sorter
Sorter achieves PSE >90%
If <90%, examine all sorted grids in
samples assigned to sorter since
last achieving proficiency (i.e.,
PSE>90%). Sorter loses
"experienced" status and must
again show proficiency by
achieving PSE >90% in 5
consecutive samples. If the sorter
shows marked improvement in
their sorting efficiency prior to
completion of the next five
samples, the Sorting QC Officer
may, at his/her discretion, consider
this individual to be "experienced"
and check only 1 in the next 10
samples.
External QC
Coordinator
evaluates grid and
quarter data to
determine if the
sample was well
mixed as
demonstrated by
consistency in
counts between
grids (or quarters)
All grids and
quarters within
each sample
Sorter demonstrates relative
consistency for 90% of
assigned samples
If <90%, evaluate whether: 1) the
sorter's consistency is similar to
other sorters; or 2) few samples
were assigned the sorter. If neither
explanation applies, EPA's External
QC Coordinator contacts the
laboratory to discuss possible
corrective action (e.g., resorting of
sorter's samples)
IDENTIFICATION
Duplicate
identification by
Internal Taxonomy
QC Officer
1 in 10 samples per
taxonomist,
PTD <15%
If PTD >15%, reidentify all samples
completed by that taxonomist
since last meeting the acceptance
criteria, focusing on taxa of
concern
Independent
identification by
outside, expert,
taxonomist
All uncertain taxa
Uncertain identifications to
be confirmed by expert in
particular taxa
Record both tentative and
independent IDs
55
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 56 of 224
External QC
Use of
widely/commonly
accepted
taxonomic
references by all
NRSA labs
Prepare reference
collection
DATA VALIDATION
Taxonomic
"reasonable-ness"
checks
10% of all samples
completed per
laboratory
For all
identifications
Each newtaxon
per laboratory
All data sheets
PDE < 5%
PTD < 15%
All keys and references used
by each lab must be on
bibliography prepared by
one or more additional
NRSA labs or in WQX (see
Section 4.4.1 for retrieval
instructions). This
requirement demonstrates
the general acceptance of
the references by the
scientific community.
Complete reference
collection to be maintained
by each individual
laboratory
Taxa known to occur in
given rivers or streams or
geographic area
If PDE > 5%, implement
recommended corrective actions.
If PTD > 15%, implement
recommended corrective actions.
If a lab proposes to use other
references, the lab must obtain
prior permission from Project QA
Officer before submitting the data
with the identifications based upon
the references.
Internal Taxonomy QC Officer
periodically reviews data and
reference collection to ensure
reference collection is complete
and identifications are accurate
Second or third identification by
expert in that taxon
4.13 References
Epler, J.H. 2001. Identification manual for the larval chironomidae (Diptera) of North and South
Carolina. A guide to the taxonomy of the midges of the southeastern United States, including Florida.
Special Publication SJ2001-SP13. North Carolina Department of Environment and Natural Resources,
Raleigh, NC, and St. Johns River Water Management District, Palatka, FL. 526 pp.
Merritt, R.W., K.W. Cummins, and M.B. Berg (editors). 2008. An introduction to the aquatic insects of
North America, 4rd edition. Kendall/Hunt Publishing Company, Dubuque, Iowa.
Stribling, J.B., S.R. Moulton, and G.T. Lester. 2003. Determining the quality of taxonomic data. Journal
of the North American Benthological Society 22(4):621-631.
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):
392-3.
USEPA. 2004. Wadeable Stream Assessment: Benthic Laboratory Methods. EPA841-B-04-007. U.S.
Environmental Protection Agency, Office of Water and Office of Research and Development,
Washington, DC.
QC
CO
QC
LJJ
O
QC
u
u
56
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 57 of 224
5.0 FECAL INDICATOR: ENTEROCOCCI
5.1 Scope & Application
This document describes the application of Draft EPA Method 1606 for the processing and qPCR analysis
of water sample concentrates from rivers and streams for the purpose of determining water quality by
Real-Time Quantitative Polymerase Chain Reaction (qPCR) assays that determine the concentration of
bacteria such as the fecal indicator, Enterococcus, by measuring the concentration of their DNA in the
water sample.
This method facilitates the microbiological determination of water quality of water bodies at remote
locations from which collected water samples cannot feasibly be analyzed for the enumeration of viable
(culturable) indicator bacteria because they cannot be transported to an analytical laboratory within 6
hours of collection time for analysis by membrane filtration and / or selective media inoculation and
incubation (e.g. MPN broth analysis) methods (EPA method 1600). Prior to qPCR analysis of the water
samples, the bacterial cells present in a water sample will have been concentrated by "field" filtration
within 6 hours after collection of the samples. The filter retentate preserved by freezing of the sample
filters on dry ice and in < -20ฐC freezers will be subjected to DNA extraction (e.g. bead-beating) and
purification processes leading up to qPCR analysis. This processing can be completed up to 1 year after
cell concentration if the sample filter retentates are maintained frozen at -20 to -SOT.
5.2 Summary of Method
Each sub-sample has previously been filtered aseptically and folded inward in half three times to form
an umbrella or in half and rolled up and then inserted into sterile sample extraction tubes containing
sterile glass beads or Roche MagNA Lyser Green BeadsTM (actually siliconized white ceramic beads in a
green capped tube). Extraction tubes containing filter concentrates (retentates) have been stored on dry
ice until transport to the analytical laboratory by air courier. Filter concentrates will be shipped by air
courier on dry ice from the field to the analytical team at EPA New England Regional Laboratory. Filter
concentrates received by NERL staff will be subjected to DNA extraction procedures and subsequently
analyzed by Draft EPA Method 1606 or 1607 for Total Enterococcus along with modifications to the
QA/QC procedures described below. The laboratory methods are summarized in Table 5.4 of Section
5.18.
5.3 Definitions of Method
Batch Size: The number of samples that will be processed by filter extraction with the same batch
(volume) of SAE buffer and analyzed by the same qPCR assay(s) using the same batch of qPCR master
mix. A batch is covered for quantitation purposes by the same "batch" calibrator samples, a minimum of u
three, analyzed during the same week. O
O
Bottle Blank: Analyte-free water is collected into a sample container, of the same lot number as the cฃ
containers used for collection of the environmental samples. Analysis of this sample is performed to ^
evaluate the level of contamination, if any, introduced into the environmental and control samples from "M
the sample container(s) from a common vendor's lot. O
DNA: Deoxyribo-Nucleic Acid, double-stranded genetic molecules containing sequences of the four y
nucleotide bases, adenine, th\
involved in protein synthesis.
nucleotide bases, adenine, thymine, guanidine, and cytosine that encode rRNA, mRNA, and tRNA ^
57
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 58 of 224
Field Filter Blank: A volume of sterile PBS, free of target organisms (i.e. Enterococcus) filtered through a
sterile filter and processed in parallel with all other samples to serve as a sentinel for detection of
reagent contamination or contamination transferred between samples by processing and analysis.
Field Replicates: Samples collected from rivers and streams that are collected at the same sampling site
one right after the other with only slight temporal variation. They are not "splits" of the same sample
volume.
Filtrate: Sample liquid or buffer rinsate passing through the filter into the vacuum flask.
Laboratory Quality Samples: Mock samples created in the lab such as lab blanks, lab-fortified blanks
(LFBs), and Lab-Fortified Matrices (LFMs) used to assure lack of sample contamination and to measure
analytical recovery during performance of sample processing and analysis methods.
Performance Testing (PT) / Performance Evaluation Sample (PES): Calibrator samples (filters spiked
with E.faecalis grown in Brain Heart Infusion Broth) and Laboratory Fortified Blanks (Phosphate
Buffered Saline; PBS) spiked with Enterococcus faecalis cells from BHI Broth suspension) will be assayed
by EPA Method 1600 and Draft EPA Method 1606 to ascertain method performance. Ball-T Bioballsฎ
which contain a specified number of E. faecalis cells may also be acquired to determine the performance
of the Relative Quantitation Method. Purified E.faecalis DNA acquired from the American Type Culture
Collection and TIB Mol Biol Inc. is used to test the performance of the Absolute Quantitation Method.
Retentate: The sample residue retained by the filter after the sample is vacuum-filtered. The retentate
contains particulates, microbiota, and macrobiota from which the DNA is extracted into buffer by bead-
beating for subsequent qPCR analysis.
Rinsate: The volume of phosphate buffered saline (PBS) applied to a sample's filter retentate in order to
"wash" any residual fine particles, smaller than the filter's nominal pore size, through the retentate and
the filter.
Sample Processing Control (SPC): A surrogate homologue analyte (e.g. Salmon DNA) spiked into each
sample to determine the recovery of target analyte and/or detect assay inhibition caused by matrix
effects.
Standards: Known amounts or numbers of copies of Enterococcus genomic DNA analyzed by the
Enterococcus qPCR assay to generate a Standard Curve (Log Copy Number vs. Crossing Point Value) in
order to determine Enterococcus genomic copy numbers in "Unknown" test sample extracts by Absolute
Quantitation Method.
5.4 Interferences
Low pH (acidic) water
Humic and fulvic acid content u
O
Suspended solids (e.g. fecal matter) and particulates (sand, dirt) <=>
Excessive algal growth 2j
-z.
5.5 Health & Safety Warnings ฃ
o
All proper personal protection clothing and equipment (e.g. lab coat, protective eyewear/goggles) must ^
be worn or applied. ^
When working with potential hazardous chemicals (e.g. 95% ethanol) or biological agents (fecally- ~
contaminated water) avoid inhalation, skin contact, eye contact, or ingestion. If skin contact occurs u
58
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 59 of 224
remove clothing immediately and wash/ rinse thoroughly. Wash the affected skin areas thoroughly with
large amounts of soap and water. If available consult the MSDS for prompt action, and in all cases seek
medical attention immediately. If inhalation, eye contact or ingestion occurs, consult the MSDS for
prompt action, and in all cases seek medical attention immediately.
5.6 Personnel Qualifications
All laboratory personnel shall be trained in advance in the use of equipment and procedures used during
the sample extraction and qPCR analysis steps of this SOP. All personnel shall be responsible for
complying with all of the quality assurance/quality control requirements that pertain to their
organizational/technical function. All personnel shall be responsible for being aware of proper health
and safety precautions and emergency procedures.
5.7 Equipment and Supplies
Clean powderless latex or vinyl gloves
Goggles or Face Shield
Roche MagNA Lyser
Roche MagNA Pure LC (automated nucleic acid isolation and purification platform)
High Speed Microfuge
Micropipettors
Semi-conical, screw cap microcentrifuge tubes (PGC, #506-636 or equivalent) pre-filled with 0.3
+ 0.02 g Acid-washed glass beads (Sigma, # G-1277 or equivalent). Filled tubes are autoclaved
15-min. Liquid Cycle (Slow Exhaust) OR
Roche MagNA Lyser Green Bead tubes (Roche Applied Science, #03-358-941-001) sterile,
siliconized 3-mm diameter ceramic beads in a siliconized 2-mL microfuge tube.
Roche MagNA Lyser Rotor Cooling Block
2-mL tube racks
Permanent marking pens (fine point and regular point) for labeling tubes
Bench Sheets & Printouts of Computer Software Sampling Loading Screen
5.8 Reagents & Standards
Qiagen AE buffer (Qiagen 19077)
Salmon DNA (Sigma D1626)
Frozen tubes of Enterococcus faecalis (ATCC #29212) calibrator cell stock
Purified Enterococcus faecalis (ATCC #29212d) genomic DNA u
ABITaqManฎ Universal PCR Master Mix (ABI #4304437) g
Enterococcus PCR primers and TaqManฎ probe LU
Sketa PCR primers and TaqManฎ probe ^
Bovine Serum Albumen (BSA) Sigma Cat. #B-4287) g
Roche MagNA Pure LC DNA Isolation Kit III for Fungi & Bacteria
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 60 of 224
5.9 Preparations Prior to DNA Extraction & Analysis
Determine/estimate the sample batch size (number of samples) for one-week of sample processing and
qPCR analysis. The batch size is the number of samples that will be processed by filter extraction with
the same batch (volume) of SAE buffer and analyzed by the same qPCR assay(s) using the same batch of
qPCR master mix. A batch is covered for quantitation purposes by the batch calibrator samples, (a
minimum of three) whose 5-fold and 25-fold diluted extracts are analyzed at the outset of the week
along with a reagent blank. The lab will fill out a batch sample analysis bench sheet.
1. Micropipettors are calibrated annually and tested for accuracy on a weekly basis. Follow
manufacturer instructions for calibration check. Measure three replicate volumes per pipettor
and keep log book of their weights on a calibrated balance scale.
2. Preparation of stock Salmon Sperm (SS) DNA: Dissolve Salmon DNA in PCR grade water at a
concentration of ~10 u.g/mL Determine concentration of Salmon testes DNA stock by OD26o
reading in a spectrophotometer. A DNA solution with an OD26oof 1.0 has a concentration equal
to approximately 50 u.g/mL depending on the GC content of the DNA's sequence(s).
3. Dilute Salmon testes DNA stock with AE buffer to make 0.2 u.g/mL Salmon DNA Extraction Buffer
(SAE). Extraction buffer may be prepared in advance and stored at 4 ^C for a maximum of 1
week.
Note: Determine the total volume of Salmon DNA Extraction Buffer required for each day or
week by multiplying the volume (600 u.L) times the total number of samples to be analyzed
including controls, water samples, and calibrator samples. For example, for 18 samples, prepare
enough Salmon/DNA extraction buffer for 24 extraction tubes (18 ) / 6 = 3, therefore, 3 extra
tubes for water sample filtration blanks (method blanks) and 3 extra tubes for calibrator
samples). Note that the number of samples is divided by 6 because you should conduct one
method blank for every 6 samples analyzed. Additionally, prepare excess volume to allow for
accurate dispensing of600u,L per tube, generally 1 extra tube. Thus, in this example, prepare
sufficient Salmon DNA Extraction Buffer for 24 tubes plus one extra. The total volume SAE
needed per sample is 600 u,L. Hence for the SAE volume for 25 sample tubes is equal to 15,000
juL Dilute the Salmon DNA working stock 1:50, for a total volume needed (15,000 ^L) 50 = 300 ^L
of 10 u,g/mL Salmon DNA working stock. The AE buffer needed is the difference between the
total volume and the Salmon testes DNA working stock. For this example, 15,000 u,L - 300 u,L =
14,700 ^LAE buffer needed.
4. Make Dilution Series of Enterococcusfaecalis purified genomic DNA for use as internal standards
in individual qPCR runs and to generate the weekly Enterococcus qPCR Standard Curve for
quantitation purposes. y
5. Enterococcusfaecalis DNA for Standards. u
O
6. Frozen Reference Stock (20-u.L) at 2.89 x 10s GEQs per ul. gj
7. Dilute 10-u.L of the Frozen Reference stock 363-fold to a final volume of 3,630 ul AE buffer. ^.
Aliquot 20-u.L volumes into many 200-u.L microfuge tubes and store frozen at -20 5Q The net ^
concentration of Enterococcus GEQs is 8,000 / ul. Each week perform a series of 10-fold and 4- H
fold dilutions from one thawed tube of the 8,000 GEQ/ul standard solution to create 800 ^
GEQ/ul, 80 GQ/ul and 20 GEQ/ul standard solutions. The analyst performs Enterococcus qPCR ?
i
upon duplicate 5-u.L volumes of each of the four standards yielding a Standard Curve of Log <
60
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 61 of 224
GEQs ENT versus Ct value from which the assays "efficiency" is subsequently calculated in the
Relative Quantitation EXCEL Spreadsheet.
a. Make Enterococcusfaecalis calibrator filter samples:
i. Assemble calibrator positive control samples by thawing tubes of E. faecalis cell stocks,
diluting their contents (10-u.L) up to 1-mL AE buffer and spotting 10-u.L on sterile PC
filter previously folded and inserted into a pre-chilled Green Bead tube.
ii. Spot a sufficient number of calibrator filter samples for the entire study to insure
uniform, consistent relative quantitation of study samples. Store the calibrator filter
samples in -20^C freezer and thaw individual calibrators (three per week) for extraction
with each week's batch of samples.
8. The calibrator sample filters are spotted with 104 or 10s Enterococcusfaecalis cells and this
number is incorporated into the Relative Quantitation EXCEL spreadsheet.
9. Prior to and after conducting work with cells and / or genomic DNA standards, disinfect and
inactivate (render non-amplifiable) DNA in the Sample Extraction Hood, the qPCR Cabinet, and
the qPCR Sample Loading Hood with 10% bleach and >_15-min. exposure to high intensity
germicidal (254 nm) ultraviolet light.
5.10 Procedures for Processing & qPCR Analysis of Sample Concentrates.
5.10.1 Sample Processing (DNA Extraction)
Typically, 100-mL volumes of surface water are filtered according to EPA Method 1606 for processing
and analysis by PCR assays. Due to the limitations of field crew sampling time and the performance
limitations of the manually-operated vacuum pumps used in the field sampling operations, only 50-mL
surface water samples were filtered. Lower volumes (< 50-mL) are acceptable if suspended particulates
hinder the filtering of the standard 50-mL volume but equivalent volumes for each filter replicate were
requested. Filtration of lower sample volumes necessitated modifications to Method 1606 which are
directed by the Analysis Decision Tree (ADT; Section 5.18.1).
In accordance with the ADT, if < 40-mL of a water sample is filtered per filter replicate, then the
laboratory analyst extracts two replicate filters in parallel and combines equivalent volumes of the filter
extracts to form one composite filter extract. Each individual filter is extracted with only 300-u.L of SAE
Extraction Buffer instead of the usual prescribed 600-u.L volume of SAE buffer. Halving the SAE buffer
volume enables the analyst to maintain an equivalent Method Detection Limit and maintain a similar
Sample Equivalence Volume (SEQ; i.e. water sample volume per extract volume) in the extract volumes
(e.g. 5-u.L) of each sample filter concentrate added to the PCR reactions.
1. Pre-chill MagNA Lyser Rotor Cooling Block in -20ฐC freezer. Label 1.7-mL sterile microfuge tubes y
with sample ID number to match them with Green Bead Tubes. Two supernatant recovery tubes u
and one "5-fold" dilution tube is needed per sample and should be labeled accordingly. The g
dilution tube shall be filled with 80-u.L AE buffer using a micropipettor. ~z.
2. To extract sample filters, uncap green bead tube (cold) and add 0.6-mL (600-u.L) SAE Buffer &
(Qiagen AE Buffer spiked with Salmon DNA). Re-cap tubes tightly. H
3. Insert Green Bead tubes of samples into MagNA Lyser and bead-beat for 60-sec (1-min) at 5,000 ^
rpm at Room Temperature. Transfer sample tubes to microfuge. Spin tubes at 12,000 rpm for 2-
61
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 62 of 224
min. Being careful to move filter aside, recover and transfer up to 400-u.L of supernatant (sans
debris) to new tube with a P-200 or P-1000 micropipettor.
4. Spin the supernatant tubes for 5-min at 14,000 rpm at Room Temperature. Recover >350-ul
supernatant and transfer to new 1.7-mL tube. When all samples in a batch have been extracted
transfer dilute 20-u.L of DNA extract (2nd supernatant) five-fold (5X) in 80-u.L AE buffer (sans SS-
DNA) and store at 4ฐC for qPCR assays. (If supernatant, 5X and even 25X sample dilutions
possess dark pigment and exhibit severe qPCR inhibition in Sketa assays, consider extracting
replicate filters of samples using the Modified MagNA Pure LC DNA Isolation Protocol (see
Section 5.18.2).
5.10.2 Sample Analysis by Enterococcus qPCR
5.10.2.1 Preparation ofqPCR assay mix
1. To minimize environmental DNA contamination, routinely treat all work surfaces with a 10%
bleach solution, allowing the bleach to contact the work surface for a minimum of 15 minutes
prior to rinsing with sterile water. If available, turn on UV light for 15 minutes.
2. Using a micropipettor with aerosol barrier tips, add PCR grade water to the lyophilized primers
and probe from the vendor to create stock solutions of 500 u.M primer and 100 u.M probe and
dissolve by extensive vortexing. Pulse centrifuge to coalesce droplets. Store stock solutions at -
202Q
3. Prepare working stocks of Enterococcus, and Salmon DNA primer/probe mixes by adding 10 ul
of each Enterococcus or Salmon DNA primer stock and 4 ul of respective probe stock to 676 ul
of PCR grade water, and vortex. Pulse centrifuge to create pellet. Use a micropipettor with
aerosol barrier tips for all liquid transfers. Transfer aliquots of working stocks for single day use
to separate tubes and store at 45Q
4. Using a micropipettor, prepare assay mix of the Enterococcus, and Salmon DNA reactions in
separate, sterile, labeled 1.7 ml microcentrifuge tubes as described in Table 5.1.
5. Finger vortex the assay mix working stocks; then pulse microcentrifuge to coalesce droplets.
Return the primer/probe working stocks and other reagents to the refrigerator.
6. Thaw and finger vortex sample extract (dilution) tubes that will be assayed in PCR run.
Microfuge a few seconds to coalesce droplets. Finger mix and spin the standards and calibrator
samples (dilutions). Temporarily store all samples in 4ฐC refrigerators until use in assay or return
to long term storage at -20ฐC. Discard disposable gloves and put on a new pair.
7. Set 32 Smart tubes in Cepheid Racks in PCR cabinet along with micro-pippetors and expose to y
germicidal UV lamp for 15-min. u
O
8. Pipette 20-uL of respective Master Mix into each labeled Smart tube. Transfer Smart tubes ^
(racks) from PCR cabinet to disinfected Sample Loading Fume Hood. -z.
LJJ
9. Using P-10 or P-20 micro-pipettor load each Smart tube with 5-uL volume of respectively ^
designated sample extract (dilution), standard, or buffer blank (SAE). Cap each sample's Smart i-
tube after loading. ^
10. Check to make sure each Smart tube is properly labeled and identifiable by sample number or I- ?
i
core position (e.g. A4). Insert loaded Smart tubes into Smart Tube microfuge. Close lid and spin <
62
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 63 of 224
5-sec. Pop lid to stop. Remove Smart Tubes from microfuge and insert into proper position in
SmartCycler.
Enterococcus (Ludwig) and Salmon (Sketa) qPCR assays (EPA Method 1606) will be performed upon 5-uL
aliquots of un-diluted & 5X diluted extracts of sample unknowns, calibrator, field blank, and lab blank. A
"No Template Controls" (NTC) shall be analyzed on an ongoing basis to ensure that the Master Mix PCR
reagents are not contaminated. To minimize the number of Enterococcus qPCR reactions needed to be
performed upon samples, Sketa qPCR assays will be performed upon the 5-fold diluted DNA extracts of
samples before any Enterococcus qPCR assays are run in order to screen samples for the presence and
dilution of PCR inhibitors by comparison with the undiluted and 5-fold dilution DNA extract of the
calibrator samples and unused portions of SAE buffer. Each sample's lowest dilution DNA extract not
exhibiting PCR inhibition in the Sketa qPCR assay will be re-assayed by the Enterococcus qPCR assay and
it's results will be used for quantitation of Enterococcus DNA sequences and CCEs.
Detection of reduced levels of Salmon DNA (higher instrument Ct values) is indicative of technical error
during extract dilution or excessive levels of PCR inhibitors or nuclease activity which could impact
detection of the Enterococcus DNA target sequences in the Enterococcus PCR assay. Alternatively, the
high Sketa Ct value may be indicative of the occurrence of a technical error during extract dilution. If a
test sample's Ct value is less than 3 cycles different than the blank negative control and calibrator
samples, indicating only negligible or marginal inhibition (the Sketa Assay is more sensitive to inhibitors
than the ENT Assay), an aliquot of its five-fold diluted extract is analyzed in the Enterococcus Assay. If an
abundance of PCR inhibitors or DNA nucleases are present in a sample extract which are causing a
greater increase in an extract's Ct value (> 3 cycles increase), then the extract is diluted an additional
five-fold (net 25-fold dilution) and re-assayed by both the Sketa and ENT assays. If the inhibition is not
ameliorated by the additional dilution, which should restore the Sketa Ct value to that of the 25-fold
diluted calibrator samples' extracts, the following actions are taken by the analyst. First, the analyst re-
dilutes the sample's undiluted DNA extract five-fold and re-analyzes the dilution with the Sketa PCR
assay to confirm that Ct variance is not due to a dilution error. If the Ct difference is not attributed to a
dilution error, replicate sample filters of the "inhibited" samples are subjected to DNA extraction and
purification by the MagNA Pure LC automated platform loaded with the Roche DNA Kit III (Bacteria;
Fungi) reagents (see Section 5.18.2).
The EPA Modified MagNA Pure LC extraction process which includes the spiking of the Lysis Binding
Buffer with the Salmon (IPC) DNA is more effective, but more costly, than EPA Method 1606 in
neutralizing severe levels of PCR inhibitors and DNA nucleases present in some environmental samples,
especially those containing high levels of algae or phytoplankton. The purified DNA extract yielded by
MagNA Pure extraction of the few (<5%) "severely inhibited" samples is subsequently analyzed by the
Sketa and Enterococcus qPCR assays and the number of Enterococcus CCEs per 100-mL determined by
the delta Ct and delta delta Ct Relative Quantitation Methods. While the MagNA Pure LC extraction y
method is not 100% conservative (no partitioning or recovery issues) like EPA Method 1606, it typically Q
exhibits DNA recoveries in the range of 25-50%. DNA recoveries and Enterococcus CCE concentrations ง
are calculated using only the Delta-Delta Ct Relative Quantitation Method. The relative DNA recoveries i-
are determined by comparison of the Sketa results from purified DNA eluates of each test sample with "-"
those of the extracted lab blank and calibrator samples. The absolute DNA recovery is calculated by Q
comparison of the former Sketa results with those of elution buffer spiked with an amount of Salmon
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 64 of 224
The "Unknown" and "Control" sample extracts whether processed using the SAE buffer or MagNA Pure
LC Kit III reagents are analyzed according to the Cepheid SmartCycler Enterococcus and Sketa qPCR
protocols described in Appendix A of the Draft EPA Method 1606 with Ct determination made by the
software using Manual Determination (equivalent of Fit Points Method of Roche LightCycler) with the
fluorescence threshold set at 8.0 units which enables uniform analysis and comparability of all samples'
qPCR results.
5.10.3 Sample analysis sequence for SmartCycler
Example: For analyses on a single 16-position SmartCycler, calibrator samples and water samples are
analyzed in separate runs and a maximum of 6 water samples (or 2 replicates of 3 samples) are analyzed
per run, as described in Table 5.2 and Table 5.3 of Section 5.18.
Enterococcus and Sketa (Salmon DNA = SPC) qPCR results are exported to an EXCEL spreadsheet in which
relative quantitation calculations are performed by analysts. The Method 1606 results are reported in
terms (units of measure) of Number of Enterococcus Sequences and Number of Enterococcus Calibrator
Cell Equivalents (CCEs) per 100-mL sample volume. The qPCR results are converted to this standardized
unit of measure based on the volume of water sample actually filtered (e.g., 10-mL, 25-mL, or 50-mL).
Non-detects are reported as below the reporting limit (RL) which varies proportionally to the volume of
sample filtered by each sample crew at a specific site. Reporting limits and Method Detection Limits
(MDLs) will be higher among samples for which a volume of water <50-mL was filtered.
Enterococcus qPCR results are flagged if some part of the sample collection, hold-time, processing,
shipment, storage, sample extraction, or qPCR analysis are compromised and did not meet the
requirements of the Sampling and Analysis SOPs.
5.11 Storage & Timing of Processing/Analysis of Filter Concentrates
When a sufficient number of water sample filter concentrates (filters and retentates) have been
received by NERL and qPCR analytical reagents have been obtained the samples will be logged into LIMS.
Sample processing and qPCR will commence and results will be entered into the LIMS upon completion
of analysis.
5.12 Chain of Custody
Follow the Sample Control Procedures, Field Sampling Form/Enterococci Filtration/Sample Processing
Standard Operating Procedures.
Field Sampling forms and NRSA 2013-2014 Sample Tracking EXCEL Spreadsheet shall be consulted to
determine if a sample has been properly preserved during collection and transport prior to analysis and
that it has passed all criteria permitting its analysis. The qPCR results of samples exceeding established
criteria or whose associated field/lab blanks had positive Enterococcus qPCR detections of DNA shall be u
flagged. O
O
5.13 Quality Assurance/Quality Control (QA/QC) Procedures 25
The Data Quality Objectives and the Laboratory QC Procedures are listed and summarized in Table 5.5 LU
and Table 5.6 of Section 5.18. g
The number of field blanks (dilution buffer only) shipped by field crews performing the resampling of 91 ^5
re-visited rivers and streams represents a frequency of 5-10% of the total number of samples extracted g
and analyzed by qPCR. All field blanks (negative controls) will be extracted and analyzed by qPCR for the 3
64
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 65 of 224
detection of Enterococcus. The blanks will be analyzed in these cases to insure that positive detections in
field samples are not due to contamination by sampling crews.
One Lab / Method Blank (LB; sterile filters) will be run per batch week in order to insure the sterility
(lack of DNA contamination) in the SAE buffer and pipette tips used to process all of the samples. The LB
sample will be processed and diluted like all other "Unknown" samples.
Up to four replicate filter concentrates (retentates) derived from the field filtration of 50-mL (in some
cases 10-mL and 25-mL) sample volumes of every sample will be received by NERL and stored at -20 to -
SOT. One filter retentate of each sample (and duplicates for 10% of samples) will be extracted to obtain
DNA lysates for Enterococcus qPCR analysis. The remaining filter concentrates will be archived for
possible extraction and analysis at a later time if needed.
Enterococcus and Sketa qPCR analysis will be performed upon 5-u.L volumes of the non-diluted and 5-
fold diluted (in AE buffer) extracts which will be added to 20-u.L qPCR Master Mix volumes and analyzed
in the Cepheid SmartCycler qPCR instrument in accordance with draft EPA Method 1606.
Duplicate Enterococcus and Sketa qPCR assays will be performed upon 10% of the sample extracts
(diluted and un-diluted) each week (batch) to determine qPCR assay variance.
5.14 Method Performance
Method Performance will be determined by the use of Performance Testing (PT)/Performance
Evaluation Samples (PES). Calibrator samples (filters spiked with frozen stocks of E. faecalis grown in
Brain Heart Infusion Broth) and Lab-Fortified Matrices (LFMs; duplicate sample filters spiked with frozen
stocks of E. faecalis grown in Brain Heart Infusion Broth) will be extracted and assayed by EPA Method
1606 Enterococcus and Sketa qPCR assays in order to ascertain method performance. The LFMs are
performed upon several samples (approx. 5% frequency) per batch, typically samples exhibiting non-
detection of Enterococcus, in order to determine method performance and also to insure that non-
detects are not due to poor DNA recovery caused by matrix effects.
5.15 Record Keeping & Data Management
Laboratory analysts shall follow the EPA OEME Laboratory Data Management SOP. Each lab analyst shall
record all details pertaining to sample processing and analysis in a designated, bound laboratory
notebook. Pertinent sample collection and analysis data shall be entered into the Laboratory
Information Management System (LIMS) and SeaGate Crystal Reports shall be generated as required by
the EPA (TOPO).
An EXCEL spreadsheet of sample analysis data and associated calculations used to derive a field sample's
or control sample's Enterococcus genomic DNA (GEQ) and Cell Equivalent (CEQ) concentration shall be
uploaded to the NRSA 2013-2014 database stored on a computer server in Corvallis, Oregon. u
O
5.16 Waste Management & Pollution Prevention o
Cฃ
I | I
During the sample processing procedures there may be hazardous waste produced. The waste must be i-
handled and disposed of in accordance with federal, state, and municipal regulations. All recyclable and ^
non-recyclable materials for disposal will be properly sorted for their respective waste streams and Q
placed into proper containers for janitorial staff to collect and process according to EPA guidelines.
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 66 of 224
filter forceps to sterilize the latter and to prevent ethanol spillage during transport between sampling
sites.
After the DNA extract is recovered from the sample filter after bead-beating in buffer and
centrifugation, the filter and bead-tube will be discarded in autoclave bags and sterilized for 30-min at
121ฐC/30 psi to inactivate any potential pathogens that may be associated with the samples.
5.17 Literature Cited
USEPA Region 1 (New England) OEME NERL Standard Operating Procedure for the Collection of
Chemical & Biological Ambient Water Samples (ECASOP-Ambient Water Sampling 2; January 31, 2007)
USEPA Draft Method 1606: Enterococci in Water and Wastewater by TaqManฎ Quantitative Polymerase
Chain Reaction (qPCR) Assay. December 2006 (12/15/06 a)
USEPA NERL OEME Draft Bench SOP for Real-Time PCR Method Quantifying Enterococci in Recreational
Water Samples (August 2006)
5.18 Tables, Diagrams, Flowcharts, Checklists, and Validation Data
Table 5.1 Enterococci: PCR assay mix composition (according to draft EPA method 1606)
Volume/Sample (multiply by #
Reagent samples to be analyzed per day)
Sterile H O
2
Bovine Serum Albumen (20 mg/mL)
TaqManฎ master mix
Primer/probe working stock solution
1.5 uL
2.5 uL
12.5 uL
3.5 uL*
Note: This will give a final concentration of 1 u.M of each primer and 80 nM of probe in the reactions. Prepare
sufficient quantity of assay mix for the number of samples to be analyzed per day including calibrators and negative
controls plus at least two extra samples. It is strongly recommended that preparation of assay mixes be performed
each day before handling of DNA samples.
Table 5.2 Enterococci: batch calibrator & enterococcus standards PCR run - 7 samples
Quantity Quantity PCR
Sample Description* Samples PCR Assay Master Mix Reactions
3 Calibrators (5- and/or 25-fold dilution)
3 Calibrators (5- and/or 25-fold dilution)
4 Enterococcus faecalis DNA Standards
No template control (reagent blank)
3
3
4
1
Salmon DNA (Sketa)
Enterococcus
Enterococcus
Enterococcus
6
6
8
1
* Diluted equivalently to the water samples
Table 5.3 Enterococci: sub batch test sample PCR run - 26 samples & 1 method blank
Sample Description"
Water samples, (5-fold dilution)
Quantity Quantity PCR
Samples PCR Assay Master Mix Reactions
u
u
8
O
cc
QC
O
B
Q
26
Enterococcus
26
66
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 67 of 224
Method blank or Sample PCR Reaction Duplicate,
(1- or 5-fold dilution)
Non-diluted SAE Buffer
Water samples, (1- or 5-fold dilution)
Method blank or Sample PCR Reaction Duplicate,
(1-& 5-fold dilution)
1
1
26
1
Enterococcus
Enterococcus
Salmon DNA
Salmon DNA
1
1
26
1
* Use of 5-fold diluted samples for analysis is currently recommended if only one dilution can be analyzed. Analyses
of undiluted water sample extracts have been observed to cause a significantly higher incidence of PCR inhibition
while 25-fold dilutions analyses may unnecessarily sacrifice sensitivity.
Table 5.4 Enterococci: laboratory methods
Variable or
Measurement
Sample
Collection
Sub-sampling
Sub-sample
(& Buffer Blank)
Filtration
Preservation &
Shipment
DNA Extraction
(Recovery)
Method 1606
(Enterococcus &
SPCqPCR)
QA
Class
C
N
N
C
C
C
Expected
Range and/
or Units
NA
NA
NA
-40Cto+40 C
10-141%
<60 (RL) to
>100,000 ENT
CCEs/100-mL
Summary of Method
Sterile sample bottle submerged to collect
250-mL sample 6-12" below surface at 1-m
from shore
4 x 50-mL sub-samples poured in sterile 50-
mL tube after mixing by inversion 25 times.
Up to 50-mL sub-sample filtered through
sterile polycarbonate filter. Funnel rinsed
with minimal amount of buffer. Filter folded,
inserted in tube then frozen.
Batches of sample tubes shipped on dry ice
to lab for analysis.
Bead-beating of filter in buffer containing
Extraction Control (SPC) DNA. DNA recovery
measured
5-|aL aliquots of sample extract are analyzed
by ENT & Sketa qPCR assays along with
blanks, calibrator samples & standards. Field
and lab duplicates are analyzed at 5%
frequency. Field blanks analyzed along with
test samples.
References
NRSA Field
Operations Manual
2008
NRSA Laboratory
Methods Manual
2008
NRSA Laboratory
Methods Manual
2008
NRSA Laboratory
Methods Manual
2008
EPA Draft Method
1606 Enterococcus
qPCR
EPA Draft Method
1606 Enterococcus
qPCR
NERL NRSA 2008
qPCR Analytical SOP
u
u
8
o
cc
C = critical, N = non-critical quality assurance classification.
.
O
B
67
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Table 5.5 Enterococci: parameter measurement data quality objectives
Laboratory Operations Manual
Page 68 of 224
Expected
Variable or QA Range and/or
Measurement Class Units Summary of Method References
DNA Extraction
C
(Recovery)
Enterococcus & SPC C
qPCR
SPC & ENT DNA
RSD =
sequence numbers 30%
of Calibrators &
Standards by AQM
ENT CCEs by dCt
RQM
RSD =
55%
10-141%
Bead-beating of filter in buffer
EPA Draft Method
containing Extraction Control (SPC) 1606 Enterococcus
DNA. DNA recovery measured
qPCR
<60to 5-nLaliquots of sample extract are EPA Draft Method
>10,OOOENT analyzed by ENT &SketaqPCR assays 1606 Enterococcus
CEQs/100-mL
80%
40%
ENT CCEs by ddQ RSD = 50%
RQM
55%
along with blanks, calibrator samples & qPCR; NERL NRSA
standards. Field and lab duplicates are 2008 2009 qPCR
analyzed at 5% frequency. Field blanks Analytical SOP
analyzed at end of testing only if
significant detections observed.
95%
95%
95%
(QAPP)
C = critical, N = non-critical quality assurance classification.
* AQM = Absolute Quantitation Method; RQM = Relative Quantitation Method;
SPC = Sample Processing Control (Salmon DNA /Sketa); CCEs = Calibrator Cell Equivalents
Table 5.6 Enterococci: laboratory QC procedures - enterococci DNA sequences
Check or Sample
Description Frequency Acceptance Criteria Corrective Action
SAMPLE PROCESSIN
Re-process sub-
samples
(duplicates)
G
10% of all
samples
completed per
laboratory
Percent Similarity >70%
If <70%, re-process additional sub-
samples
qPCR ANALYSIS
Duplicate analysis
by different
biologist within
lab
Independent
analysis by
external
laboratory
10% of all
samples
completed per
laboratory
None
Percent Congruence <30% RSD
Independent analysis TBD
If >30%, determine reason and if
cause is systemic, re-analyze all
samples in question.
Determine if independent analysis
can be funded and conducted.
u
u
8
o
cc
QC
O
B
Q
68
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 69 of 224
Use single stock of
E. faecalis
calibrator
ForallqPCR
calibrator
samples for
quantitation
All calibrator sample Cp (Ct)
must have an RSD < 30%
If calibrator Cp (Ct) values exceed
an RSD value of 30% a batch's
calibrator samples shall be re-
analyzed and replaced with new
calibrators to be processed and
analyzed if RSD not back within
range.
DATA PROCESSING & REVIEW
100% verification
and review of
qPCR data
AllqPCR
amplification
traces, raw and
processed data
sheets
All final data will be checked
against raw data, exported
data, and calculated data
printouts before entry into
LIMS and upload to Corvallis,
OR database.
Second tier review by contractor
and third tier review by EPA.
5.18.1 Enterococcus qPCR Analysis Decision Tree (ADT)
Confirm water volume filtered for NRSA sample
filter to be processed and analyzed by qPCR
Extract Sample Filters with 600-uL SAE
buffer and bead beating (EPA Mtd 1606)
Perform Sketa qPCR assay upon 5-uL of
5 fold dilution of DMA extracts
Add 300-uL SAE Buffer to
each of 2 equiv. filter
replicates; Bead-Beat
If Florescence
Cp>3
Standard
Deviations
Re-dilute Sample 5-
fold & 25-fold, Repeat
Sketa assays to
confirm dilutions and
results.
lf5X&25X
Sketa Cp still
unacceptable
5X&25X Sketa
Cps Acceptable
Dilute sample S-fold
more to 25-fold and
analyze for Sketa
Analyze 5-uL of 5-
fold dilution of
sample DNA by
Enterococcus qPCR
assay
Cp >3 S.D.s
Enter Sketa and ENT qPCR
Ct value, Sample Vol &
Dilution Factor in Calc.
Template; Calculate ENT
CCEspei 100-ml
srform Sketa qPCR
jted/diluted purified
A eluate to contain
<500 ng DNA
Analyze most cone. D
of sample DNA with
significant PCR inhib
with ENT qPCR assay
EXCEPTION:-ifSketaC
DNA recovery is low or
inhibition is excessive.
5-uL of the 3-fold dilutio
Enterococcus qPCR ass
>
ilution
10
tion
p value
of mean,
:>CR
Analyze
i by the
ay.
If Cp value
23 Standard
Deviations
X
Cp<3
S.Ds
1
Repeat last step
f dilution has not
exceeded 9-fold
x 1
Dilute eluate
3-fold and
re -assay by
Sketa qPCR.
Created 10/25/07
Updated 1/2/08
Revised 11/05/08
Figure 5.1 Enterococci: qPCR analysis decision tree (ADT)
5.18.2 "Modified" MagNA Pure LC DNA Purification Kit III Protocol
1. Pre-warm the MagNA Pure LC DNA Isolation Kit III Lysis Buffer to 65 ?C in waterbath. Quickly
pipette 260-u.L of warm Lysis Buffer (un-amended) into each "Green Bead" tube with filter
(preserved after filtration temporarily on ice or during long-term storage in freezer). Shake tube
u
u
8
o
cc.
.
ง
69
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 70 of 224
5 to 10 seconds to mix buffer with beads and filter. Let stand at RT until batch of 16 samples
(including positive control LFB or LFM and negative control LB samples) have all had Lysis Buffer
and had their caps sealed tight. Leave water bath on to use during 30-minute Proteinase K
treatment period.
2. Load the 16 samples into MagNA Lyser Rotor Plate and insert into MagNA Lyser. Tighten the
three handscrews of the locking mechanism. Close the lid tightly. Set controls to shake for 60-
sec at 5,000 rpm. Press the start button.
3. When the shake cycle has ended press the Open Lid Button. Open the lid and unlock the locking
mechanism screws. Remove tube plate and set on bench top MagNA Lyser tube ring hub.
Remove tubes, insert into tube styrofoam water bath float and cool tubes in ice water for 2-min.
or place directly into 24-place microfuge rotor, pre-chilled in freezer.
4. Insert tubes into centrifuge rotor symmetrically in order to balance rotor. Close lid of centrifuge.
Set spin parameters for 3,000 rpm for 1-min at 4^C. Press Start button. Centrifuge to collect
drops and foam off of cap down into tube.
5. When centrifuge stops, open lid and remove tubes from rotor. Uncap tubes in order and add 40-
u.L of Proteinase K (dissolved in Lysis Buffer Elution Buffer). Re-cap tubes and mix lysate by
inversion. Do not vortex. Knock beads and filter down from cap into bottom of tube by tapping
tubes on bench countertop.
6. Insert tubes into styrofoam floating rack. Incubate tubes 30-min at 65^C in water bath. Set timer
for 15-min. At end of 15-min remove rack from water bath and inverts several times to mix
samples and tap beads and filter back down into tube. Re-place rack in 65^C waterbath for 15-
min. for total of 30-min.
7. Repeat steps 3 to 8 to process 16 more samples in parallel for loading MagNA Pure LC sample
cartridge with 32 DNA extracts for downstream processing in the robotic platform.
8. After 30-min in 65 ^c waterbath remove tubes from water bath and place in MagNA Lyser Bead
Beater for 15 seconds at 5,000 rpm. After 15 seconds of bead-beating, place in ice bath for 5-
min to cool.
9. Insert tubes in centrifuge rotor and spin 3-min at 12,000 rpm and 4 ^C to pellet sediment and
cell debris. When spinning is complete, open lid of centrifuge and rotor and mark side of outer
side of cap where pellet should have formed.
10. Carefully remove rotor from centrifuge and set on bench. Remove tubes one at a time from
rotor and use 200-u.L pipettor and sterile aerosol-proof tips to transfer approximately 150ul
lysate supernatant from tube to wells in MagNA Pure LC Sample Cartridge in pre-designated y
order. Q
11. When all 16 sample supernatants transferred to sample cartridge put adhesive film over cz
cartridge to prevent contamination and evaporation. Put sample cartridge in ice water bath or ^
LJJ
fridge to maintain 4 2C. ^
12. Repeat steps 9 to 13 for second batch of 16 samples (lysates). Re-cover sample cartridge with H
adhesive film for storage. Centrifuge sample cartridge opposite a balance cartridge for 75-sec (1- y
min, 15-sec) at 2800 rpm in IEC centrifuge (or equivalent) with rotor adaptors for microtiter ^
plates in place. Insert the film-covered sample cartridge in MagNA Pure LC platform. <
70
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 71 of 224
13. Load the MagNa Pure LC platform with volumes of extraction kit reagents prescribed by MagNA
Pure LC computer software for the number of samples being extracted. Before closing the
platform' lid and starting the extraction process add 1.34ul of 9.3u.g/mL Salmon DNA Stock (10
u.g/mL nominal concentration) per ImL Lysis Binding Buffer (blue soapy solution) as the Sample
Processing Control (SPC). If the amount of Salmon DNA stock to be added is less than 10-u.L,
dilute the Salmon DNA stock so that a volume > 10-u.L can be pipetted into the Lysis Binding
Buffer. Rinse pipette tip up and down three times in Lysis Binding Buffer.
14. Remove film from top of sample cartridge and re-insert in Roche MagNA Pure LC platform set up
with DNA Purification Kit III (Fungi; Bacteria) reagents in tubs, tips, tip holders, and processing /
elution cartridges. Close platform lid and after checking off checklist of loaded items (e.g.
reagents, tips) lock the lid and start the automated DNA III Extraction Protocol which purifies
each sample's DNA and elutes it into 100-u.L Elution Buffer.
15. When extraction process is complete, unlock the MagNA Pure LC platform lid and remove the
sample eluate cartridge. Cover the cartridge with adhesive film and store at 4 C until qPCR
analysis. Store cartridge at < -20 ^c for long term preservation.
16. Prepare Elution Buffer Control from 9.3u.g/mL Salmon DNA Stock by diluting a small volume to
37.2pg/1000ul (1-mL). This control sample is only analyzed by the Sketa qPCR assay. The Ct
value obtained represents that value expected in Sketa qPCR assays of each MagNA Pure LC
purified sample if 100% of the Salmon DNA was recovered and detected. Vortex to mix on low
speed briefly prior qPCR analysis. Centrifuge for 1.5-min to coalesce droplets. Remove film to
aliquot sub-samples and re-place with new film cover to restore at cool temperatures.
u
u
8
o
cc.
.
O
B
71
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 72 of 224
6.0 FISH VOUCHER SPECIMENS
This chapter describes the procedures for taxonomic identification of fish voucher specimens collected
from river and streams as part of the EPA's National Rivers and Streams Assessment. Two types of
voucher samples are collected and sent to a laboratory.
The first type is the QC voucher sample prepared by the field crew at sites identified by EPA. The QC
voucher sample includes specimens (preserved or digital images) of all types of fish, identified to the
lowest practicable taxon level at the site by the field taxonomist. QC voucher samples are sent to the QC
laboratory to assess the accuracy of the field identifications.
The second type is the Unknown (UNK) voucher sample that includes physical specimens or digital
images for fish that the field fish taxonomist could not positively identify during the site visit. Field crews
also label as "UNK" specimens for fish collected outside their usual or known range. In most cases, the
field fish taxonomist will retain the UNK samples for further study and identification. However, in some
instances, the field crew will opt to send the UNK voucher sample to the QC laboratory for identification.
This chapter describes the procedures and data reporting for the QC laboratory. The Field Operations
Manual describes the procedures for the field fish taxonomist. In the following discussion, the first three
sections summarize the procedure; health and safety concerns; and required resources. Section 6.4
describes the sample receipt and login procedures. Section 6.5 describes the identification steps.
Section 6.6 describes laboratory assistance visits. Section 6.7 identifies the sample and record retention
requirements. Section 6.9 provides a summary of the quality control requirements. The final section
provides references to aid in identifications.
6.1 Summary of Procedures
Each field crew includes a field fish taxonomist approved by EPA based upon a review of their
qualifications and experience. At each site with sufficient fish, field crews collect a minimum of 500 fish
of length greater than 25 millimeters (mm). The fish taxonomist on the field crew ("field fish
taxonomist") quickly identifies and enumerates the fish, and then returns them to the water m.
In accordance with quality control requirements, EPA selected sites ("QC sites") for independent,
taxonomic confirmation. At each QC site, the field fish taxonomist prepares a QC voucher sample that
includes specimens, preserved or digital images, for each taxon of fish collected at the site. The field fish
taxonomist identifies each fish taxon to the lowest practicable level. Instead of specimens, digital images
are required for listed or trophy game specimens that must be released alive, specimens that are too
large to preserve, or very common and easily identified fish types. EPA uses the QC taxonomists'
identifications of the QC voucher samples as a "gold standard" in determining the accuracy of the field
identifications.
In addition to providing vouchers for QC evaluations, the field fish taxonomist may opt to send the QC
taxonomist any specimen, or its image, that cannot be identified in the field ("UNK voucher sample").
The QC taxonomist uses the same identification procedures, regardless of whether the specimens are g
from QC or UNK voucher samples. ^
u
6.2 Health and Safety Warnings ฃ
In addition to the laboratory's usual requirements, laboratories must adhere to the following health and ^
safety procedures: ^
O
1. Laboratory facilities must properly store and dispose of solutions of 10% formalin and/or 75-
95% ethanol.
72
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 73 of 224
2. Laboratory personnel must wear proper personal protection clothing and equipment (e.g. lab
coat, protective eyeware, gloves).
3. When working with potential hazardous chemicals (e.g. formalin, ethanol), laboratory personnel
must avoid inhalation, skin contact, eye contact, or ingestion. If skin contact occurs, remove
clothing immediately. Wash and rinse the affected skin areas thoroughly with large amounts of
soap and water.
6.3 Definitions and Required Resources (Personnel, Taxonomy Laboratories, and
Equipment)
This section provides definitions and required resources for using the procedure. Section 6.3.1 defines
the terms used throughout the procedure. Section 6.3.2 describes the taxonomic expertise required for
each QC taxonomist using the procedure. Section 6.3.3 describes the roles and responsibilities of the
personnel involved in the procedure. Section 6.3.4 identifies the equipment necessary to apply the
procedure in identifying fish voucher specimens in samples.
6.3.1 Definitions
The following terms are used throughout the procedure:
Bench Sheet: Optional form used by the QC taxonomist to record information about the voucher
specimen. See Figure 6.1 for an example.
Dissecting microscope: Microscope that is configured to allow low magnification of three-dimensional
objects that are larger or thicker than the compound microscope can accommodate.
External QC Coordinator is an EPA staff person. For some activities, the External QC Coordinator may
be supported by a contractor that is not involved with field sampling. The External QC Coordinator is
responsible for arranging for laboratory assistance visits; comparing fish voucher identifications by the
field and QC taxonomists; and assessing other quality control issues.
NARS: National Aquatic Resource Surveys. The National Rivers and Streams Assessment (NRSA) is part of
the NARS program.
NARS Information Management System (NARS IM): The IM system established to support all surveys,
including NRSA, in the NARS program. The NARS IM system is used to track the samples from field
collection to the laboratory.
NRSA: National Rivers and Streams Assessment. The samples will be collected during the field stage of
NRSA.
Photovoucher: Digital image of the fish specimen.
QC Site: site where a QC voucher specimen is prepared.
QC Voucher: Fish voucher sample collected at the QC site. ~z.
Reference Collection: A collection of voucher specimens and digital images that provide examples of TT
each taxon identified from the NRSA samples sent to the laboratory. A reference collection is used to ฃ
help with future identifications of unknown specimens, and to provide physical documentation of cc
reported identifications. n:
v u
Tagged lot: A group of voucher specimens believed to represent a single taxon based on field o
identifications. Tagged lots are maintained separately in a voucher sample, and are associated with a -,-
unique voucher tag number. ฃ
73
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 74 of 224
UNK voucher specimen: A voucher specimen of fish that could not be positively identified in the field.
Voucher collection: an assortment of preserved specimens maintained for validating taxonomic
identifications. For each selected QC site, the field crew submits up to 20 examples of each taxon of fish
identified at the site. If the fish is endangered, rare, or too large to preserve, the field crew will provide
digital images instead of physical specimens.
Voucher sample: a collection of voucher specimens from a single site.
Voucher Specimens: Preserved specimens or digital images of one or more individual fish used to
identify the taxa collected at a site.
Voucher Specimen Tag: A pre-numbered paper tag included in the mesh bag with each fish voucher
specimen (or included with each digital image). Tags are used to distinguish between tagged lots in a
sample.
6.3.2 General Requirements for Taxonomists and Taxonomy Laboratories
The procedures may be used by any fish taxonomist and associated taxonomy laboratory that has
expertise in each of the following areas:
Taxonomic expertise. To demonstrate its expertise, the organization with the QCtaxonomists shall
provide EPA with one or more of the following:
1. Memorandum that identifies the relevant services that the organization or its taxonomists
provided for a large national survey in the past five years.
2. A vitae, resume, or equivalent documentation detailing the taxonomic expertise of the
organization and its taxonomists, including coursework, professional certifications, membership
in professional societies, job experience specifically related to fish taxonomy (especially with
taxa that are difficult to identify), and any experience with the curation of museum or other
voucher collections.
3. Quality assurance and quality control requirements. To demonstrate its expertise in quality
assurance and quality control procedures, the organization shall provide EPA with copies of the
quality-related documents relevant to the procedure. Examples include Quality Management
Plans (QMP), QAPPs, and applicable Standard Operating Procedures (SOPs).
4. To demonstrate its ongoing commitment, the person in charge of quality issues for the
organization shall sign the NRSA QAPP Certification Page.
6.3.3 Personnel
QC Taxonomist: A systematic ichthyologist having considerable training and considerable experience in
identifying fish taxa from large regions of the U.S., and experience with difficult groups of taxa. A QC
taxonomist maintains contact with other taxonomists through professional societies and other
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 75 of 224
Fine-tipped forceps (watchmaker type, straight and curved)
Dissecting microscope (3.5-12X magnification)
Dividers and/or calipers for obtaining measurement and proportional measurement data from
specimens
Scalpel
List of standard common and scientific names used in NRSA (Appendix D)
Taxonomic keys or published descriptions in up-to-date, regional faunal works including online
resources
Keys or descriptions for the most recent, accepted taxonomic revisions of individual groups
Descriptions in other taxonomic studies, especially original descriptions (preferably published).
Voucher samples
o Sample tracking form that accompanies each shipment of samples
o Site location
o Physical specimens or image file
o Access to additional site information upon request to EPA
Reporting data template file
6.4 Sample Receipt
Field crews ship the QC and UNK voucher specimens to a central facility ("batching laboratory") or the
State's QC laboratory. Periodically, the batching laboratory ship voucher specimens to the QC
laboratory.
Under U.S. regulations, samples preserved in formalin are classified as "Dangerous Goods" and must be
shipped according to hazardous material shipping requirements and regulations. Personnel receiving
the shipment must be certified to handle hazardous material.
Because EPA initiates tracking procedures designed to recover any missing shipment, start the following
login steps within 24 clock hours of receiving a delivery.
1. Report receipt of QC voucher samples (including digital images) in the NARS IM sample tracking
system (within 24 clock hours).
2. Inspect each QC voucher sample THE SAME DAY THEY ARE RECEIVED:
a. Verify that the sample IDs on the jar labels and the tag numbers in the shipment match
those recorded on the:
i. Chain of custody forms when the batching laboratory sends the samples to the QC
laboratory; or
ii. Sample tracking form if the field crew sends the shipment directly to the State QC (s>
laboratory. ^
b. Verify that all jars for each sample are included in the shipment (i.e., there may be cases u
LJJ
where multiple jars are required to properly preserve specimens collected at a site). 5i
c. Complete the Condition Code for each sample: LU
i. OK: Sample is in good condition ^
ii. C: Sample container was cracked >
iii. L: Sample container is leaking ^
75
-------
Laboratory Operations Manual
Page 76 of 224
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
iv. ML: Sample label is missing
v. NP: Sample is not or insufficiently preserved
vi. 5: Sample shipping problem
vii. Q: Other quality concerns, not identified above
d. A few samples may be preserved with ethanol or isopropyl alcohol rather than formalin. If
the crew used either of these, replace the preservative with formalin.
3. Store preserved samples at room temperature until identification begins.
4. Maintain the chain of custody or sample tracking forms with the samples.
5. Transfer any photovoucher images included in the shipment to a suitable office computer for
viewing.
a. Organize image files by site ID and then by file sequence.
b. The computer should have the graphics capability to view images in high-resolution, and
regularly scheduled backup of files.
6. If necessary to support the identifications, request site information (e.g., site coordinates, state,
and river) for the samples from the NARS IM staff. You should receive a tabular file with this
information.
Table 6.1 Fish voucher: required data elements- login
FORMAT DESCRIPTII
DATE RECEIVED
SITE ID
MMDDYY
text
Name or abbreviation for QC laboratory
Date sample was received by lab
NRSA 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 (and on sample label)
TAG NUMBER
numeric
Tag number as used on field sheet (and 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
OK
ML
NP
Definition
Sample is in good condition
Sample container is cracked
Sample or container is leaking
Sample label is missing
Sample is not or insufficiently preserved
Sample shipping problem (explain in QA_COMMENTS field)
Other quality concerns, not identified above
6.5 QC Identification
The QC taxonomist identifies the common name at the lowest practical level, of the fish voucher
specimens, or photovouchers, as described in the following steps. For each sample, the QC taxonomist
will record the identifications on the bench sheet or in an electronic spreadsheet file. Figure 6.1 provides
an example of a bench sheet that may be used. Before submitting the results to EPA, the laboratory staff
must provide the required information using EPA's data template (required codes are provided in Table
6.2). The steps are described below:
u
LJJ
CL
l/l
U
z>
o
I
l/l
76
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 77 of 224
1. Process the QC voucher sample by voucher tag.
2. For preserved specimens:
a. Remove a bag of specimens from the jar. Record the voucher specimen tag number on the
bench sheet. Transfer the specimens from the bag to a dissecting tray. Arrange specimens
by size and/or potential taxon (if you think the tagged lot includes more than one taxon).
b. View each specimen to identify necessary diagnostic characters using:
i. Well-lighted conditions; and
ii. A dissecting microscope, magnification 3.5X to 12X (larger fish may not require
magnification).
c. Vary lighting (reflected or transmitted) and specimen conditions (wet or dry), as needed, to
maximize the ability to observe different characters.
d. Dissect the specimen, if necessary, to observe internal characters. For example:
i. Remove pharyngeal teeth; or
ii. Incise abdomen to observe peritoneum or viscera.
3. For photovouchers:
a. For images of whole fish, examine size, color, and external characters. Use zoom feature to
enlarge specific areas of the image if necessary.
b. Some specimens may have multiple images that include close-ups of specific morphological
features (e.g., mouthparts, fins, etc.).
c. Each image of a whole fish should have some kind of measurement scale reference (e.g.,
ruler). Use dividers to estimate proportional measurements based on the scale represented
in the image.
4. Unless thoroughly familiar with all regional species in the family (or genus), select appropriate
taxonomic literature to use as references in identifying the specimens based upon the
characters observed in the preceding steps. Appropriate references (see Walsh and Meador
[1998] for examples) include:
a. Keys or descriptions in up-to-date, regional faunal works (including online resources), such
as the references provided in Section 6.9 as examples;
b. Keys or descriptions in the most recent, accepted taxonomic revisions of individual groups;
c. Descriptions in other taxonomic studies, especially original descriptions.
d. Comparison with museum collections, if expert taxonomists consider the museum's
identifications to be reliable.
e. If controversy exists for a particular taxon in the scientific literature, use the identification
consistent with recent publications.
CO
5. Use the written descriptions and illustrations in determining the taxon. For example, consider if "z.
the specimen has: ^
u
a. Diagnostic and other characters consistent with known characters for the taxon. ฃ
CO
b. Size within known size ranges. cc
LJJ
c. Site information data (e.g., geographic coordinates, drainage basin, stream or river name) J
indicate the collecting locality that falls within the known range of the taxon. O
77
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 78 of 224
6. If identifications are problematic or difficult (e.g., specimens or images do not contain fully
diagnostic characters), even after consulting the references in Step 4, consider the following
aspects:
a. Mature males in breeding condition present colors and/or tuberculation that can be used to
identify the taxon. Assume that their taxon identification applies to non-breeding male and
female specimens with similar characters.
b. Mature specimens that have a more developed morphology can be used to identify the
taxon. Assume that their taxon identification applies to less developed specimens with
similar characters.
c. Location can be used as the basis of identification if the taxon is well-studied or allopatric
(e.g., speciation that occurs when populations of the same species become isolated from
each other such that they do not occur in any one place together.)
d. Hybrids are typically detected by presence of diagnostic characters of two closely related
taxa.
e. Commonly-stocked hybrids, such as muskellunge x northern pike (tiger muskie), or striped
bass x white bass (wiper), are diagnosed in many keys.
f. Backcrosses (i.e., progeny of crosses of hybrids and a parent species) may not be
distinguishable from the parent species on the basis of visual and partially dissected
characters. Because the evidence is consistent with non-hybrid characters, assume that the
specimen is not a hybrid.
7. If you cannot confidently identify a specimen, consult with another taxonomist who specializes
in the group. If necessary, provide them with the specimen(s) and associated information.
8. Record the final identification(s) on the bench sheet or data submission worksheet.
a. Use common or scientific names from the NRSA master taxa list (APPENDIX D: OTHER
PERTINENT ATTACHMENTS).
b. For taxa that are not included on the master taxa list, provide the accepted name. Use the
common name if available and well-recognized, scientific name if a common name is not
available or if it is less likely to be confused with other taxa. Provide the taxonomic
reference for the reported name in the Citation column of the submission worksheet e.g.,
Nelson et al. (2004), FishBase.org).
c. For hybrids that do not have accepted standard names (see S.d.i above), record the
common name of both taxa (e.g., green sunfish x bluegill, cutthroat trout x rainbow trout).
Avoid using non-specific terms.
9. If a tagged lot from the field turns out to include multiple taxa (assuming multiple individuals
CO
were provided): g
a. Record the name and count for one taxon on the current line of the bench sheet or ^
u
submission worksheet ฃ
i/i
b. Record the revised name for the second taxon as a new line on the bench sheet or the oc
LJJ
submission worksheet. jj
i. Record the original existing specimen tag number on the next available blank line of the o
submission worksheet. i
l/l
ii. Mark as New Taxon. Record the new name. u-
78
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 79 of 224
c. Provide a comment for any taxa that could not definitively be identified using the list
provided by EPA. Here are examples of situations that require comments:
i. Specimen cannot be identified to the lowest level provided in the master taxa list
because:
1) State of preservation prevented observation of diagnostic characters (e.g., loss of
diagnostic characters prior to preservation).
2) Inconsistencies among or between diagnostic characters and other aspects (e.g.,
other characters, size, range, habitat).
3) Incomplete taxonomic or faunal knowledge for group in generally accepted
references (keys).
4) Other factors, explained in Comments column.
ii. Identification is tentative and based upon:
1) Review of digital images (this qualifier is only necessary if the digital image did not
provide enough information to make a definitive identification).
2) Identification was based upon other specimens (e.g., breeding males) in the sample
with similar characters.
3) Location or habitat information provided from NARS IM staff.
10. Check and correct the final common (or scientific) names recorded on the bench sheet or
submission worksheet for the following common problems.
a. Abbreviations
b. Extra information identifiers (e.g., sp., spp.,, nr., cf., species A)
c. Extra characters (e.g., "?")
d. Extraneous words (e.g., "probably", "prob", "may be")
e. Common misspellings
f. Names not in the master list. Provide a taxonomic reference for these.
g. Incorrect citation or old name
11. Return the specimens to the bag along with the voucher tag. Place the bag back into the sample
jar. See Section 6.7 for retention requirements.
12. Select another bag and repeat Steps 1-10.
13. If using bench sheets, transfer the information to the data submission worksheet file that EPA
has provided. The worksheet can be found at https://nars.sharefile.com/ in the NRSA 2013-
2014/Data Templates folder. Table 6.2 identifies the contents and formats used by EPA's file.
14. Save the submission worksheet file using the following convention: Taxonomist
name+affliation-abbreviation+submission date (yyyymmdd).xlsx (e.g.,
JSmith_ABCfirm_20130615.xlsx). |
15. Prepare a list of primary and secondary technical literature used in completing the ^
identifications (Step 4). Provide complete citations in bibliographic format, including authors' ฃ
i/i
names, date of publication, title of document, name of journal or publisher, volume and page oc
numbers, or ISBN number, as appropriate. If using on-line resource, include a screen capture of jj
the URL/data source. Keep the references on file, and provide to EPA if requested. O
79
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Table 6.2 Fish voucher: required data elements- data submission worksheet
Laboratory Operations Manual
Page 80 of 224
FIELD FORMAT DESCRIPTION
LAB ID
DATE RECEIVED
SITE ID
VISIT NUMBER
SAMPLE ID
TAG NUMBER
DATE COLLECTED
TAXONOMIST NAME
DATE IDENTIFIED
NEW TAXON
COMMON OR
SCIENTIFIC NAME
NUMBER OF
INDIVIDUALS
CONDITION CODE
DATA FLAG (if
appropriate)
text
text
text
numeric
numeric
text
MMDDYY
text
MMDDYY
Text (Y/blank)
text
numeric
text
text
Name or abbreviation for QC laboratory
Date sample was received by lab
NRSA site ID code as recorded on sample label or tracking form
Sequential visits to site (1 or 2)
6-digit Sample ID number as recorded on sample jar or tracking form
Voucher tag number (01, 02, etc.) included with tagged lot (bag in
sample jar) or digital image
Date sample was collected
Name of QC taxonomist
Date first specimen identified in sample
Indicates a new taxon identified from multiple taxon presented in a
single tagged lot in the sample. This may or may not represent a new
taxon to the NRSA master taxa list.
Unique common name from NRSA master taxa list (Appendix D). For
taxa not included on the NRSA master taxa list, record the common or
scientific name (whichever is more widely recognized and/or less
ambiguous). Provide a citation for the reported name in the Citation
column.
The number of individuals of taxa noted
Condition codes associated with the condition of the sample upon
arrival at the laboratory.
Flag
OK
C
L
ML
NP
S
UN
Q
Definition
Sample is in good condition
Sample container is cracked
Sample or container is leaking
Sample label is missing
Sample is not or insufficiently preserved
Sample shipping problem (explain in QA_COMMENTS field)
Unknown. Specimen has been sent to expert taxonomist for
definitive identification.
Other quality concerns, not identified above
Data qualifier codes associated with specific identifications of voucher
samples. These codes provide more information that those used when
reporting receipt of samples. A QC taxonomist may use alternative or
additional qualifiers if definitions are provided to the external QC
Coordinator as part of the submitted data package (e.g., as a separate
worksheet page of the data submission file).
Flag
CF
DD
IM
L
NP
Definition
Tentative identification due to inconsistent characters; no
comprehensive reference for genus; etc. (explain in COMMENTS
field)
Damaged Specimen
Young-of year or too young/small to identify
Identification based upon location
Not enough preservative used
80
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 81 of 224
COMMENTS
CITATION
text
text
UN
Q
Specimen/ or tagged lot could not be positively identified, even
after consultations with other taxonomic specialists
Other quality concerns, not identified above
Explanation for data flag(s) (if needed) or other comments regarding the
taxon lot.
Citation for reference used to identify organism, if taxon not present in
NRSA list offish names (APPENDIX D: OTHER PERTINENT
ATTACHMENTS)
6.6 Assistance Visits
The External QC Officer may arrange for an assistance visit to one or more QC taxonomists. Assistance
visits are intended to familiarize EPA with actual procedures being implemented by different QC
taxonomists; and to ensure a clear and consistent understanding of procedures and activities by both
EPA and QC taxonomists. If EPA decides to conduct an assistance visit, a qualified EPA scientist or
contractor will administer a checklist based upon the steps described in this chapter. A checklist will be
developed and attached to the LOM before conducting an assistance visit.
6.7 Sample and Record Retention
The laboratory shall retain:
1. The sample materials for a minimum of 3 years from the date the EPA publishes the final report.
During this time, the laboratory shall store the materials in a cool location away from sunlight.
The laboratory shall periodically check the sample materials for degradation and refill jars with
preservative if necessary.
2. Original records, including laboratory notebooks and the reference library, for a minimum of 10
years from the date that EPA publishes the final report.
After the stated time periods, the laboratory shall follow its internal protocols for disposal.
6.8 Summary of QC Requirements for Fish Voucher Specimens
Percent taxonomic disagreement (PTD): measure of taxonomic precision comparing the number of
agreements (positive comparisons, comppos) of the field taxonomist and QC taxonomist. In the following
equation, N is the total number of specimens in the larger of the two counts. PTD should be <15%.
PTD =
1-
comppos
N
xlOO
Table 6.3 Fish voucher: measurement data quality objectives
Variable or Measurement Precision Accuracy Completeness
Identification 85%a 85%a 99%
a As measured by (100%-PTD)
u
LJJ
CL
l/l
QC
U
z>
o
I
l/l
81
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Table 6.4 Fish Voucher: quality control -taxonomic identification
Laboratory Operations Manual
Page 82 of 224
Check or Sample
Description
Use
widely/commonly
accepted taxonomic
references
Independent
identification by
outside, expert,
laboratory fish
taxonomist ("QC
taxonomist")
External QC
Calculate average
PTD for field
taxonomist
Conduct assistance
visit
All identifications
When field taxonomist
cannot identify
specimen
Approximately 10% of
all sites sampled by
each field taxonomist
Each sample
submitted to the QC
taxonomist
EPA may choose to
visit any laboratory
Acceptance Criteria
All keys and references used
must be on bibliography
prepared by the field and QC
taxonomists
Identification by QC
taxonomist (who must be a
different individual than the
field taxonomist)
PTD < 15%
PTD < 15%
Visit conducted using checklist
Corrective Action
For all field crew identifications,
EPA will convert field crew's use
of common names to taxonomic
references
Replace field crew's "unknown"
identification with
determination by QC
taxonomist
If PTD > 15%, review data for
possible explanations;
otherwise, insert data qualifier
for field crew identifications
If PTD > 15%, consult with NARS
QA Officer for appropriate
action.
Performance and any
recommended improvements
described in debrief with
laboratory staff
Table 6.5 Fish voucher: data validation
Check or Sample
Description
Taxonomic
"reasonable-ness"
checks
Frequency
All data sheets
Acceptance Criteria
Genera known to occur in
given rivers/streams or
geographic area
Corrective Action
Data qualifiers on data that fail
reasonableness check. No
further corrective action steps.
6.9 References
Froese, R. and D. Pauly. Editors. 2011.FishBase. World Wide Web electronic publication.
www.fishbase.org, version (10/2013).
Nelson, J. S., E. J. Grossman, H. Espinosa-Perez, L T. Findley, C. R. Gilbert, R. K. Lea, and J. D. Williams.
2004. Common and Scientific Names of Fishes from the United States Canada and Mexico. Sixth edition.
Special Publication 29, American Fisheries Society, Bethesda, Maryland.
Stribling, J. B., K. L. Pavlik, S. M. Holdsworth, and E. W. Leppo. 2008. Data quality, performance, and
uncertainty in taxonomic identification for biological assessments. Journal of the North American
Benthological Society 27:906-919.
Walsh, S. J. and M. R. Meador. 1998. Guidelines for quality assurance and quality control of fish
taxonomic data collected as part of the National Water-Quality Assessment Program. Water-Resource
Investigations Report 98-4239, US Geological Survey, Raleigh, North Carolina.
u
LJJ
CL
l/l
U
z>
o
I
l/l
82
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 83 of 224
Laboratory Information Sample Information
Labname
QC Taxonomist
Project ID
Internal Sample ID
Sample ID
Site ID
Visit Number
Date Collected
Taxonomist Name
Date 1st Specimen Identified in Sample:_
Tag New Condition Data Comment Including Citation if
No. Taxon? Common or Scientific Name Code Flag Appropriate
Figure 6.1 Fish voucher: example QC fish taxonomy bench sheet (optional)
Additional Comments
u
LJJ
CL
l/l
CC.
U
z>
o
I
l/l
83
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 84 of 224
7.0 FISH TISSUE FILLET (WHOLE FISH COLLECTION)
Laboratory Methods incorporated in OST Manuals.
u
0)
o
u
_QJ
O
CO
i/l
i/l
84
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
8.0 FISH TISSUE PLUG
Laboratory Operations Manual
Page 85 of 224
This procedure is applicable to the analysis of mercury in fish tissue plugs. The method is performance
based. Laboratories may use any method that meets the requirements below to analyze the fish tissue
samples (for example, EPA Method 1631). Example SOPs are provided in APPENDIX F: EXAMPLE SOPs
FOR MERCURY IN FISH TISSUE PLUG ANALYSES.
Table 8.1 Fish tissue plug: measurement data quality objectives
Variable or Measurement MDL
Quantitation Limit
Mercury
0.47 ng/g 5.0 ng/g
Table 8.2 Fish tissue plug: quality control
Activity
Demonstrate competency
for analyzing fish samples to
meet the performance
measures
Evaluation/Acceptance Criteria Corrective Action
Demonstration of past
experience with fish tissue
samples in applying the
laboratory SOP in achieving the
method detection limit
EPA will not approve any laboratory for NRSA
sample processing if the laboratory cannot
demonstrate competency. In other words, EPA
will select another laboratory that can
demonstrate competency for its NRSA
samples.
Check condition of sample
when it arrives.
Sample issues, such as punctures
or rips in wrapping; missing
label; temperature; adherence
to holding time requirements;
sufficient volume for test. All
samples should arrive at the
laboratory frozen.
Assign appropriate condition code identified in
Appendix 3.
Store sample appropriately.
While stored at the
laboratory, the sample must
be kept at a maximum
temperature of -20ฐ C.
Check the temperature of the
freezer per laboratory's standard
operating procedures.
Record temperature of sample upon arrival at
the laboratory. If at any other time, samples
are warmer than required, note temperature
and duration in comment field.
Analyze sample within
holding time
The test must be completed
within the holding time (i.e., 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. EPA expects that the
laboratory will exercise every effort to perform
tests before the holding time expires.
Maintain quality control
specifications from selected
method/SOP (that meets
the measurement data
quality objectives)
Data meet all QC specifications
in the selected method/SOP.
Maintain the required MDL
If data do not meet all QC requirements, rerun
sample or qualify data. If the lab believes the
data are to be qualified without rerunning
sample, the lab must consult with the EPA
Survey QA Lead before proceeding.
Evaluate for each sample
If MDL could not be achieved, then provide
dilution factor or QC code and explanation in
the comment field.
(D
i
CL
CO
i/l
i/l
Use consistent units for QC
samples and field samples
Verify that all units are provided
in wet weight units and
consistently
If it is not possible to provide the results in the
same units as most other analyses, then assign
a QC code and describe the reason for
different units in the comments field of the
database.
85
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 86 of 224
Maintain completeness Completeness objective is 95% Contact the EPA Survey QA Lead immediately
for all parameters. if issues affect laboratory's ability to meet
completeness objective.
(D
i
CL
CO
i/l
i/l
86
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 87 of 224
9.0 PERIPHYTON
This procedure is applicable to the analysis of the diatom and soft algal components of algal samples
collected for the National Rivers and Streams Assessment (NRSA) 2013-2014. NRSA field crews collect
composite samples of the algae present on benthic and vegetative surfaces; preserve samples with
formalin to obtain a pH of 7; and ship the samples to a central holding facility or directly to a state
laboratory. Each 50mL composite sample contains both diatoms and soft algae from benthic and
vegetative surfaces. This procedure describes the steps for identifying and enumerating periphyton
organisms in the samples and has been adapted from Protocols for the analysis of algal samples
collected as part of the U.S. Geological Survey National Water-Quality Assessment program (Charles et
al., 2002).
In the following discussion, Sections 9.1, 9.2, and 9.3 summarize the procedure; health and safety
concerns; and required resources. Section 9.4 provides the steps for acknowledging sample receipt.
Section 9.5 provides the steps for subsampling and preparing the vials for the diatom and soft algal
analyses. Section 9.6 provides the steps for the diatom cleaning and slide mounting. Section 9.7
describes the steps for diatom and all algal analyses. Sections 9.8 and 9.9 provide the steps for the data
entry, and sample and record retention. Section 9.10 describes EPA's external quality control evaluation.
Section 9.11 identifies references used in developing the procedure. The Attachment summarizes the
quality control measures.
9.1 Summary of Procedure
The procedure describes the extraction and separate analysis of the two subsamples of: 1) diatoms and
2) composite algae consisting of soft algae and diatoms. Figure 9.1 provides a graphical summary of the
procedure.
As described in Section 9.5, laboratory personnel subsample the liquid portion by volume and separate
the heavier material that is difficult to suspend by mass. This step removes (oxidizes) organic matter
from the siliceous frustules of diatoms and other material such as the soft algae in the subsample.
Removing the organic matter is necessary so that all details of diatom structures essential to taxonomic
identification are clearly visible.
As described in Section 9.6, laboratory personnel prepare slides by cleaning and mounting materials for
identification and enumeration of diatoms. The laboratory technician mounts the cleaned material
between a microscope slide and coverslip using a mounting medium with a high refractive index. With
slides prepared with the mounting medium, the features of diatom frustules or valves are clearly visible
at high magnification. The distribution of specimens on the final mounted coverslips will represent the
samples contained within the cleaned material vials.
Also as described in Section 9.6, taxonomists spend a maximum of 8 hours to perform the diatom
subsample analysis to estimate the proportion of diatom taxa found in a count of 600 valves (i.e., one-
half of an individual diatom cell). For each observed species in the diatom subsample analysis, the
taxonomist must verify that the laboratory's NRSA library includes the average biovolume measurement
and a photograph.
As described in Section 9.7, the taxonomists identify, count, document, and measure all algae. Often
this protocol is referred to as the soft-algal count because of the emphasis on identification of non-
diatom algae. For this reason, this document refers to "soft algae" instead of "all algae." The procedure
is designed to provide data on algal densities and amount of algal biovolume. It is the same procedure
used by NRSA in 2008-2009.
LJJ
CL
87
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 88 of 224
Sample Receipt:
* 250 ml cornposiie algal
sample
Figure 9.1 Periphyton: summary of procedure for identifying soft algae and diatoms
9.2 Health and Safety Warnings
In addition to the laboratory's usual requirements, personnel must implement the following health and
safety procedures for this procedure:
1. Wear or apply proper personal protection clothing and equipment (e.g. lab coat, protective
eyewear, gloves).
2. When working with potential hazardous chemicals (e.g. a mounting medium with a high
refractive index such as Naphrax) or biological agents (algae and sediments), avoid inhalation,
skin contact, eye contact, or ingestion. If skin contact occurs, remove clothing immediately,
wash, and rinse the affected skin areas thoroughly with large amounts of soap and water.
O
LJJ
CL
88
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 89 of 224
9.3 Definitions and Required Resources (Laboratory, Personnel, and Equipment)
This section provides definitions and required resources for using this procedure. Section 9.3.1 defines
the terms used throughout the procedure. Section 9.3.2 describes the expertise required for each
laboratory using the procedure. Section 9.3.3 describes the roles and responsibilities of the personnel
involved in the procedure. Section 9.3.4 identifies the equipment necessary to apply the procedure in
preparing, sorting, and identifying algal samples.
9.3.1 Definitions
The procedure uses the following terms:
ANC: Academy of Natural Sciences
Biovolume: refers to the volume of algal cells.
Burn Mount: Rarely used. Refers to a procedure where a known portion of the untreated sample is
dripped onto a coverslip and allowed to dry at room temperature and then placed on hot plate.
DCF: Dilution/Concentration Factor.
Digestion refers to the solubilization of organic material by strong acid oxidation.
Frustules are the siliceous, inorganic component (SiO2) of diatom cells. Each diatom cell is composed of
a frustule and organic cytoplasm. The frustule is composed of two valves. The taxonomist uses the
features of the frustule to identify the taxon.
NARS: National Aquatic Resource Surveys. The National Rivers and Streams Assessment (NRSA) is part of
the NARS program.
NARS Information Management (IM) System: The NARS IM system established to support all surveys,
including NRSA, in the NARS program. The NARS IM system is used to track the samples from field
collection to the laboratory.
Natural counting unit. Algae grow unicellularly or in multicellular filaments and colonies. Each natural
grouping of algae (i.e., each individual filament, colony, or isolated cell) is defined as a natural counting
unit. Diatoms are an exception; each diatom cell is always considered a natural counting unit, even if
attached to other cells. The main purpose of using 'natural counting units' is to prevent a colonial or
filamentous form from dominating a count. It also facilitates the counting of algal forms which have
linked cells that may be hard to distinguish.
NRSA: National Rivers and Streams Assessment. The samples were collected during the field stage of
NRSA.
Organic matter: Material that is capable of decay or the product of decay (e.g., leaves, sticks, algae).
Palmer-Maloney Fraction. A diluted or concentrated sample derived from the original sub-sample and
used for the Palmer-Maloney count described in Section 9.7.
Reverse Osmosis (RO) is a membrane-technology filtration method that removes many types of large
molecules and ions from solutions by applying pressure to the solution when it is on one side of a
selective membrane.
Percent Difference (PctDiff) is a measure for soft algae that compares the enumerations for the taxa
within a sample, as reported by the primary taxonomist (a) and secondary taxonomist (b) as follows:
LJJ
CL
89
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
PctDiff =
species
1-
# species
Laboratory Operations Manual
Page 90 of 224
X 100
Percent disagreement in enumeration (PDE) is a measure of taxonomic precision comparing the
number of organisms, nlt counted in a sample by the primary taxonomist with the number of organisms,
n2, counted by the secondary taxonomist.
n, -
PDE = - -xlOO
ซ! +n2
Percent taxonomic disagreement (PTD) is a measure of taxonomic precision comparing the number of
agreements (positive comparisons, comppos) of the primary taxonomist and secondary taxonomists. In
the following equation, N is the total number of organisms in the larger of the two counts.
PTD =
comP
pos
N
xlOO
Primary laboratory: The laboratory that first receives the sample for analysis. The primary laboratory is
responsible for: 1) preparing the subsamples and diatom slides; and 2) providing the original
identification of diatoms and soft algae.
Secondary laboratory: The laboratory that performs a second, independent, quasi "gold standard"
evaluation of a sample, as part of the external quality control evaluation.
Subsample: Portion of the sample obtained by randomly extracting volume from the composite sample
collected by the field crew.
Target taxonomic level: species
Taxonomic nomenclature: All laboratories must identify taxon using the information provided in the
BioData. The algae list is provided at the following website:
https://aq uatic.biodata.usgs.gov/domainDownloads. action.
9.3.2 Laboratory
To participate in NRSA, each laboratory first must demonstrate to EPA that it has appropriate
qualifications as follows:
1. Analytical work. To demonstrate its expertise, the laboratory shall provide EPA with one or more
of the following:
2. Memorandum that identifies the relevant services that the laboratory provided for the National
Aquatic Resource Surveys in the past five years.
3. Memorandum that describes the laboratory's participation in relevant round robin studies
and/or performance studies.
4. Report of relevant findings from an on-site technical assessment or audit.
5. Quality procedures.
6. To demonstrate its expertise in quality assurance and quality control procedures, the laboratory
shall provide EPA with copies of the quality-related documents relevant to the procedure.
Examples include Quality Management Plans (QMP), Quality Assurance Project Plans (QAPPs),
and applicable Standard Operating Procedures (SOPs).
o
LJJ
CL
90
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 91 of 224
7. To demonstrate its ongoing commitment, the person in charge of quality issues for the
laboratory shall sign the NRSA QAPP Certification Page.
8. Reporting standardized data.
9. To demonstrate its expertise, the laboratory shall provide EPA with a memorandum that
confirms that the laboratory has a computerized Laboratory Information Management System
(LIMS) that is routinely used to track samples and record laboratory results.
10. To demonstrate its ongoing commitment to use LIMS, the memorandum also shall confirm that
the laboratory will use LIMS to record and report results from the procedure.
9.3.3 Lab Personnel
Any person who has received training in processing and identification of algae may use the procedure;
however, the work is generally parsed out to several personnel:
Quality Control (QC) Officers provide oversight of daily operations, sample processing, monitors QC
activities to determine conformance, and conducts performance and systems audits of the procedures.
The procedure has separate responsibilities for the:
Internal QC Taxonomist is a senior taxonomist at the laboratory who has at least 10 years of
experience in analyzing algal samples.
External QC Coordinator is an EPA staff person who manages the QC contractor. Because the
assigned duties are primarily administrative in nature, the External QC Coordinator is not
required to have laboratory experience, although such experience would be preferable.
Taxonomists are trained, and have considerable experience, in identifying algae, 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. Each laboratory must submit the resume or curriculum vitae for the
taxonomists who analyze NRSA samples.
Algal Analysts are taxonomists who specialize in the identification of algal specimens.
Diatom Analysts, or diatomists, are taxonomists who specialize in the identification of diatoms.
Lab Technicians are technicians who have basic training in laboratory procedures.
9.3.4 Equipment/Materials
The procedure requires the following equipment and materials for sample preparation (subsampling)
and taxonomic identifications:
9.3.4.1 Subsampling Equipment/Materials (Section 9.5)
Distilled (DW) or reverse osmosis (RO) water
Dispenser bottle for DW or RO water
Beakers
Beaker holding box (24 slots)
Graduated cylinders
Vials with 1 mL intervals marked on it for measuring volumes
Positive-draw fume hood
LJJ
CL
91
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 92 of 224
Screen cloth (210-um mesh)
Screening apparatus
Large plastic disposable weighing boats
Analytical balance, capacity to 500 g, 0.2 g accuracy
Spatulas
Plastic disposable pipettes (tips of plastic pipettes can be cut)
Small turkey baster
9.3.4.2 Diatoms
9.3.4.2.1 Cleaning Equipment/Materials (Section 9.6 Diatom Cleaning and Mounting)
See equipment and materials identified in ANS Protocol P-13-42 "Diatom Cleaning by Nitric Acid
Digestion with a Microwave Apparatus" (http://diatom.ansp.org/nawqa/pdfs/P-13-42.pdf)
9.3.4.2.2 Preparation of Diatom Slides (Section 9.6)
Corning ceramic-top hot plate with temperature control
Positive-draw chemical hood
Aluminum drying plate (25.5 x 20.0 x 0.5 cm, solid aluminum; lines forming 48 squares, each 3.2
cm on a side, are etched on the surface. Each square is etched with an identifying number)
Glass microscope slides (1x3 inches; 2.5 x 7.5 cm)
Glass coverslips (18 mm x 18 mm or 22 mm x 22 mm) - No. 1 thickness, stored in covered glass
jar filled with 100 % ethanol.
Mounting medium with a high refractive index (e.g., Zrax, Naphrax)
Diamond scribe
Disposable plastic pipettes
Adjustable pipettor (0 - 250u.l); adjustable pipettor (200 - 1000u.l)
Pipette tips for adjustable pipettors
Round-style tooth picks
Forceps
Polished, rounded wooden splints
Wash bottle filled with distilled (DW) or reverse osmosis (RO) water
Single-edged razor blades
Ethanol, 70%
Acetone
Tissues (e.g., Kimwipeฎ)
Slide labels
Wax (the kind commonly used for candle making and canning foods)
10% HCI
9.3.4.2.3 Analysis of Diatoms (Section 9.7Analysis of Diatoms and Soft Algae) Q
Compound microscope with transmitted light including: >
o Objective lenses of at minimum (lOx, 40x, lOOx) E:
o Oil immersion objective (lOOx) with a minimum numerical aperture of 1.3 ^
92
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 93 of 224
o Substage condenser lens with a minimum numerical aperture of 1.3
o Ocular lenses of 10-15x
o DIG (differential interference contrast) illumination
o Diamond objective marker mounted on the objective turret
o 100 watt halogen illumination, or equivalent
o Digital camera
9.3.4.3 Soft algae (Section 9.7)
Compound microscope with 20x objective for a total system magnification of 200x and 40-45x
objectives for a total system magnification of 400-450x.
Glass microscope coverslips, rectangular, 26 x 60 mm, #1 thickness.
Glass, wide-bore pipettes > 1 mm inside diameter, or eye-dropper.
Two Palmer-Maloney Counting Cells with ceramic chamber (chamber depth of 0.4 mm; volume
of 0.1 ml) or metal chambers
Glass Pasteur pipettes, 5.25 inch, < 1 mm diameter.
Glycerin
Digital camera
9.4 Sample Receipt
Under U.S. regulations, shippers must classify samples preserved in formalin as "Dangerous Goods" and
handle according to hazardous material shipping requirements. At the laboratory, only personnel
certified to handle hazardous material can receive the hazardous samples. Because EPA initiates tracking
procedures designed to recover any missing shipment, the laboratory personnel start the following login
steps within 24 clock hours of receiving a delivery.
1. Record receipt of samples in the NARS IM system (within 24 clock hours) and the Laboratory's
Information Management System (LIMS). Assign the appropriate chronological bench number to
each sample.
2. Inspect each jar THE SAME DAY THEY ARE RECEIVED:
a. Verify that the site identification and sample number on the label also appear on the chain
of custody form in the shipment.
b. Notify the NARS IM team if any jars were broken and/or if there are discrepancies between
the custody form and the 50 mL bottles.
c. Verify that the bottles contain enough formalin. If not, then:
i. Add 2 mL of a 10% formalin solution
ii. Continue to add, if necessary, increments of 2 mL of the solution. Check after each
addition to determine if additional solution is required.
iii. Record the amount of solution added to the bottle in the Comments_Arrival field of
the Login spreadsheet (Table 9.1).
3. Maintain the chain-of-custody form with the samples; it will be needed if the samples are ^
transported to any other location (e.g., for taxonomic identification, external QC evaluation). ฃ:
4. Verify that the login information includes the required data elements in Table 9.1. After ^
Cฃ
completing all required elements, provide the information to the data entry personnel. ^
93
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Table 9.1 Periphyton: required data elements - login
Laboratory Operations Manual
Page 94 of 224
FIELD FORMAT DESCRIPTION
LABJD
DATE_RECEIVED
SITEJD
VISIT_NUMBER
SAMPLEJD
DATE_COLLECTED
CONDITION_CODE
FORMALIN ADDED
COMMENTS_ARRIVAL
text
MMDDYY
text
numeric
numeric
MMDDYY
text
numeric
text
Name or abbreviation for QC laboratory
Date sample was received by lab
NRSA site id as used on sample label
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.
CODE
OK
C
HM
L
TL
ML
PA
0
Definition
Sample is in good condition
Sample container is cracked
Sample contains heavy amounts of sand, silt, or other heavy
material that may interfere with the algal analysis
Sample or container is leaking
Too little sample left for the procedure
Sample label is missing
Preservative added, add amount in comments field
Other quality concerns, not identified above
If the laboratory added formalin to the bottle, provide the amount in
milliliters. If no formalin was added, then the value should be zero or
blank.
Any comments about the condition of the sample upon arrival.
9.5 Sample Preparation (Subsampling)
This section describes the steps to prepare subsamples from samples that have relatively small amounts
of sand, silt, or other heavy material. EPA modified the following steps from ANS Protocol P-13-48
"Subsampling Procedures for USGS NAWQA Program Periphyton Samples"
(http://diatom.ansp.org/nawqa/pdfs/P-13-48.pdf). If a sample contains large amounts of sand, silt, or
other heavy material that the technicians determines could interfere with algal analysis, the technician
should consult ANS P-13-48 for additional guidance (see Step 7.5).
In preparing the subsamples, the laboratory technician shall:
1. Abide by the following Safety Precautions:
a. Wear safety glasses, protective gloves and lab coats at all times when handling concentrated
samples with formalin.
b. Use a positive-draw fume hood at all times to avoid exposure to formalin.
2. Keep the laboratory room and bench surfaces as clean as possible and free of debris to avoid
cross-contamination of samples.
3. Verify that all necessary supplies and equipment are available. See Section 9.3.4 for
recommended supplies and equipment.
4. Examine the condition of the sample. If it leaked in transit, and thus, the bottle contains less
than the required 50 mL:
a. If more than 20 mL remains:
O
LJJ
CL
94
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 95 of 224
i. Reduce the volume sub-sampled for soft algal analysis as required. The minimum for
any sub-sample, either for diatom side preparation of soft algae analysis, is 10 ml.
ii. Advise the algae analyst of any reductions in sample volume on handover of the
samples.
b. Otherwise, report the sample as 'lost' at login (Section 9.4).
5. Decide how much to subsample for soft algae and diatoms. This is somewhat of a judgment call,
but, generally, for:
a. Diatoms: Subsample at least 20 ml.
b. Soft algae: Subsample at least 10 ml for soft algae. If the sample is very silty, it will be better
to subsample a minimum of 10 ml for soft algae, whereas if the sample looks clear, at least
20 ml is recommended
c. Always save a small portion of the initial sample, even for soft algae, in case the taxonomist
needs a "burn mount" as described in Section 9.6.
d. Record the volumes in the vials.
6. Select graduated cylinders of appropriate size for measuring subsamples. Verify that:
a. the desired subsample volume will be at least one-third the capacity of the graduated
cylinder; and
b. the units of the graduated cylinder will allow estimation to the nearest milliliter.
7. Separate the sample into 2 subsamples as follows:
a. Suspend the algal material by shaking or swirling the sample
b. Obtain two subsamples by selecting a method that allows a representative subsample. Two
possible methods include:
i. Carefully pour the determined amount of subsample into a graduated cylinder.
Ensure the sample stays well mixed during subsampling.
ii. Use a wide-bore pipette or turkey baster, which allows pumping the pipette or
baster to mix the sample, while withdrawing an aliquot of sample. Take multiple
aliquots with the turkey baster to ensure a representative draw. Filaments and
debris will block pipettes with small openings. Careful pouring or turkey basters may
be more appropriate.
1. Transfer each subsample to a vial. Label each of the two vials (i.e., one for diatoms and the other
for soft algae) with a different Vial ID# using the sample number plus a "D" or "S." For each vial,
record the Vial ID, subsample volume, and intended analysis (i.e., diatom or soft algae).
2. If the laboratory intends to ship any vial to another location, pack it carefully for shipping and
include the appropriate custody forms.
Verify that the subsampling information includes the required data elements in -^
O
LJJ
CL
95
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 96 of 224
Table 9.2. After completing all required elements, provide the information to the data entry personnel.
O
LJJ
CL
96
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Table 9.2 Periphyton: required data elements - subsampling
Laboratory Operations Manual
Page 97 of 224
FIELD FORMAT DESCRIPTION
LABJD
SITEJD
VISIT_NUMBER
SAMPLEJD
VIALJD
SAMPLE_VOLUME
SUBSAMPLE_VOLUME
PURPOSE
DESTINATION
COMMENTS_SUBSAMPLE
text
text
numeric
numeric
numeric or text
numeric
numeric
D/S
text
text
Name or
abbreviation for QC laboratory
NRSA site id as used on sample label
sequential visits to site (1 or 2)
sample id as used on field sheet (on sample label)
Laboratory assigned ID to the vial holding the subsample
Total volume of sample before any processing (from the sample
label)
Volume of subsample in the vial
Codes indicating whether the vial will be used for the diatom or
soft algal analyses .
CODE
D
S
Definition
Diatom Analysis
Soft Algal Analysis
Name of the receiving laboratory (i.e., if the vial will be shipped
to another location)
any comments about the subsampling
9.6 Diatom Cleaning and Mounting
The diatom cleaning steps remove both extracellular and intracellular organic material by digesting it
with nitric acid. After removing the organic matter, all details of diatom structures essential to
taxonomic identification should be clearly visible. To clean the diatoms, the laboratory technician may
follow the cleaning steps in ANS Protocol P-13-42 "Diatom Cleaning by Nitric Acid Digestion with a
Microwave Apparatus" (http://diatom.ansp.org/nawqa/pdfs/P-13-42.pdf) or the following nitric acid
cleaning method:
1. Place the subsample of material for diatoms in a 250 mL beaker.
2. Place the beaker in a positive draw hood. Carefully add 50 mL of concentrated nitric acid.
3. Simmer on a hot plate or warmer for about 3 hours or until organic matter has dissolved. If small
bits of coarser debris remain after acid-cleaning, they can be rinsed and removed at a later time
with forceps.
4. Rinse the diatoms in the cleaned sample repeatedly to remove the nitric acid by repeatedly
adding deionized water to the cleaned diatom sample to fill the beaker, letting the diatoms
settle to the bottom, and then siphoning the water from the beaker before refilling again. This
method is similar to instructions by ANSP, but draw from center of the water column, not the
top. Also gently spin beakers and sprinkler water on the surface with a squirt bottle about
midway through settling to dislodge diatoms adsorbed onto the water surface and sides of
beakers (remember surface tension). We recommend using 1200 mLtall beakers.
5. Let samples in these beakers settle for a total of 8 hours or more.
6. After settling and no additional disturbance of the diatoms on the bottom of the beaker, slowly
and carefully siphon water with a fine tip glass pipette, like a Pasteur pipette. Usually, 20 mL
should be left above the settled diatoms in a 1200 mL tall beaker to minimize siphoning and
discarding diatoms that were adsorbed to the beaker sides or bottom, or even the water
surface. Be certain to avoid siphoning diatoms from the bottom. Watch for diatoms to move
O
LJJ
CL
97
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 98 of 224
from the water surface, sides or bottom of the beaker toward the pipette tip. Quickly remove
the pipette from the water if clouds of suspended diatoms move toward the pipette tip. Rinse
the pipette with a squirt bottle between samples.
7. Concentrate the cleaned diatom suspension to a volume appropriate for mounting on slides by
settling into smaller and smaller containers.
After cleaning the materials, the laboratory technician produces high-quality diatom mounted slides
from the cleaned diatom material. EPA modified the following cleaning steps in ANS Protocol P-13-49
"Preparation of Diatom Slides Using Naphrax Mounting Medium"
(http://diatom.ansp.org/nawqa/pdfs/P-13-49.pdf).
To produce the slides, the laboratory technician shall:
1. Abide by the following Safety Precautions:
a. Wear safety glasses, protective gloves and lab coats at all times when handling liquid and/or
solidified mounting medium. For example, Naphraxb is a hazardous substance because it
contains toluene, which volatilizes readily when heated.
b. Use a positive-draw fume hood at all times for heating of the mounting medium.
c. Use extreme care when manipulating slides on the hot plate and when working close to the
hot plate. Hot plate temperatures required for this procedure are high enough to cause
severe burning of exposed skin.
2. Follow lab practices similar to those used for sterile experiments (bacteriological plating, etc.) to
reduce the risk of cross contamination of samples. At a minimum:
a. Keep the laboratory room and bench surfaces as clean as possible and free of debris.
b. Use disposable pipettes, stirrers, etc. where feasible.
c. Rinse glass (non-disposable) pipettes, stirrers, etc. in DW or RO water at least three times
after each sample. (Explanatory note: at times, tap water, because of algal blooms and use
of diatomaceous earth filters, may contain diatoms.)
d. Dry all equipment before storing it. This step prevents growth of algae and fungi.
3. Verify that all necessary equipment is available. See Section 9.3.4 for recommended supplies.
4. Determine if the vial's contents will produce satisfactory slides. Consider the following:
a. The ideal density to be achieved on the final mount is somewhat subjective and is based on
the amount of debris in the sample, the preferences of the slide analyst, and the way in
which the slide is to be used (e.g., counting, documentation).
b. Generally, between 10 and 20 diatom valves or frustules should be present in a single high
power microscope field (1000X).
c. The number of specimens per field will need to be reduced if samples contain considerable
amounts of silt.
d. In many cases, analysts will request both a "heavy" slide (~40 cells/field at 400 - 450x
magnification) and a "light" slide (~30 cells/field at 400 - 450x magnification).
O
.
b Naphrax is a commercially-available toluene-based mounting medium with high refractive index. ^J
98
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 99 of 224
5. If the vial's contents will not produce satisfactory slides, because the number of diatoms is too
sparse (this could be the result of too few diatoms in the sample and/or the sampled habitat),
then:
a. Determine (i.e., judgement call) if concentrating the vial's contents 2-5 times will be
sufficient to produce satisfactory slides. If yes, then increase the concentration of cleaned
diatom material by two to five times as follows:
i. Allow the vial to settle for approximately 4 hours.
ii. Spin the vial carefully while still resting on the tabletop. This step dissociates
diatoms that have adsorbed onto the side of the vial so they will settle.
iii. Allow the vial to settle for approximately 4 more hours (total of approximately 8
hours)
iv. Use a micropipettor to remove the required amount of water from the vial of
material. Keep track of volume removed from the vial.
v. Record the SUBSAMPLE_DCF (i.e, subsample dilution/concentration factor).
b. If a concentration of cleaned material greater than two to five times is required, then re-
subsample the original sample following the steps in Section 9.5. Use the entire remaining
sample only if necessary. Digest the subsample as described previously (i.e., using ANS
Protocol P-13-42).
c. If the concentration is still not sufficient, then combine two vials of cleaned subsample
materials and concentrate as described in the previous two steps. Also revise the amount
recorded for SUBSAMPLE_VOLUME.
d. If, after following the steps above to concentrate the cleaned material, the density of
diatoms on a coverslip still does not meet the criteria of 30 to 40 cells per field at 400 - 450x
magnification, proceed to make the densest slide possible and consult with a diatom analyst
to determine whether it is practical to analyze the sample. This evaluation includes the
following steps:
i. Scan the slide in its entirety under lOOx magnification, and estimate the total
number of individuals on the slide.
ii. Determine if the slide is countable (i.e., 100 specimens or more can be counted
within 4 hours), considering factors such as:
1) Density of diatoms.
2) Evidence of dissolution
3) Amount of debris (silt, clay, broken remains of diatoms and other siliceous
organisms) that would make it difficult to identify specimens accurately.
iii. Determine if evidence suggests that lightly silicified diatoms in the sample may not
have survived the digestion process in ANS Protocol P-13-42. If so, prepare a "burn
mount" to view later to determine whether diatoms existed in the original sample
and can be identified. (Burn mounts are seldom necessary.)
1) If necessary, prepare a burn mount using the 1973 EPA procedure (USEPA
1973). It contains the following steps:
a) Drip a known portion of the untreated sample onto a coverslip.
b) Dry the coverslip at room temperature.
99
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 100 of 224
c) When the sample is dry, place the coverslip onto a hot plate for about 30
min at approximately 570ฐC.
6. Deposit cleaned material on coverslip as follows:
a. Use forceps to remove single coverslips from the storage container.
b. Carefully clean each by wiping with a tissue (e.g., Kimwipeฎc).
c. Place each coverslip on a marked space. (Be sure this space is clean and dry to avoid cross-
contamination.)
d. Add enough diatom suspension to cover the entire surface of the coverslip, including the
extreme edges. Usually, 1.0 ml is about right for a 22x22 mm coverglass. Add 10% HCI to
the cleared slurry (diatom suspension) (2 drops per 20mL of material in vial) to achieve a
more even distribution on the coverslip. Calculate and record the DRIP_DCF. Note:
i. If the intended drip count will be less than sufficient to cover the coverslip because
the sample is dense (e.g., 1.0 ml of distilled water diatom suspension on a 22 x 22
mm coverslip or 600 ul of acidified suspension), then:
1) Drip an amount of distilled water onto the coverslip with a disposable pipette
that will be sufficient to form a thin layer of water over the entire coverslip
when the diatom suspension is added.
2) Agitate the sample vial to homogenize the diatom suspension.
3) Using an adjustable pipettor, quickly withdraw the required amount from near
the central portion of the sample.
4) Eject this material smoothly and carefully into the layer of distilled water
already on the coverslip.
5) Repeat until achieving a homogeneous suspension on the coverslip.
6) Record the subsample amount ejected.
ii. In the case where more thanl.O ml of original sample is required, eject and mix the
sample directly on the coverslip (i.e., the addition of distilled water is not
necessary).
iii. If the coverslip overflows, discard the coverslip, wipe the drying area, and repeat
the procedure with a freshly cleaned coverslip.
iv. Discard the pipette tip when finished with each vial.
e. Once the marked space is loaded with coverslip preparations, do not disturb until the
coverslips are dry.
f. Dry the coverslips as follows:
i. Leave them undisturbed at room temperature for several hours;
OR
ii. Use gentle heat (warm to the touch only) to hasten evaporation using a:
1) Crook-neck lamp with incandescent light bulb placed 15 - 30 cm over the
coverslips;
2) Slide maker that provides heat;
c Kimwipeฎ is a commercially-available product. ^J
100
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 101 of 224
3) Or some other device that will gently dry the slides. Avoid any device that will
rapidly evaporate the suspension. Rapid evaporation could produce strong
patterns of diatoms settling on coverslips.
iii. Once completely dry, put the coverslips on the hot plate preheated to 250 to 30Q5F.
iv. Leave coverslips on hot plate for 3-5 minutes. Verify that:
1) Heat has driven nearly all water from the material on the coverslips; and
2) Diatom frustules adhere to the surface of the glass.
v. Remove the coverslips from the hotplate and inspect them. Consider:
1) If the pattern of diatoms distributed on any coverslip is not even and smooth,
discard and prepare a new coverslip.
2) If coverslip distributions are still unsatisfactory after repeated attempts, consult
an algal analyst.
7. Mount coverslip on slide in a POSITIVE-DRAW FUME HOOD using the following steps.
a. Use a rounded wooden splint or disposable pipette to transfer a small amount of the
mounting medium (volume equivalent to two to four drops of water) to the central portion
of the etched side of the microscope slide.
b. Remove the appropriate coverslip from the aluminum plate with forceps, being careful to
handle the coverslip only at the extreme corners.
c. Invert the coverslip and place it gently on the portion of the slide covered with the mounting
medium so the diatoms are in the medium between the coverslip and the microscope slide.
d. Place the slide (coverslip up) on the hotplate and apply gentle heat until the evolution of
bubbles resulting from the evaporation of the toluene solvent first occurs, and then
significantly diminishes. Heating the slide will allow the mounting medium to flow under the
coverslip in a later step.
e. Remove the slide from the hot plate.
f. Gently position the coverslip using the rounded toothpicks.
g. Bring the edges of the coverslip parallel to the edges of the microscope slide.
h. Press the coverslip to form a uniform, thin layer of mounting medium beneath the entire
coverslip that spreads to the edges of the coverslip. Take care at this stage to press gently to
prevent breakage, warping of the coverslip, or dislodging diatoms. As this procedure is
taking place, the mounting medium is "setting up" (becoming hard), and the ability to move
the coverslip will diminish rapidly.
i. Set aside the mount to finish cooling.
1. Use a single-edge razor blade to carefully trim any excess mounting medium which has been
squeezed out from beneath the coverslip. Take great care to avoid "lifting" the coverslip by
inadvertently allowing the edge of the blade to move between the coverslip and the microscope
slide.
2. After removing and discarding most of the excess mounting medium, and while still working
under the hood, place the mount in successive baths of acetone, and then ethanol for no more
than 10 or 15 seconds each.
3. Wipe the mount clean with a tissue (e.g., Kimwipeฎ).
4. Add a paper label to slides before analyzing the slides.
101
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 102 of 224
5. Mark the sample ID number on the side of the slide without a label with a diamond pencil.
6. Provide the slide to the Internal QC Taxonomist to verify that the slide has been properly made
(i.e., does not have any obvious problems such as trapped bubbles). If the Internal QC
Taxonomist identifies any problems with the slide, the laboratory discards the slide and the
laboratory technician follows the above steps in preparing a replacement slide.
7. Preserve and store cleaned material.
8. Record the data elements described in Table 9.3. Comments should include information such as:
Observations about any interfering materials (sand, silt, etc.)
Rationale for using a sparsely populated slide.
Information on all burn mount attempts, successful or unsuccessful.
Table 9.3 Periphyton: required data elements - diatom cleaning & mounting
FIELD
LAB ID
SITEJD
VISIT NUMBER
SAMPLEJD
VIALJD
SUBSAMPLE_DCF
SLIDEJD
SLIDE_DATE
TECHNICIAN
MOUNT_MEDIUM
INTERNAL QC TAXONOMIST
SLIDE_CHECK
EJECT_AMOUNT
EJECT_DCF
DESTINATION
COMMENTS_DIATOM_PREP
FORMAT
text
text
numeric
numeric
numeric or text
numeric
numeric or text
date
text
text
text
Y/N
numeric
numeric
text
text
DESCRIPTION
Name or abbreviation for QC laboratory
NRSA site id as used on sample label
sequential visits to site (1 or 2)
sample id as used on field sheet (on sample label)
Laboratory assigned ID of the diatom vial used for the
cleaning
Dilution or concentration factor. Record as 1 if sample was not
diluted or concentrated.
Laboratory assigned ID for the slide
Date that slide preparation was completed
Name or initials of technician who prepared the slide
Mounting medium used (e.g., Zrax, Naphrax)
Name or initials of QC Taxonomist who reviewed the slide
Is slide acceptable for diatom analysis? If no, still record the
slide number, but note in the comments that the slide has
been destroyed.
Final amount of cleaned diatom material ejected on coverslip
Dilution or concentration factor. Record as 1 if ejected
amount was not diluted or concentrated separately from any
dilution or concentration of the subsample.
Name of the receiving laboratory (i.e., if the slide will be
shipped to another location)
any comments about the diatom cleaning and mounting
9.7 Analysis of Diatoms and Soft Algae
The taxonomist should complete the analyses of properly concentrated and prepared samples as
described below within 8 hours or 6 transects (i.e., whichever limit is reached first) for each diatom
slide, and within 4 hours for the soft algae subsample. However, EPA does not consider any research
related to unfamiliar taxa to be part of the time limit. Within these time limits, the taxonomist should
count 600 diatoms and 300 natural counting units of soft algae.
O
LJJ
CL
102
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 103 of 224
9.7.1 Taxonomic Nomenclature and Photographic Specifications
For both analyses of diatoms and soft algae for NRSA, EPA requires that all laboratories use the same
sources for taxon names. EPA also requires photographs of each unique taxon. The following steps
provide EPA's specifications:
1. Identify the species (i.e., the target level). Where it is not possible to identify the species, then
identify the specimen to the lowest practical taxon level:
a. If the species/taxon is included in BioData
(https://aq uatic.biodata.usgs.gov/doma in Down loads. action), then select the records
corresponding to "Current" in TaxonConceptState and record:
i. NADED_ID for the taxon identification number
ii. BiodataTaxonName for the taxon name
iii. BenchTaxonNameReferenceCode, if provided in BioData. Otherwise, cite the
reference used to identify the specimen. The reference only needs to be cited once
for each taxon.
b. If the species/taxon is not included in BioData, then record:
i. LAB_TAXON_ID: an identifier assigned by the laboratory to provide a unique
number for each taxon identified for NRSA
ii. LAB_TAXON_NAME: taxon name
iii. LAB_REFERENCE: reference used to identify the specimen. The laboratory only
needs to cite a reference for the first identification of the taxon.
c. Photograph each species/taxon one or more times for the laboratory's NRSA library. The
image must demonstrate the diagnostic feature(s) of the species/taxon so that they are
distinguishable and clear to an algal taxonomist. The image should be:
i. Positioned vertically
ii. Tightly cropped
iii. Scaled with a 10 u.m scale bar in the lower right corner of the image
iv. Saved with a tiff format
v. Have a filename of following elements in the order listed below:
NRSA2 (for the second NRSA conducted in 2013-2014)
Laboratory name (or abbreviation)
Sample number
Taxon name
NADEDJD (from BioData) or Laboratory's ID number for the taxon
Date (format YYYYMMDD) that the photograph was taken.
Last character is 'e' for an edited version of the photograph
For example, on September 8, 2013, laboratory ABC identified the specimen in
sample 1234 to be a Homoeothrixjanthina, for which the laboratory ID code is
HJ0001, and took a digital photograph at a resolution of 40x, with a 10 u.m scale z
bar present, and then cropped the photograph to eliminate extraneous i-
material. The filenames of the original and edited photographs would be:
LJJ
CL
103
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 104 of 224
NRSA2_ABC_1234_homoeothrixjanthina_HJ0001_40x_20130908.tiffand
NRSA2_ABC_1234_homoeothrixjanthina_HJ0001_40x_20130908e.tiff.
9.7.2 Analysis of Diatoms
This section describes the diatom subsample analysis used to estimate the proportion of diatom taxa
found in a count of 600 valves (one-half of an individual diatom cell). The diatom analysis also includes
biovolume measurements and imaging of all taxa encountered, including unknown taxon. In developing
this section, EPA modified and expanded upon the steps on ANS Protocol P-13-39 "Analysis of Diatoms
on Microscope Slides Prepared from USGS NAWQA Program Algae Samples"
(http://diatom.ansp.org/nawqa/pdfs/P-13-39.pdf).
To analyze the diatoms, the taxonomist shall:
1. Verify that all necessary equipment is available. See Section 9.3.4 for recommended supplies.
2. Verify that the slides can be used for diatom analysis:
a. Review comments from the subsampling, cleaning, and mounting steps for any deviations
that may affect the outcome of diatom analysis.
b. Scan slides at low to medium magnification (lOOx to 450x) to confirm that diatoms are:
i. Evenly distributed on the coverslip.
ii. Present at a density appropriate for efficient counting. At high magnification
(lOOOx), each field should have 5-10 diatoms.
1) If diatoms on the slides are very sparse, refer to procedures in Section 9.6 for
handling low-density samples.
c. If there are problems with dispersion or density that would compromise the quality and
accuracy of the analysis, have new slides made. Always save any count data generated for a
sample, even if the number of valves or frustules is low (e.g., <100).
3. Create the transect and count the diatoms as follows:
a. Secure the slide in the mechanical stage and use the microscope's diamond scribe to etch a
horizontal or vertical line (depending on personal preference) on the coverslip to mark the
edge of the first row to be counted. Clearly demarcate the areas of a slide scanned during a
count. This is an important step because another taxonomist may need to recount the slide
for QA/QC purposes.
i. If etching the slide before counting:
a) Etch at least three lines on the diatom slide.
b) Locate a starting point near one end of the etched line and make a circle with
the scribe. This denotes the starting point of the count.
ii. If etching during the count:
a) Etch a line for each row counted. Rows are narrow rectangular areas (strips) of
the slide adjacent to the scribed line, with width equal to the field of view. Start
rows far enough from the coverslip edge to avoid optical distortion, and end
them near the opposite coverslip edge where diatoms are no longer clearly ~z.
O
visible (see diagram below). ฃ;
b) Etch a circle around the last field counted in the first row and at the start and a.
cr*
end of all other rows. ฃj
104
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 105 of 224
iii. Verify that the etching is clearly visible so that another taxonomist can easily locate
the circles and lines.
L.
1
!
_J
b. Focus the first field under oil immersion.
c. On a bench sheet and the upper right hand corner of the coverslip, record in mm the
transect start coordinates from the microscope stage. Transects are narrow rectangular
areas (strips) of the slide with width equal to the field of view. The transect width should be
the maximum width common to all participating taxonomists (at least 90 microns).
d. For each species/taxon, count diatom valves along the transect using the following rules:
i. One valve is:
a) A complete valve; or
b) A partial valve that is more than 50% of the valve. The valve must extend at
least halfway into the transect (i.e., the center of the valve must be in the
transect).
ii. Avoid counting valves in any disrupted areas of the mount, particularly edges that
have optical aberrations.
iii. Identify and count 600 valves or as many as possible to complete the entire analysis
within 8 hours or 6 transects (i.e., whichever limit is reached first).
e. Record the level of magnification used to view the diatoms.
f. Verify that photographs of the valves for the species/taxon exist in the NRSA library. If the
valve is the first occurrence of the species/taxon or the valve is particularly notable, take a
photograph following the specifications in Section 9.7.1.
g. On a bench sheet, record the transect end coordinates from the microscope stage.
4. Calculate the total transect length and width (units are mm) evaluated for the sample. Record as
TRANS_LENGTH and TRANS_WIDTH in the database.
5. For each species (or lowest practicable taxon), estimate the average biovolume for the
species/taxon using information from one of the following sources:
a. Preferably, measurements reported in:
i. NRSA 2008-2009;
ii. Well-respected literature;
iii. Previous laboratory analyses for which the taxonomist is relatively confident provides a
good estimate of the average biovolume for the taxon; or
iv. Databases with known sources. z
O
b. When no other reasonable source is readily available, base the taxon's average biovolume ฃ:
on the measurements of the cells on the slide. Perform the following steps: o.
i. Determine the number of cells to measure: 2j
CL
105
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 106 of 224
a) For each abundant taxon (i.e., occurring in more than 5% in any one sample),
measure the biovolume of 10 or more cells.
b) For each common taxon (i.e., occurring 2-5% in any one sample), measure the
biovolume for one or more cells.
c) For each rare taxon, use the biovolume measurements from literature descriptions
of taxa or measure one or more cells.
ii. Measure the biovolume (BV,) of each cell / as follows:
a) Select a simple geometric figure that matches the shape of the cell as best as
possible, and measure its dimensions.
b) If a dimension of cannot be measured (e.g. depth), develop an estimate based on
sizes of cells of the same taxon with similar known dimensions. To estimate depth,
focus on tops and bottoms of cells.
c) Estimate the average biovolume by calculating the arithmetic average of the
measured biovolumes of the cells on the slide:
total number of cells measured
6. Verify that the SUBSAMPLE_DCF value reflects all dilution and concentration of the subsample.
Make any necessary corrections to the value.
7. Record the required data elements in described in Section 9.7.4 (
O
LJJ
CL
106
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 107 of 224
8. Table 9.4).
9.7.3 Analysis of Soft Algae
This section describes the steps for identifying, counting, documenting, and measuring soft algae using
the Palmer-Maloney counting cell method. The steps include re-processing a small portion of the
subsample using final dilution/concentration factors. EPA adapted the steps from ANS Protocol P-13-50
"Preparation of USGS NAWQA Program Algal Samples for Analysis Using Palmer-Maloney Cells"
(http://diatom.acnatsci.org/nawqa/pdfs/P-13-50.pdf) and ANS Protocol P-13-52 "Analysis of USGS
NAWQA Program Phytoplankton Samples" (http://diatom.ansp.org/nawqa/pdfs/P-13-52.pdf).
In analyzing the soft algae, the taxonomist shall:
1. Follow lab practices similar to those used for sterile experiments (bacteriological plating, etc.) to
reduce the risk of cross contamination of samples. At a minimum:
a. Keep the laboratory room and bench surfaces as clean as possible and free of debris.
b. Use disposable pipettes, stirrers, etc. where feasible.
c. Rinse glass (non-disposable) pipettes, stirrers, etc. in DW or RO water at least three times
after each sample. (Explanatory note: at times, tap water, because of algal blooms and use
of diatomaceous earth filters, may contain diatoms.)
d. Dry all equipment before storing it. This step prevents growth of algae and fungi.
2. Verify that all necessary equipment is available. See Section 9.3.4 for recommended supplies.
3. Prior to taking a small portion of the subsample from the vial, verify that the sample is
thoroughly mixed. If there are visible macro forms in the soft-algae subsample (i.e., visible
filaments or colonies), break them up using scissors or small blender. This does not include
maceration or grinding. The purpose of this step is to suspend algae so that fractions will have
proportions of algal taxa equal to those in the original sample.
4. Concentrate the sample, if necessary. The original sample should usually be concentrated prior
to adding to a Palmer-Maloney cell. The initial concentration should be approximately 5-10
times the original whole-water, leaving about 20 ml of concentrate for analysis. Concentrate the
sample as follows:
a. Only concentrate/dilute a portion (10 ml or less) of the original subsample (i.e., from the
vial).
b. Settle and decant (settle for at least 2 days) or use centrifugation (1000 g for 20 min)
c. Revise the subsample DCF using the original and final volumes, before and after
concentration.
5. For samples with high amounts of sand and/or silt, dilute the sample if necessary, but do not
dilute any sample to more than 20 % of its original volume. This step is valid ONLY for samples
with large amounts of inorganic sand and silt. No settling should occur with samples that
contain organic detritus or at any time during the dilution process.
LJJ
CL
107
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 108 of 224
a. Determine if dilution is necessary by placing a portion of the subsample in the Palmer-
Maloney Cell for assessment.
b. Dilute if necessary and recheck sample. If large inorganic particles are present (but NOT
organic detritus) and blocking view of algal cells, again thoroughly mix the Palmer-Maloney
fraction.
c. Allow the sample to stand while slowly counting to 5 or 6 (approximately 4-5 seconds).
d. Re-load the Palmer-Maloney cell with the subsample according to instructions below and
count the sample or dilute again as necessary.
6. Use the concentrated/diluted fraction instead of the original in the following steps.
7. Calculate the subsample dilution/concentration factor (SUBSAMPLE_DCF) by dividing the final
diluted or concentrated subsample volume by the initial volume. For example, a 10 ml
subsample concentrated to 1 ml would have a subsample DCF=0.1; and a 10 ml subsample
diluted to 100 ml would have a subsample DCF=10.0.
8. Prepare Palmer-Maloney counting cell as follows:
a. Place a rectangular coverslip (#1 thickness, 22 x 50 mm) at 45ฐ to the counting cell, covering
about l/3rd of the chamber, but not across the center of the cell.
b. Thoroughly mix the Palmer-Maloney fraction and draw into the micro-pipette 100 u.1 (plus
suitable additional amount to allow for displacement from the chamber by the coverslip and
prevent the occurrence of air bubbles).
c. Quickly add the 100 ul fraction, into the center of the chamber.
d. When the surface tension starts to draw the coverslip across the chamber, adjust the sides
of the coverslip so that ends of the chamber are covered and the coverslip hangs over both
sides of the ceramic portion of the counting cell.
e. Add glycerin to the area where the coverslip extends past the ceramic portion. This seals
the coverslip to the counting cell temporarily (without excess heat or vibration, the counting
cell can be used for a week or more).
9. Evaluate the Palmer-Maloney Fractions for suitability for the analysis:
a. Assess 5 fields of view to determine if 15 - 30 natural counting units (both diatom and soft
algae are visible per field of view.
b. Determine if it is appropriate to concentrate or dilute the subsample in the vial to create a
new fraction. Such decisions are subjective and thus made on a case-by-case basis. Consider
the following:
i. If the number exceeds 30 natural counting units, dilution may be appropriate.
ii. If the number is less than 15 natural counting units, concentration may be
appropriate.
iii. In samples with high silt/detritus levels, even if there are < 15 natural units per field of z
view, consider dilution. h-
i
a.
Cฃ.
LJJ
CL
108
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 109 of 224
iv. Avoid concentrating samples were the silt/detritus level would be raised to a level too
high to complete a successful count. Include a comment in the database (see Table
9.4) if the silt/detritus levels prohibit concentration or lead to a dilution.
c. Once the Palmer-Maloney cell is prepared, allow chamber to stand for up to 10 minutes to
allow contents to settle.
d. Revise the SUBSAMPLE_DCF if any step in this section required additional concentration or
dilutions of the subsample. Because EPA will use the SUBSAMPLE_DCF in estimating whole
sample values, it is extremely important that the recorded value for SUBSAMPLE_DCF
reflects all of the dilution and concentration steps.
10. Use transects of varying length to enumerate 300 natural counting units.
a. Position the microscope stage so that the first field of view is in the top left hand area of the
Palmer-Maloney cell at, but not overlapping, the cell edge.
b. Use transects, of varying length, with a width of 0.54 mm (or width of the field of view) scan
up to, but not exceeding, a total transect length of 115.7 mm (or equivalent). Every other
transect, moving down the Palmer-Maloney cell, is to be enumerated, i.e. each transect
enumerated should be separated by a width of one field of view. This will prevent field of
view overlap. For microscopes where the 40x field of view differs from 0.54 mm calculate
the maximum transect length required. The full length of each transect should be scanned
for algal enumeration. Record the start and end coordinates for each transect for the
purpose of total transect length calculation on the Soft Algae Bench Sheet.
c. Identify and enumerate all soft algal forms within the field of view.
d. Record the level of magnification used to view the algal forms.
11. Enumerate 300 natural algal units and identify to species (or, if not possible, then the lowest
practicable taxonomic level) as follows:
a. Using the pattern developed in the previous step, move the microscope stage to a new
position in the pattern. Make all movements of the microscope stage without looking
through the objectives.
b. 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.
c. Differentiate diatoms as "living" or "dead" at type of collection. If there is any protoplast
material in the frustules, consider the diatom to have been living when collected.
i. Identify all non-diatom taxa to species (or, if not possible, then the lowest practicable
taxonomic level).
ii. Group all diatoms into one category - undifferentiated diatoms - instead of
identifying their taxa. z
d. Count the number of algal cells (living and dead) comprising each multicellular counting unit h-
until reaching 300 natural algal units. Only "living" diatoms are part of the required 300 J
natural algal units. 2j
CL
109
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 110 of 224
e. Record the number field or total transect length used for counting, the species
identifications, and number of natural cells for each species.
f. For the undifferentiated diatoms, record their count and label the species "Dead diatoms"
and leave all other taxonomic fields blank.
12. Enumerate larger, rarer taxa. There is an additional required 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 um). Using a low-power objective (10-15x), scan 20
fields or 4 transects. Count the larger, rarer taxa. Enumerate as natural units and estimate the
number of cells in each. Record the counts of each of the taxa, 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. Record
that number as the total count for that taxon.
13. Calculate and record the estimated number of cells within a 'typical' natural unit. (This need
only be a rough estimate.)
14. Verify that photographs exist in the NRSA library for the species/taxon. If this species/taxon is
the first occurrence or particularly notable, take a photograph following the specifications in
Section 9.7.1.
15. For each species (or lowest practicable taxon), estimate the average biovolume for the
species/taxon using information from one of the following sources:
a. Preferably, measurements reported in:
i. NRSA 2008-2009;
ii. Well-respected literature;
iii. Previous laboratory analyses for which the taxonomist is relatively confident provides
a good estimate of the average biovolume for the taxon; or
iv. Databases with known sources.
16. When no other reasonable source is readily available, base the taxon's average biovolume on
the measurements of the cells. Perform the following steps:
a. Determine the number of cells to measure:
i. For each abundant taxon (i.e., occurring in more than 5% in any one sample), measure
the biovolume of 10 or more cells.
ii. For each common taxon (i.e., occurring 2-5% in any one sample), measure the
biovolume for one or more cells.
iii. For each rare taxon, use the biovolume measurements from literature descriptions of
taxa or measure one or more cells.
b. Measure the biovolume (BVi) of each cell / as follows:
i. Select a simple geometric figure that matches the shape of the cell as best as possible, z
and measure its dimensions. When length within a filament cannot be determined, h-
then use a standard length of 10 u.m per cell. Note in the comments field (Table 9.4) J
when this standard length is applied. 2j
CL
110
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 111 of 224
ii. If a dimension of cannot be measured (e.g. depth), develop an estimate based on sizes
of cells of the same taxon with similar known dimensions. To estimate depth, focus on
tops and bottoms of cells.
iii. Estimate the average biovolume by calculating the arithmetic average of the
measured biovolumes of the cells on the slide:
, _ _ _
AvgBiovolume measured
17. Verify that the SUBSAMPLE_DCF value reflects all dilution and concentration of the subsample.
Make any necessary corrections to the value.
9.7.4 Internal Quality Control
For each sample, the laboratory must perform internal quality control evaluations. The Internal QC
Taxonomist must randomly select 10% of the diatom slides and 10% of the soft algae subsamples for an
independent count and identification. Calculate the PctDiff, PDE, and PTD using the equations in Section
9.3. If any samples do not meet the QA requirements listed in the Attachment, perform a third count
and reidentification for the sample.
9.7.5 Required Data Elements for Diatom and Soft Algae Analyses
Table 9.3 identifies the required data elements for the analyses described in Section 9.7.2 (diatom) and
Section 9.7.3 (soft algae).
O
LJJ
CL
111
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Table 9.4 Periphyton: required data elements - analysis
Laboratory Operations Manual
Page 112 of 224
FIELD
LAB ID
SITE ID
VISIT_NUMBER
SAMPLEJD
SAMPLE_VOLUME
VIALJD
SUBSAMPLE_VOLUME
SLIDEJD
TAXONOMIST
QC_CHECK
ANALYSIS
DATE_ANALYSIS
NADED_ID
LAB_TAXON_ID
BiodataTaxonName
LAB_TAXON_NAME
BenchTaxonNameReferenceCode
LAB_REFERENCE
MAGNIFICATION
CLASS
ORDER
FAMILY
GENUS
SPECIES
TRANSECTS_COUNTED
TRANS_LENGTH
TRANS_WIDTH
CHAMBER_AREA
CHAMBER_VOLUME
COUNT
FORMAT
text
text
numeric
numeric
numeric
numeric or text
numeric
numeric or text
text
Y/N
DIA/SFT
date
numeric
numeric or text
text
text
text
text
numeric
text
text
text
text
text
numeric
numeric
numeric
numeric
numeric
numeric
DESCRIPTION
Name or abbreviation for QC laboratory
NRSA site id as used on sample label
sequential visits to site (1 or 2)
sample id as used on field sheet (on sample label)
Original sample volume measured during subsampling
step in Section 9.5. This value is used for both diatoms
and soft algae analyses.
Laboratory assigned ID of the diatom vial
Volume of the subsample in the vial
Laboratory assigned ID for the slide
Name or initials of taxonomist or Internal QC Taxonomist
analyzing the sample.
Y if results were provided by the Internal QC Taxonomist
for a sample selected for the 10% QC check.
Type of analysis: DIA=Diatoms; SFT=Soft Algae
Date when the analysis was completed
BioData's identification number for the taxon
An identifier assigned by the laboratory to provide a
unique number for each taxon identified for NRSA. If
NADEDJD is available, this field is optional.
BioData's taxon name
Taxon name. If BiodataTaxonName is available, this field
is optional.
BioData's reference for the taxon. Identify the reference
in BenchTaxonNameReferenceCode or LAB_REFERENCE
the first time the lab identifies the taxon. Otherwise, this
field is optional.
Identify the reference in
BenchTaxonNameReferenceCode or LAB_REFERENCE the
first time the lab identifies the taxon. Otherwise, this
field is optional.
Identify the magnification used to identify the taxon.
taxonomic class
taxonomic order
taxonomic family
taxonomic genus
taxonomic species. For soft algae, label undifferentiated
diatoms as "Dead diatoms" and leave other taxonomic
fields blank.
Number of transects counted
Length (mm) of transects counted
Width (mm) of transects counted
Chamber area in mm2
Volume of the chamber
Number of valves (diatoms) or natural counting units
(soft algal) identified for the taxon
112
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 113 of 224
COUNT_CENSORED
AVG BIOVOLUME
BIOVOLUME_AVG_SOURCE
BIOVOLUME_REFERENCE
LAB_REFERENCE
PHOTOGRAPH
COMMENTS, ANALYSIS
BV1
BV2
BV3
BV4
BV5
BV6
BV7
BV8
BV9
BV10
BV11
BV12
BV13
BV14
BV15
text
numeric
text
text
text
text
text
numeric
numeric
numeric
numeric
numeric
numeric
numeric
numeric
numeric
numeric
numeric
numeric
numeric
numeric
numeric
Code for a sample for which the taxonomist counted
fewer than 600 valves (diatoms) or 300 natural counting
units (soft algal)
CODE
TL
SS
OT
Average
Definition
Time limit reached
Sparse sample
Other. Identify other reason in
COMMENT_ANALYSIS
biovolume for the species/taxon
Source of the biovolume average value:
CODE
NR8
REF
SS
OT
Definition
From NRSA 2008-2009
Cited in literature
Sparse sample (i.e., fewer than 600 organisms
in the sample)
Other. Identify other reason in
COMMENT_ANALYSIS
Identify the reference that provided the average
biovolume value the first time the lab identifies the
taxon. Otherwise, this field is optional. Leave blank if the
laboratory estimated the biovolume from measuring the
cells.
The laboratory reference used to identify the taxon.
Identify the reference in
BenchTaxonNameReferenceCode or LAB_REFERENCE the
first time the lab identifies the taxon. Otherwise, this
field is optional.
Filename of relevant photograph in the NRSA library. The
filename is required the first time the lab identifies the
taxon. Otherwise, this field is optional.
any comments about the analysis
Biovolume of organism 1 (before measuring the
biovolume, consider if the average biovolume is available
from NRSA 2008-2009 or some other reliable source)
Biovolume of organism 2
Biovolume of organism 3
Biovolume of organism 4
Biovolume of organism 5
Biovolume of organism 6
Biovolume of organism 7
Biovolume of organism 8
Biovolume of organism 9
Biovolume of organism 10
Biovolume of organism 11
Biovolume of organism 12
Biovolume of organism 13
Biovolume of organism 14
Biovolume of organism 15
o
LJJ
CL
113
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
9.8 Data Entry
Table 9.1,
Laboratory Operations Manual
Page 114 of 224
O
oc
LJJ
a.
114
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 115 of 224
Table 9.2 and Table 9.3 identify the required data elements that the laboratories must provide to EPA,
in EPA's data template, available separately from EPA.
O
oc
LJJ
a.
115
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 116 of 224
Table 9.4 provides a consolidated list of required data elements. In addition to the database, the
laboratory must provide the resume or curriculum vitae for each taxonomist who performs the diatoms
and algal analyses for the NRSA samples. The resume or cv for each taxonomist is submitted once to
EPA's External QC Coordinator.
Table 9.5 Periphyton: required data elements - all
RECORDED
DURING:
LOGIN
LAB ID
DATE RECEIVED
SITE ID
VISIT NUMBER
SAMPLE ID
DATE COLLECTED
CONDITION CODE
FORMALIN ADDED
COMMENTS ARRIVAL
FORMAT DESCRIPTION
text
MMDDYY
text
numeric
Name or abbreviation for QC laboratory
date sample was received by lab
NRSA site id as used on sample label
sequential visits to site (1 or 2)
numeric sample id as used on field sheet (on
sample label)
MMDDYY
text
date sample was collected
Condition codes describing the condition
of the sample upon arrival at the
laboratory.
CODE Definition
OK
HM
TL
ML
PA
Sample is in good condition
Sample container is cracked
Sample contains heavy amounts
of sand, silt, or other heavy
material that may interfere with
the algal analysis
Sample or container is leaking
Too little sample left for the
procedure
Sample label is missing
Preservative added, add amount
in comments field
Other quality concerns, not
identified above
numeric If the laboratory added formalin to the
bottle, provide the amount in milliliters.
If no formalin was added, then the value
should be zero or blank.
text
any comments about the condition of the
sample upon arrival.
SUB-SAMPLING VIAL ID
numeric or Laboratory assigned ID to the vial holding
text the subsample
SAMPLE VOLUME
numeric
SUBSAMPLE VOLUME
numeric
PURPOSE
D/S
Total volume of sample before any
processing (from the sample label)
Volume of subsample in the vial
Codes indicating whether the vial will be
used for the diatom or soft algal analyses
CODE
D
Definition
Diatom Analysis
Soft Algal Analysis
116
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 117 of 224
DIATOM
CLEANING AND
MOUNTING
ANALYSIS
DESTINATION
COMMENTS_SUBSAMPLE
SUBSAMPLE_DCF
SLIDEJD
SLIDE_DATE
TECHNICIAN
MOUNT_MEDIUM
INTERNAL QC_TAXONOMIST
SLIDE_CHECK
EJECT_AMOUNT
EJECT_DCF
DESTINATION
COMMENTS_DIATOM_PREP
TAXONOMIST
QC_CHECK
ANALYSIS
DATE_ANALYSIS
NADEDJD
LAB_TAXON_ID
BiodataTaxonName
LAB_TAXON_NAME
text
text
numeric
numeric or
text
date
text
text
text
Y/N
numeric
numeric
text
text
text
Y/N
DIA/SFT
date
numeric
numeric or
text
text
text
Name of the receiving laboratory (i.e., if
the vial will be shipped to another
location)
any comments about the subsampling
Dilution or concentration factor. Record
as 1 if sample was not diluted or
concentrated.
Laboratory assigned ID for the slide
Date that slide preparation was
completed
Name or initials of technician who
prepared the slide
Mounting medium used (e.g., Zrax,
Naphrax)
Name or initials of QC Taxonomist who
reviewed the slide
Is slide acceptable for diatom analysis? If
no, still record the slide number, but note
in the comments that the slide has been
destroyed.
Final amount of cleaned diatom material
ejected on coverslip
Dilution or concentration factor. Record
as 1 if ejected amount was not diluted or
concentrated separately from any
dilution or concentration of the
subsample.
Name of the receiving laboratory (i.e., if
the slide will be shipped to another
location)
any comments about the diatom cleaning
and mounting
Name or initials of taxonomist or Internal
QC Taxonomist analyzing the sample.
Y if results were provided by the Internal
QC Taxonomist for a sample selected for
the 10% QC check.
Type of analysis: DIA=Diatoms; SFT=Soft
Algae
Date when the analysis was completed
BioData's identification number for the
taxon
An identifier assigned by the laboratory
to provide a unique number for each
taxon identified for NRSA. If NADEDJD is
available, this field is optional.
BioData's taxon name
Taxon name. If BiodataTaxonName is
available, this field is optional.
117
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 118 of 224
BenchTaxonNameReferenceCod
e
LAB_REFERENCE
MAGNIFICATION
CLASS
ORDER
FAMILY
GENUS
SPECIES
TRANSECTS COUNTED
TRANS_LENGTH
TRANS_WIDTH
CHAMBER_AREA
CHAMBER_VOLUME
COUNT
COUNT_CENSORED
EST_CE LLS_I N_N AT_U N IT
AVG_BIOVOLUME
BIOVOLUME_AVG_SOURCE
text
text
numeric
text
text
text
text
text
numeric
numeric
numeric
numeric
numeric
numeric
text
numeric
numeric
text
BioData's reference for the taxon.
Identify the reference in
BenchTaxonNameReferenceCode or
LAB_REFERENCE the first time the lab
identifies the taxon. Otherwise, this field
is optional.
Identify the reference in
BenchTaxonNameReferenceCode or
LAB_REFERENCE the first time the lab
identifies the taxon. Otherwise, this field
is optional.
Identify the magnification used to
identify the taxon.
taxonomic class
taxonomic order
taxonomic family
taxonomic genus
taxonomic species. For soft algae, label
undifferentiated diatoms as "Dead
diatoms" and leave other taxonomic
fields blank.
Number of transects counted
Length
(mm) of transects counted
Width (mm) of transects counted
Chamber area in mm2
Volume of the chamber
Number of valves (diatoms) or natural
counting units (soft algal) identified for
the taxon
Code for a sample for which the
taxonomist counted fewer than 600
valves (diatoms) or 300 natural counting
units (soft algal)
CODE
TL
SS
OT
Definition
Time limit reached
Sparse sample
Other. Identify other reason in
COMMENT_ANALYSIS
Estimated number of cells in a 'typical'
natural counting unit for the
species/taxon (soft algae only)
Average biovolume for the species/taxon
Source
CODE
NR8
REF
SS
OT
of the biovolume average value:
Definition
From NRSA 2008-2009
Cited in literature
Sparse sample (i.e., fewer than
600 organisms in the sample)
Other. Identify other reason in
COMMENT_ANALYSIS
118
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 119 of 224
BIOVOLUME_REFERENCE
LAB_REFERENCE
PHOTOGRAPH
COM ME NTS_ ANALYSIS
BV1
BV2
BV3
BV4
BV5
BV6
BV7
BV8
BV9
BV10
BV11
BV12
BV13
BV14
BV15
text
text
text
text
numeric
numeric
numeric
numeric
numeric
numeric
numeric
numeric
numeric
numeric
numeric
numeric
numeric
numeric
numeric
Identify the reference that provided the
average biovolume value the first time
the lab identifies the taxon. Otherwise,
this field is optional. Leave blank if the
laboratory estimated the biovolume from
measuring the cells.
The laboratory reference used to identify
the taxon. Identify the reference in
BenchTaxonNameReferenceCode or
LAB_REFERENCE the first time the lab
identifies the taxon. Otherwise, this field
is optional.
Filename of relevant photograph in the
NRSA library. The filename is required
the first time the lab identifies the taxon.
Otherwise, this field is optional.
any comments about the analysis
Biovolume of organism 1 (before
measuring the biovolume, consider if the
average biovolume is available from
NRSA 2008-2009 or some other reliable
source)
Biovolume of organism 2
Biovolume of organism 3
Biovolume of organism 4
Biovolume of organism 5
Biovolume of organism 6
Biovolume of organism 7
Biovolume of organism 8
Biovolume of organism 9
Biovolume of organism 10
Biovolume of organism 11
Biovolume of organism 12
Biovolume of organism 13
Biovolume of organism 14
Biovolume of organism 15
9.9 Sample and Record Retention
After the taxonomist analyzes the vials for a sample, retain the vials for possible selection into the
external quality control evaluation. When the external evaluation is complete, process the vials
containing the remaining acid-cleaned material for long-term storage. The lab technician shall:
1. Work under a fume hood to add two to four drops of 100% buffered formalin to each vial.
2. Add glass beads to the vials to help preserve the diatoms.
3. Tightly cap the vials and seal them by immersing the top 1/3 of the vial in melted wax.
4. Transfer the vials to long-term storage.
The laboratory shall retain:
O
LJJ
CL
119
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 120 of 224
1. The sample materials, including vials and slides, for a minimum of 3 years from the date the EPA
publishes the final report. During this time, the laboratory shall store the materials in a cool
location away from sunlight. The laboratory shall periodically check the sample materials for
degradation and refill jars and vials with formalin if necessary.
2. Original records, including laboratory notebooks and the reference library, for a minimum of 10
years from the date that EPA publishes the final report.
After the stated time periods, the laboratory shall follow its internal protocols for disposal.
9.10 External Taxonomic Quality Control Evaluation
EPA requires that all NRSA laboratories ("primary laboratories") participate in the External Taxonomic
Quality Control Evaluation. Each taxonomist must participate in the QC evaluation, even if the
taxonomist is under subcontract with, or consulting for, another firm.
In contrast to the internal QC evaluation in the earlier sections that verify adherence to the procedures
and ensures in-laboratory consistency between taxonomists, the purpose of the external QC evaluation
is to ensure consistency between laboratories and taxonomists. To achieve this objective, EPA compares
the primary laboratory results to those from a second laboratory, considered a quasi "gold standard" for
taxonomic evaluations.
The External QC Coordinator, who is an EPA staff member, is responsible for selecting and managing the
"QC contractor." To eliminate the appearance of any inherent bias, the QC contractor must be dedicated
to QA/QC functions, and thus, must not be a primary laboratory or a field sampling contractor for NRSA.
The QC contractor is responsible for complying with instructions from the External QC Coordinator;
obtaining and managing the secondary laboratory; coordinating and paying for shipments of the QC
samples between locations; comparing sample identifications by different laboratories; facilitating
reconciliation teleconferences; and preparing brief summary reports.
The External QC Coordinator will arrange for the QC contractor to conduct a minimum of two QC
evaluations in 2014. To the extent practicable, the External QC Coordinator and QC contractor will
schedule batch evaluations evenly throughout the project period.
Each QC evaluation consists of the following steps:
1. In consultation with the QC contractor, the External QC Coordinator determines an appropriate
time to conduct the evaluation based upon the total number of samples assigned to the
laboratory, the delivery schedule, processing schedule, and the following constraints:
a. Availability of samples from other laboratories. For example, if three state laboratories are
each processing less than 30, the External QC Coordinator might combine their samples into
one batch for the QC evaluation.
b. If a primary laboratory is responsible for processing 100 samples or more for the NRSA, the
External QC Coordinator will split their samples into several batches (e.g., each 50 to 100
samples) so that EPA can evaluate and correct performance on an ongoing basis.
2. The External QC Coordinator provides the QC contractor with a list of laboratories and O
processed samples. Sample identification includes the site identification code, sample number, ^
CL
and taxonomist who performed the identifications. E
LJJ
CL
120
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 121 of 224
3. The QC contractor randomly selects 10% of the samples from each NRSA laboratory, subject to
the following constraints:
a. If the primary laboratory received fewer than 30 samples, then the QC contractor randomly
selects three samples for the evaluation.
b. For each taxonomist identified on the list, the QC contractor ensures that one or more of
his/her samples are selected.
4. The QC contractor provides a list of the QC samples, and instructions, to the External QC
Coordinator and each primary laboratory participating in the evaluation. Although the External
QC Coordinator and QC contractor may tailor the instructions for the participating taxonomists'
preferences, the instructions are likely to specify the following:
a. Pack and ship the QC samples to the central holding facility designated by the QC
contractor. Instructions are likely to require that the:
i. Shipments contain chain-of-custody documentation for all slides and containers.
ii. Containers (e.g., slides, vials) include the site identification code and sample number.
iii. Containers cannot be marked in any way that might identify the taxonomic
classification for any organism.
b. Use the NARS IM database to track the QC samples (see Section 9.4). Because of the
potential impact to the QC contractor operations, the External QC Coordinator must
approve any requests to use another tracking method than NARS IM.
c. Email a spreadsheet with the data for the QC samples to the External QC Coordinator. (EPA
requires that all labs use its spreadsheet template for recording the taxonomic data.)
5. The QC contractor reviews the condition of the QC samples (e.g., verifies that the containers do
not identify taxon for any organism) and ships the samples to the secondary laboratory along
with instructions and the EPA template for reporting data.
6. Within 24 hours of receipt, the secondary laboratory:
a. Uses the NARS IM database to log in the sample (see Section 9.4);
b. Faxes or emails any additional receipt records, including discrepancies, within 24 hours; and
c. Completes any other instructions from the QC contractor.
7. The secondary laboratory:
a. Re-identifies and re-counts following the procedures in the Method.
b. Verifies that the secondary laboratory's NRSA library includes photographs for the
species/taxon. If this species/taxon is the first occurrence or particularly notable, the
secondary taxonomist takes a photograph following the specifications in Section 9.7.1.
c. Records the required data elements in Section 9.8. z
d. Enters the data using EPA's spreadsheet template for the taxonomic data. H
e. Emails the completed spreadsheet to the QC contractor. J
LJJ
CL
121
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 122 of 224
8. The QC contractor compares the original taxonomic results (i.e., data) generated by the primary
laboratory to the taxonomic results generated by the secondary laboratory for each sample. As
part of this evaluation, the QC contractor calculates PctDiff, PDE, and PTD using the equations in
Section 9.3 and compares their values to the QC requirements in the Attachment.
9. If any samples exceed the PctDiff, PDE, or PTD limits in the QAPP, the QC contractor consults
with the External QC Coordinator to determine if reconciliation calls are necessary to resolve
differences. The External QC Coordinator may decide that a reconciliation call is unnecessary if
there appears to be an obvious explanation for differences, few samples are affected, or other
reasons.
10. The QC contractor schedules and facilitates reconciliation teleconferences with EPA and the
laboratories.
a. In preparation for the teleconferences:
i. The QC contractor provides the participants with a spreadsheet that includes:
ii. List of samples and taxon identifications for discussion;
iii. Relevant data from the primary and secondary laboratories; and
iv. PctDiff, PDE, and PTD values.
v. The primary and secondary laboratories provide participants with the relevant
reference (or citation) and photograph for each taxonomic identification for the
discussion.
vi. The QC contractor emails a meeting announcement for a convenient time for all
participants. The email identifies instructions for accessing the External QC
Coordinator's toll-free teleconference line.
b. Within a week after the teleconference, the QC contractor sends an email to the External
QC Coordinator and other teleconference participants that summarizes:
i. Agreements to use common nomenclature for discrepancies;
ii. Commitments to reevaluate identifications by reexamining samples;
iii. Application of changes that are appropriate for all samples, not just the QC samples
(e.g., common nomenclature)
iv. Items that will not be resolved for some reason (e.g., sample degraded during
shipment).
11. After completing the reconciliation calls, the participants complete the following steps:
a. Secondary laboratory:
i. Reexamines samples as deemed necessary during the reconciliation call
ii. Updates its database with changes to:
1) QC samples per reexamination and other items in the QC contractor email; and
2) Non-QC samples as appropriate (e.g., nomenclature changes apply to all
samples, not just QC samples). 5
iii. Provides database to QC contractor. >
b. QC contractor confirms that the secondary laboratory (i.e., its subcontractor) completed its
assignments before allowing the secondary laboratory to move to the next step. a!
122
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 123 of 224
c. Secondary laboratory stores its original records, including laboratory notebooks and the
reference library, for a minimum of 10 years from the date that EPA publishes the final
report.
d. Secondary laboratory and QC contractor follow steps 4 and 5 above to return the samples to
the primary laboratory.
e. After receiving the samples (and tracking per step 4), the primary laboratory:
i. Reexamines samples as deemed necessary during the reconciliation call;
ii. Updates its database with changes to:
1) QC samples per reexamination and other items in the QC contractor email; and
2) Non-QC samples as appropriate (e.g., nomenclature changes apply to all
samples, not just QC samples)
iii. Provides the revised database to the External QC Coordinator (not the QC contractor).
It also confirms that it has completed all relevant items identified in the QC
contractor's email summary of the teleconferences (from Step 10.b).
f. QC contractor provides EPA with a report or memorandum that:
i. Identifies the participating laboratories, with the following information about each
laboratory:
1) Laboratory name
2) Address
3) Contact person (name, telephone, and email)
ii. Quantifies the taxonomic precision (PctDiff, PDE, and PTD) as they were prior to the
reconciliation call;
iii. Assesses data acceptability;
iv. Highlights taxonomic problem areas;
v. Identifies any discrepancies for which the External QC Coordinator determined that a
reconciliation teleconference was not necessary;
vi. Identifies primary and secondary laboratory commitments to change its identifications
or provide additional review of any organisms; and
vii. Provides recommendations for improving precision for other samples not included in
the QC evaluation.
12. After review, the External QC Coordinator:
a. Submits the report, and draft technical direction with next steps for the laboratory, to the
EPA staff managing or coordinating with the primary laboratory.
b. Determines if significant differences within the batch of QC samples warrant re-
identification of samples by the primary laboratory and a second QC evaluation by the
secondary laboratory. If deemed necessary, EPA will instruct the primary laboratory to
include the samples for review with the next batch of QC samples.
O
As an additional verification on the generation of the data, EPA may conduct assistance visits at the >
laboratories. If EPA decides to conduct an assistance visit, a qualified EPA scientist or contractor will ^
CC.
LJJ
CL
123
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 124 of 224
administer a checklist based upon the steps described in this chapter. EPA will develop the checklist and
append it to the LOM prior to conducting assistance visits. The objective of the visit would be to:
Confirm the laboratory is properly implementing the steps in the method.
Assist with questions from laboratory personnel.
Suggest corrections if any errors are made.
9.11 References
Acker, F., B. Russell, and E. Hagan. 2002. Protocols for the analysis of algal samples collected as part of
the U.S. Geological Survey National Water-Quality Assessment Program: P-13-42 Diatom Cleaning by
Nitric Acid Digestion with a Microwave Apparatus. The Academy of Natural Sciences. Report No. 02-06.
Retrieved December 2, 2013, from http://diatom.ansp.org/nawqa/pdfs/P-13-42.pdf.
Charles, D. F., C. Knowles, and R. Davis. 2002. Protocols for the analysis of algal samples collected as part
of the U.S. Geological Survey National Water-Quality Assessment Program. Patrick Center for
Environmental Research Report No. 02-06. Patrick Center for Environmental Research, The Academy of
Natural Sciences, Philadelphia, Pennsylvania. Retrieved December 2, 2013, from
http://water.usgs.gov/nawqa/protocols/algprotocol/algprotocol.pdf.
Moulton, S.R., II, J.G. Kennen, R.M. Goldstein, J.A. Hambrook. 2002. Revised protocols for sampling algal,
invertebrate, and fish communities in the National Water-Quality Assessment program, U.S. Geological
Survey Open-File Report 02-150. Retrieved December 2, 2013, from http://pubs.usgs.gov/of/2002/ofr-
02-150/pdf/ofr02-150.pdf.
PCER, ANSP. 2002. Analysis of Diatoms in USGS NAWQA Program Quantitative Targeted-Habitat (RTH
and DTH) Samples. Protocol No. P-13-39. Retrieved December 2, 2013, from
http://diatom.ansp.org/nawqa/pdfs/P-13-39.pdf.
PCER, ANSP. 2002. Analysis of Soft Algae and Enumeration of Total Number of Diatoms in USGS NAWQA
Program Quantitative Targeted-habitat (RTH and DTH) Samples. Protocol P-13-63. Retrieved December
2, 2013, from http://diatom.ansp.org/nawqa/pdfs/P-13-63.pdf.
PCER, ANSP. 2002. Analysis of USGS NAWQA Program Phytoplankton Samples. Protocol No. P-13-52.
Retrieved December 2, 2013, from http://diatom.ansp.org/nawqa/pdfs/P-13-52.pdf.
PCER, ANSP. 2002. Diatom Cleaning by Nitric Acid Digestion with a Microwave Apparatus. Protocol No.
P-13-42. Retrieved December 2, 2013, from http://diatom.ansp.org/nawqa/pdfs/P-13-42.pdf.
PCER, ANSP. 2002. Preparation of Diatom Slides Using Naphrax Mounting Medium. Protocol No. P-13-
49. Retrieved December 2, 2013, from http://diatom.ansp.org/nawqa/pdfs/P-13-49.pdf.
PCER, ANSP. 2002. Preparation of USGS NAWQA Program Algal Samples for Analysis Using Palmer-
Maloney Cells. Protocol No. P-13-50. Retrieved December 2, 2013, from
http://diatom.ansp.org/nawqa/pdfs/P-13-50.pdf.
PCER, ANSP. 2002. Subsampling Procedures for USGS NAWQA Program Periphyton Samples. Protocol
No. P-13-48. Retrieved December 2, 2013, from http://diatom.ansp.org/nawqa/pdfs/P-13-48.pdf.
USEPA. 1973. Biological Field and Laboratory Methods for Measuring the Quality of Surface Waters and
Effluents. EPA-670/4-73-001. US. EPA Office of Research and Development. Cincinnati, OH. O
i-
USEPA. 2009. National Rivers and Streams Assessment: Laboratory Methods Manual. EPA 841-B-07-010. i
r\
Retrieved December 2, 2013, from ^
124
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 125 of 224
http://water.epa.gov/type/rsl/monitoring/riverssurvev/upload/NRSA200809 LabMethodsManual 2009
1229 2 web.pdf.
LJSGS. 2013. BioData-Aquatic Bioassessment Data for the National available at
http://aquatic.biodata.usgs.gov. Version 3.1.2. Accessed 12/20/2013
O
LJJ
CL
125
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Attachment: Summary of QC Requirements
Laboratory Operations Manual
Page 126 of 224
Percent disagreement in enumeration (PDE): measure of taxonomic precision for diatoms comparing the number
of organisms, nt, counted in a sample by the primary taxonomist with the number of organisms, n2, counted by the
secondary taxonomist.
PDE =
n.-n.
xlOO
+n
Percent taxonomic disagreement (PTD): measure of taxonomic precision for diatoms comparing the
number of agreements (positive comparisons, compp0s) of the primary taxonomist and internal or
external QC taxonomists. In the following equation, N is the total number of organisms in the larger of
the two counts.
PTD =
1-
comppos
N
xlOO
Percent Difference (PctDiff): measure of difference for soft algae that compares the enumerations for
the taxa within a sample, as reported by the primary taxonomist (a) and secondary laboratory (b), as
follows:
PctDiff =
i=total # species
1-
in species
Table 9.6 Periphyton: measurement data quality objectives - diatoms
X 100
Variable or Measurement Precision Accuracy Completeness
Enumeration
Identification
75% a
75% a
85% b
85% b
99%
99%
a As measured by (100%-PTD);b As measured by (100%-PDE)
Table 9.7 Periphyton: measurement data quality objectives - soft bodied algae
Variable or Measurement Precision Accuracy Completeness
Identification 50%a 50%b
a As measured by PctDiff
Table 9.8 Periphyton: quality control - all activities
Internal QC Taxonomist
verifies that diatom
slide is appropriate for
diatom analysis
Duplicate identification
by Internal QC
Taxonomists
requency
All samples
Acceptance C
1 in 10 samples per
taxonomist, with a
minimum of 1 NRSA
sample
No obvious problems such
as bubbles under the
coverslip
PctDiff<50% (soft algae)
PDE < 15% (diatoms)
PTD < 25% (diatoms)
Slide is discarded and replaced
with a new slide
If any criterion is exceeded,
perform a third count and
reidentification for the sample.
O
LJJ
CL
Independent
identification by
All uncertain taxa
Uncertain identifications
to be confirmed by expert
in particular taxa
Record both tentative and
independent IDs
126
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 127 of 224
outside, expert,
taxonomist
External QC
Use of
widely/commonly
accepted taxonomic
references by all NRSA
labs
Prepare reference
collection
10% of all samples
PctDiff<50% (soft algae)
Attempt to reconcile results
completed per PDE < 15% (diatoms) | during a conference call with
laboratory
For all identifications
Each new taxon per
laboratory
PTD < 25% (diatoms)
All keys and references
EPA and the two laboratories.
Document unresolved
disagreements for the data
analyst to determine if lowest
practical level (e.g., species) is
appropriate, or if identification
should be to the level that both
labs found (e.g., class)
If a lab proposes to use other
used by each lab must be references, the lab must identify
on bibliography prepared them in the database.
by one or more additional
NRSA labs or in BioData
(see Section 9.7 for
retrieval instructions). This
requirement demonstrates
the general acceptance of
the references by the
scientific community.
Complete reference
collection to be
maintained by each
individual laboratory
Internal Taxonomy QC Officer
periodically reviews data and
reference collection to ensure
reference collection is complete
and identifications are accurate
Table 9.9 Periphyton: data validation
Check or Sample
Description
Taxonomic "reasonable-
ness" checks
Frequency
All data
Acceptance Criteria
Taxa known to occur in
given rivers or streams or
geographic area
Corrective Action
Second or third identification by
expert in that taxon
O
LJJ
CL
127
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 128 of 224
10.0 PERIPHYTON META-GENOMICS (PILOT RESEARCH EFFORT)
Laboratory methods incorporated in an ORD Quality Assurance Project Plan.
t
o
it
tc
0)
OJ
cc.
I
b.
O
z
LJJ
(D
O
LJJ
CL
128
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
11.0 WATER CHEMISTRY AND CHLOROPHYLL A
Laboratory Operations Manual
Page 129 of 224
11.1 Analytical Parameters
A total of 18 parameters are determined from each bulk water chemistry sample collected (Table 11.1).
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 11.1 Water chemistry: NRSA 2013/14 parameters
Analyte Units Comments
Conductivity
pH (laboratory)
Turbidity
Dissolved Organic Carbon
(DOC)
Ammonium (NH4)
Nitrate-Nitrite (NO3-NO2)
Total Nitrogen (TN)
Total Phosphorus (TP)
Sulfate(SO4)
Chloride (Cl)
Nitrate (NO3)
Calcium (Ca)
Magnesium (Mg)
Sodium (Na)
Potassium (K)
Silica (SiO2)
Total Suspended Solids (TSS)
True Color
Chlorophyll a
|aS/cm at 25ฐC
Standard (Std) Units
Nephelometric Turbidity Units (NTU)
mgC/L
mgN/L
mgN/L
mg/L
ugP/L
mg SO4/L
mg CI/L
mgN/L
mg Ca/L
mg Mg/L
mg Na/L
mgK/L
mg SiO2/L
mg/L
PCU
|ag/L (in extract)
11.2 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), labs
must notify the NARS IM Center (see APPENDIX A: CONTACT INFORMATION).
Upon receipt of samples, the assigned lab personnel inspects each sample and review the tracking form
that was included with the samples. The assigned lab personnel will flag samples damaged during the
shipping process in NARS IM upon receipt and inspection. The lab personnel then store samples at 4ฐC
in darkness until aliquots are ready to be prepared. If possible, the lab will prepare aliquots the same
day as samples are received, but no later than 48 hours after receipt. Labs 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). The NRSA team and water chemistry analysts do not
CL
O
Cฃ
O
U
T3
C
tc
cc
U
cc
129
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 130 of 224
reject results from samples that exceeded target holding times outright; the team will review the data
and holding times and make a decision about whether EPA will use the data in the NRSA assessment.
11.2.1 Water Chemistry Samples
Sample Receipt
4 L Bulk Sample
Inspect samples and
complete tracking form
Store at 4ฐC in darkness
.
IPCC
Filtration (0.4um)
HOPE
bottle
Acid
washed
Preserv
p with
J
Analyses
Calcium (180
days)
Magnesium
(180 days)
f. -*--s
HOPE
bottle
Not acid
washed
Store at
4 ฐC. in
J
Analyses
Chloride (28
days)
Nitrate (7 days)
Sulfate (28
days)
Silica (28 days)
v
HOPE
bottle
Acid
washed
Preserv
I p with
J
Analyses
Ammonia (28 days)
Dissolved Organic
Carbon (28 days)
J Nitrate-Nitrite (28 davs^
HOPE HOPE
bottle bottle
Acid Not acid
washe washed
d Store at
Prespr 4 T. in
Analyses
Total Phosphorus
(28 days)
^^^
^^
Analyses
pH (3 days)
ANC (7 days)
Conductivity
(28 days)
TSS (7 days)
Turbidity (3
^_.._x ^
Figure 11.1 Water chemistry: sample processing procedures
Figure 11.1 illustrates sample preparation processing for the water chemistry indicators, including
filtering and acidifying, for the various analytes.
1. Use 0.4u.m pore size polycarbonate filters for all filtration.
2. Rinse vacuum filter funnel units thoroughly with reverse-osmosis (RO) or de-ionized (Dl) water
(ASTM Type II reagent water) five times before each use and in between samples. After placing a
filter in the funnel unit, run approximately 100 ml of RO or Dl water through the filter, with
vacuum pressure, to rinse the filter. Discard the rinse water.
CL
O
Cฃ
O
u
T3
C
rc
U
130
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
3.
Laboratory Operations Manual
Page 131 of 224
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 (HNO3 or H2SO4,
depending on the analyte, see Table 11.2) 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 HNO3) in a refrigerator at 4ฐC.
Table 11.2 Water chemistry: acid preservatives added for various analytes
Preservatives
H2SO4
DOC
NH4
Total N
Total P
NO2-NO3
HNO3
Ca
Mg
Na
K
11.2.2 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 ฑ
2ฐC for no more than thirty days before analysis.
11.3 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 11.4). 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
Wilson (1986). The transition value is the value at which performance objectives for precision and bias
switch from absolute (< transition value) to relative (> transition value). For pH, the objectives are
established for samples with higher and lower pH levels.
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
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 (EPA ORD-Corvallis) are summarized in Table 11.3.
Participating laboratories may use alternative analytical methods for each target analyte as long as they
can satisfactorily demonstrate the alternative method is able to achieve the performance requirements
CL
O
Cฃ
O
U
T3
C
tc
cc
U
cc
131
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 132 of 224
as listed in Table 11.4. Information is provided by the lab to the NRSA Quality Team. The team reviews
the information to determine whether the labs meet the necessary requirements. The information from
this process is maintained in the NRSA 2012 QA files by the EPA HQ NARS QA Lead.
Table 11.3 Water chemistry: NRSA 2013/14 analytical methods (Central Laboratory, EPA ORD-Corvallis)
Analyte
pH (lab)
Specific conductance
@25ฐC
Acid neutralizing
capacity (ANC)
Turbidity
Total suspended solids
(TSS)
True color (Hach Kit)
Dissolved Organic
Carbon (DOC)g
Nitrate+Nitrite, as N
(fresh waters)
Ammonia, as N (fresh
waters)
Silica, dissolved (SiO2)
Fresh waters
Total nitrogen (TN)
Summary of Method*1
Automated, using ManSci PC-Titrate w/Titra-Sip
autotitrator and Ross combination pH electrode.
Initial pH determination for ANC titration
Electrolytic, Man-Tech TitraSip automated analysis
OR manual analysis, electrolytic
Automated acidimetric titration to pH<3.5, with
modified Gran plot analysis
Nephelometric; Man-Tech TitraSip automated
analysis,
OR
Manual analysis using Hach turbidimeter (high
turbidity samples)
Gravimetric, dried at 104 ฐC
Visual comparison to calibrated glass color disk.
UV promoted persulfate oxidation to COz with
infrared detection
Ion Chromatography
OR
FIA automated colorimetric (cadmium reduction)
FIA automated colorimetric (salicylate,
dichloroisocyanurate)
FIA automated colorimetric (molybdate, stannous
chloride)
Persulfate Digestion; FIA Automated Colorimetric
Analysis (Cadmium Reduction, sulfanilamide)
References"
EPA 150.6 (modified)
EPA 120.6
U.S. EPA (1987)
APHA 214 A, EPA 180.1 U.S. EPA
(1987)
EPA 160.2; APHA 209-C
APHA 204 A (modified), EPA
110.2 (modified), U.S. EPA (1987)
APHA5310-C
U.S. EPA (1987)
EPA 300.6; SW-846 9056A; APHA
4110B
EPA 353.2
APHA4500-NO3-N-E
Lachat 10-107-04- 1-C
Lachat 10-107-06- 3-D
EPA366.0,APHA425C
Lachat 10-114-27- 1-B
EPA353.2 (modified)
APHA 4500-N-C (modified)
ASTM WK31786
U.S. EPA (1987)
Lachat 10-107-04- 1-C (modified)
WRS SOP'
WRS 16A.O (April
2011)
WRS 16A.O (April
2011)
WRS 11A.4 (April
2011)
WRS 16A.O (April
2011)
WRS 16A.O (April
2011)
WRS 13A.3 (April
2011)
WRS 14B.4
(February 2011)
WRS 15A.3 (April
2011)
WRS21A.4(May
2011)
WRS 36A.O (April
2011
WRS40A.5(May
2011)
WRS 30A.4 (April
2011)
WRS 32A.5
(February 2010)
WRS 34A.5 (April
2011)
d FIA=Flow injection analysis. AAS=Atomic Absorption Spectrometry
e 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.
f WRS= Willamette Research Station. References are to laboratory SOP being used at central laboratory. Available
upon request, (contact the Project Lead)
8 For DOC, "dissolved" is defined as that portion passing through a 0.45 u.m nominal pore size filter. For other
analytes, "dissolved" is defined as that portion passing through a 0.4 urn pore size filter (Nucleopore or
equivalent).
CL
o
QC
O
u
T3
c
tc
cc
u
QC
132
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 133 of 224
Analyte Summary of Methodd References6 WRS SOP*
Total phosphorus (TP)
Major anions,
dissolved
chloride, nitrate,
nitrite, sulfate
Major cations,
dissolved
calcium, sodium,
potassium,
magnesium
Chlorophyll-a
(Chl-a)
Persulfate Digestion; Automated Colorimetric
Analysis (molybdate, ascorbic acid)
Ion Chromatography
Inductively-coupled Plasma Atomic Emission
Spectroscopy (ICP-AES)
OR
Flame AAS
Extraction 90% acetone analysis by fluorometry
APHA4500-P-E
USGS 1-4650-03
U.S. EPA (1987)
Lachat 115-01- 1-B (modified)
EPA 300.6; SW-846 9056A; APHA
4110B
EPA 200. 7; EPA 6010B
U.S. EPA (1987), EPA 215.1
EPA 273.1, EPA 258.1
EPA 242.1
EPA 445.0, EPA 446.0
WRS34A.5 (April
2011)
WRS40A.5(May
2011)
WRS SOP 3.04 v3
(October 2011)
WRS 50A.4 (March
2007)
WRS 71A.3 (April
2011)
11.4 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 labs.
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 NRSA 2013/14 QAPP will be followed to ensure these
LRLs are met for the NRSA 2013/14.
11.4.1 Laboratory Performance Requirements
Table 11.4 summarizes the pertinent laboratory performance requirements for the water chemistry and
chlorophyll A indicators.
11.4.2 Laboratory Quality Control Samples
Table 11.5 summarizes the pertinent laboratory quality control samples for the water chemistry and
chlorophyll A indicators.
CL
O
Cฃ
O
U
T3
C
tc
cc
U
QC
133
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 134 of 224
Table 11.4 Water chemistry & chlorophyll-a: laboratory method performance requirements
Potential Lower
Range of Reporting Transition Precision Bias
Analyte Units Samplesh Limit' Valuej Objectivek Objective1
Conductivity
pH (laboratory)
Turbidity
Dissolved
Organic Carbon
(DOC)
Ammonium
(NH4)
I^S/cm at 25ฐC
Std Units
Nephelometric
Turbidity Units
(NTU)
mgC/L
mgN/L
Nitrate-Nitrite mg N/L
(NO3-NO2)
Total Nitrogen
(TN)
Total
mg/L
ugP/L
Phosphorus
(TP)
1 to 15,000
3.5 to 10
0 to 44,000
0.1 to 109
Otol7
2.0
N/A
2.0
0.20
0.02 (1.4
Heq/L)
0 to 360 (as 0.02
nitrate)
0.1 to 90
0 to 22,000
0.02
4
20
5.75
20
<1
>1
0.10
0.10
0.10
20
ฑ 2 or ฑ10%
>5.75= ฑ0.15
<5.75 =ฑ0.07
ฑ 2 or ฑ10%
ฑ0.10 or
ฑ10%
ฑ0.01 or
ฑ10%
ฑ0.01 or
ฑ10%
ฑ0.01 or
ฑ10%
ฑ 2 or ฑ10%
ฑ 2 or 5%
>5.75 =
ฑ0.05
<5.75 =
ฑ0.15
ฑ2 or
ฑ10%
ฑ0.10 or
ฑ10%
ฑ0.01 or
ฑ10%
ฑ0.01 or
ฑ10%
ฑ0.01 or
ฑ10%
ฑ2 or
ฑ10%
h 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.
'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 method
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.
J Value at which performance objectives for precision and bias switch from absolute (< transition value) to relative
> transition value). Two-tiered approach based on Hunt, D.T.E. and A.L Wilson. 1986. The Chemical Analysis of
Water: General Principles and Techniques. 2nd ed. Royal Society of Chemistry, London, England.
k For standard samples, precision is estimated as the standard deviation of repeated measurements across batches
at the lower concentration range, and as percent relative standard deviation of repeated measurements across
batches at the higher concentration range.
For pH precision, the looser criteria applies to more highly alkaline samples. For NRSA, that is less of a concern
than the ability to measure acidic samples accurately and precisely.
1 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.
CL
o
CC
o
u
T3
c
tc
cc
u
cc
134
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 135 of 224
Potential Lower
Range of Reporting Transition Precision Bias
Analyte Units Samplesh Limit' Valuej Objectivek Objective1
Sulfate(SO4) mgSO4/L
Chloride (Cl)
Nitrate (NO3)
Calcium (Ca)
Magnesium
(Mg)
Sodium (Na)
Potassium (K)
Silica (SiO2)
Total
Suspended
Solids (TSS)
True Color
Chlorophyll a
mg CI/L
mgN/L
mg Ca/L
mg Mg/L
mg Na/L
mgK/L
mg SiO2/L
mg/L
PCD
|ag/L (in extract)
0 to 5,000 0.50 (10 2.5
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
0.01 to 100
0 to 27,000
0 to 350
0.7 to
11,000
Heq/L)
0.20 (6
Heq/L)
0.02 (4
Heq/L)
0.10(5
Heq/L)
0.10(8
Heq/L)
0.10(4
Heq/L)
0.10(2
Heq/L)
0.10
2
5
0.5
1
0.1
0.5
0.5
0.5
0.5
0.5
10
50
15
ฑ0.25 or
ฑ10%
ฑ0.10 or
ฑ10%
ฑ0.01 or
ฑ10%
ฑ 0.05 or
ฑ10%
ฑ 0.05 or
ฑ10%
ฑ 0.05 or
ฑ10%
ฑ 0.05 or
ฑ10%
ฑ 0.05 or
ฑ10%
ฑ 1 or ฑ10%
ฑ5 or ฑ10%
ฑ 1.5 or ฑ10%
ฑ0.25 or
ฑ10%
ฑ0.10 or
ฑ10%
ฑ0.01
ฑ10%
ฑ0.05 or
ฑ10%
ฑ0.05 or
ฑ10%
ฑ0.05 or
ฑ10%
ฑ0.05 or
ฑ10%
ฑ0.05 or
ฑ10%
ฑ lor
ฑ10%
ฑ5 or
ฑ10%
ฑ1.5 or
ฑ10%
CL
O
Cฃ
O
U
T3
C
tc
cc
U
cc
135
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Table 11.5 Water chemistry: quality control - laboratory samples
Laboratory Operations Manual
Page 136 of 224
QC Sample
Type and Acceptance
Description Analytes Description Frequency Criteria Corrective Action
Laboratory/
Reagent
Blank
Filtration
Blank
LT-MDL
Limit
Quality
Control
Check
Sample
(QCCS)
Calibration
QCCS
All except TSS
(For TSS, the lab
will filter a
known volume
of reagent water
and process the
filters per
method)
All dissolved
analytes
All analyses
ASTMTypell
Once per day
Control limits
Prepare and analyze new
prior to < LRL blank. Determine and
sample
analysis
Prepare once
Measured
correct problem (e.g.,
reagent contamination,
instrument calibration, or
contamination introduced
during filtration) before
proceeding with any
sample analyses.
Reestablish statistical
control by analyzing three
blank samples.
Measure archived
reagent per week 1 concentrations samples if review of other
water and archive
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 137 of 224
QC Sample
Type and Acceptance
Description Analytes Description Frequency Criteria
Laboratory
Duplicate
Sample
Standard
Reference
Material
(SRM)
Matrix
Spike
All analyses
When available
fora particular
analyte
Only prepared
when samples
Samples with potential
for matrix
interferences are
encountered
One per
batch
One analysis
Control limits
Corrective Action
If results are below LRL:
< precision Prepare and analyze split
objective from different sample
Manufacturers
(volume permitting).
Review precision of QCCS
measurements for batch.
Check preparation of split
sample. Qualify all
samples in batch for
possible reanalysis.
Analyze standard in next
in a certified range batch to confirm
minimum of
five separate
batches
One per
batch
Control limits
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.
Select two additional
for recovery samples and prepare
cannot exceed fortified subsamples.
100ฑ20% 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).
137
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
11.4.3 Data Reporting, Review, and Management
Laboratory Operations Manual
Page 138 of 224
Checks made of the data in the process of review and verification are summarized in Table 11.6. Data
reporting units and significant figures are given in Table 11.7. The NRSA 2013/14 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 11.6 Water chemistry: quality control - data validation
Activitv or Procedure
Range checks, summary statistics, and/or
exploratory data analysis (e.g., box and
whisker plots)
Requirements and Corrective Action
Correct reporting errors or qualify as suspect or invalid.
Review holding times
Ion balance:
Calculate percent ion balance difference
(%IBD) using data from cations, anions,
pH, andANC.
Qualify value for additional review
Conductivity check:
Compare measured conductivity of each
sample to a calculated conductivity
based on the equivalent conductance of
major ions in solution (Hillman et al.,
1987).
If total ionic strength <100 |aeq/L
-%IBD<ฑ25%.
If total ionic strength > 100 |aeq/L
-%IBD<ฑ10%.
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
Review data from QA samples (laboratory
PE samples, and inter-laboratory
comparison samples)
If measured conductivity < 25 |j,S/cm,
- ([measured 13 calculated] * measured) < ฑ25%.
If measured conductivity > 25 |j,S/cm,
([measured 13 calculated] -f- measured) < ฑ15%.
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 11.7 Water chemistry: data reporting criteria
No. Significant Maximum No.
Measurement Units Figures Decimal Places
DO
Temperature
PH
Carbon, total & dissolved organic
ANC
Conductivity
mg/L
ฐC
pH units
mg/L
|aeq/L
|aS/cm at 25 ฐC
2
2
3
3
3
3
1
1
2
1
1
1
CL
o
Cฃ
o
u
T3
c
tc
cc
u
QC
138
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 139 of 224
No. Significant Maximum No.
Measurement Units Figures Decimal Places
Calcium, magnesium, sodium, potassium,
ammonium, chloride, nitrate, and sulfate
Silica
Total phosphorus
Total nitrogen
Nitrate-Nitrite
Ammonium
Turbidity
True co lor
TSS
Chlorophyll a
^eq/L
mg/L
MgA
mg/L
mg/L
mg/L
NTU
PCD
mg/L
ug/l
3
3
3
3
3
3
3
2
3
3
1
2
0
2
2
2
0
0
1
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 11.1 Percent ion difference (%IBD)
(y cations - y anions) - ANC
%IBD= ^ ^ '
ANC + ^ anions + ^ cations + 2[H+\
where ANC is the acid neutralization capacity; cations are the concentrations of calcium, magnesium,
sodium, potassium, and ammonium (converted from mg/L to |o,eq/L); anions are the concentrations of
chloride, nitrate, and sulfate (converted from mg/L to |o,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 |o,eq/L are
presented in Table 11.8. For the conductivity check, equivalent conductivities for major ions are
presented in Table 11.9.
Table 11.8 Water chemistry: constants for converting major ion concentration from mg/L to u.eq/L
Analyte Conversion from mg/L to u,eq/Lm
Calcium
Magnesium
Potassium
Sodium
Ammonium
Chloride
Nitrate
Sulfate
49.9
82.3
25.6
43.5
55.4
28.2
16.1
20.8
CL
O
CC
O
U
T3
C
tc
cc
1 Measured values are multiplied by the conversion factor.
u
cc
139
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 140 of 224
Table 11.9 Water chemistry: factors to calculate equivalent conductivities of major ions."
Equivalent Conductance per mg/L Equivalent Conductance per
Ion (uS/cm at 25 ฐC) Ion mg/L (uS/cm at 25 ฐC)
Calcium
Magnesium
Potassium
Sodium
Ammonium
Chloride
2.60
3.82
1.84
2.13
4.13
2.14
Nitrate
Sulfate
Hydrogen
Hydroxide
Bicarbonate
Carbonate
1.15
1.54
3.5 x 105 ฐ
1.92 x 105
0.715
2.82
11.5 Literature Cited
Hunt, D.T.E. and A.L. Wilson. 1986. The Chemical Analysis of Water: General Principles and Techniques.
2nd ed.. Royal Society of Chemistry, London, England.
Youden, W.J. 1969. Ranking laboratories by round-robin tests. In Precision Measurement and
Calibration. H.H. Ku, ed. NBS Special Publication 300, Vol. 1. U.S. GPO Washington, D.C.
CL
O
Cฃ
O
U
T3
C
tc
cc
"From Hillmanetal. (1987).
"Specific conductance per mole/L, rather than per mg/L.
u
cc
140
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 141 of 224
APPENDIX A: CONTACT INFORMATION
O
o
u
X
Q
CL
CL
141
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
EPA HQ NRSA Project Lead Ellen Tarquinio, OW
Laboratory Operations Manual
Page 142 of 224
Contact Information
tarquinio.ellen@epa.gov
202-566-2267
EPA HQ NRSA Project QA Sarah Lehmann, OW
Lead
lehmann.sarah@epa.gov
202-566-1379
EPA HQ NRSA Laboratory Kendra Forde, OW
Review Manager
forde.kendra@epa.gov
202-564-0417
Information Management Marlys Cappaert, SRA
Center Coordinator International Inc.
cappaert.marlys@epa.gov
541-754-4467
541-754-4799 (fax)
O
O
u
X
Q
CL
CL
142
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 143 of 224
APPENDIX B: LABORATORY REMOTE EVALUATION FORMS
QC
o
o
QC
QC
o
QC
O
CD
5
CD
X
Q
143
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 144 of 224
NRSA 2013-2014 Document Request Form - Chemistry Labs
EPA and its state and tribal partners will conduct a survey of the nation's rivers and streams. This
National River and Streams Assessment (NRSA), is designed to provide statistically valid regional and
national estimates of the condition of rivers and streams. Consistent sampling and analytical
procedures ensure that the results can be compared across the country.
As part of the 2013-2014 NRSA, the Quality Assurance Team has been requested to conduct a technical
assessment to verify quality control practices in your laboratory and its ability to perform chemistry
analyses under this project. Our review will be assessing your laboratory's ability to receive, store,
prepare, analyze, and report sample data generated under EPA's 2013-2014 NRSA.
The first step of this assessment process will involve the review of your laboratory's certification and/or
documentation. Subsequent actions may include (if needed): reconciliation exercises and/or a site visit.
All labs will need to complete the following forms:
All labs will be required to complete the following forms and check the specific parameter in which
your lab will be conducting an analysis for the 2013-2014 NRSA:
D Water Chemistry, chlorophyll a, and Ash Free Dry Mass (AFDM) (all of the analytes identified in
the LOM and QAPP)
D Microcystin
D Fish Tissue Plugs
If your lab has been previously approved within the last 5 years for the water chemistry indicator:
D A signature on the attached Lab Signature Form indicates that your lab will follow the quality
assurance protocols required for chemistry labs conducting analyses for the 2013-2014 NRSA.
D A signature on the Quality Assurance Project Plan (QAPP) and the Laboratory Operations
Manual (LOM) Signature Form indicates that you will follow both the QAPP and the LOM.
If you have not been approved within the last 5 years through the lab verification process for the on
water chemistry indicator, in order for us to determine your ability to participate as a lab in the NRSA, E
we are requesting that you submit the following documents (if available) for review: 2
D Documentation of a successful quality assurance audit from a prior National Aquatic Resource Q
Survey (NARS) that occurred within the last 5 years (if you need assistance with this please ^
contact the individual listed below). <
D Documentation showing participation in a previous NARS for Water Chemistry for the same ^]
parameters/methods. O
LJJ
Additionally, we request that all labs provide the following information in support of your >
capabilities, (these materials are required if neither of the two items above are provided): o
D A copy of your Laboratory's accreditations and certifications if applicable (i.e. NELAC, ISO, state g
certifications, NABS, etc.). ^
D An updated copy of your Laboratory's QAPP. ^
D Standard Operating Procedures (SOPs) for your lab for each analysis to be performed (if not x-
covered in 2013-2014 NRSA LOM). g
CL
CL
144
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 145 of 224
D Documentation attesting to experience running all analytes for the 2013-2014 NRSA, including
chloraphyll a and Ash Free Dry Mass (AFDM).
This documentation may be submitted electronically via e-mail to forde.kendra@epa.gov with a cc: to
tarquinio.ellen@epa.gov and lehmann.sarah@epa.gov. Questions concerning this request can be
submitted forde.kendra@epa.gov (202-566-0417) or tarquinio.ellen@epa.gov (202-566-2267).
cc.
O
O
tc
cc.
cc.
O
cc.
O
CO
s
CO
X
Q
CL
CL
145
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 146 of 224
Lab Signature Form - Chemistry Labs
I certify that the lab,
located in , will abide by the following standards in
performing the following data analysis and reporting for the 2013-2014 National Rivers
and Streams Assessment (NRSA).
This applies to the chemistryl indicator.
1.) Utilize procedures identified in the 2013-2014 NRSA Lab Operations Manual (or
equivalent). If using equivalent procedures, please provide procedures manual
and obtain approval from EPA.
2.) Read and abide by the 2013-2014 NRSA Quality Assurance Project Plan (QAPP)
and related Standard Operating Procedures (SOPs).
3.) Have an organized IT tracking system in place for recording sample tracking and
analysis data.
4.) Provide Quality Control (QC) data for internal QC check, on a quarterly basis.
5.) Provide data using the template provided on the NARS Sharefile.
6.) Provide data results in a timely manner. This will vary with the type of analysis
and the number of samples to be processed. Sample data must be received no
later than May 1, 2014 (for samples collected in 2013) and May 1, 2015 (for
samples collected in 2014) or as otherwise negotiated with EPA.
7.) Participate in a lab technical assessment or audit if requested by EPA NRSA staff
(this may be a conference call or on-site audit).
8.) Agree to analyze for all parameters specified in the LOM for the appropriate
indicator(s) identified above, including Chlorophyll-a and AFDM for water ^
chemistry. ฐ^
u_
z.
o
I
Signature Date ^
QC
QC
O
QC
O
CD
5
CD
X
Q
CL
CL
146
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 147 of 224
NRSA 2013-2014 Document Request Form - Biology Labs
EPA and its state and tribal partners will conduct a survey of the nation's rivers and streams. This
National River and Streams Assessment (NRSA), is designed to provide statistically valid regional and
national estimates of the condition of rivers and streams. Consistent sampling and analytical
procedures ensure that the results can be compared across the country.
As part of the 2013-2014 NRSA, the Quality Assurance Team has been requested to conduct a technical
assessment to verify quality control practices in your laboratory and its ability to perform biology
analyses under this project. Our review will be assessing your laboratory's ability to receive, store,
prepare, analyze, and report sample data generated under EPA's 2013-2014 NRSA.
The first step of this assessment process will involve the review of your laboratory's certification and/or
documentation. Subsequent actions may include (if needed): reconciliation exercises and/or a site visit.
All labs will be required to complete the following forms and check the specific parameter in which
your lab will be conducting an analysis for the 2013-2014 NRSA:
D Fish Voucher
D Benthic Macroinvertabrates
D Periphyton
If your lab has been previously approved within the last 5 years for the specific parameters:
D A signature on the attached Lab Signature Form indicates that your lab will follow the quality
assurance protocols required for biology labs conducting analyses for the 2013-2014 NRSA.
D A signature on the Quality Assurance Project Plan (QAPP) and the Laboratory Operations
Manual (LOM) Signature Form indicates you will follow both the QAPP and the LOM.
If you have not been approved within the last 5 years through the lab verification process for the
specific parameters, in order for us to determine your ability to participate as a lab in the NRSA, we
are requesting that you submit the following documents (if available) for review: ,
D Documentation of a successful quality assurance audit from a prior National Aquatic Resource %
Survey (NARS) that occurred within the last 5 years (if you need assistance with this please ^
contact the individual listed below). p
D Documentation showing participation in previous NARS for this particular indicator. ^
i
<
Additionally, we request that all labs provide the following information in support of your capabilities, ^
(these materials are required if neither of the two items above are provided): H
D A copy of your Laboratory's accreditations and certifications if applicable (i.e. NELAC, ISO, state LU
QC
certifications, NABS, etc.). >
''I Q;
0 Documentation of NABS (or other) certification for the taxonomists performing analyses (if p1
applicable). oc
D An updated copy of your Laboratory's QAPP. ^
D Standard Operating Procedures (SOPs) for your lab for each analysis to be performed (if not -
covered in 2013-2014 NRSA LOM). ><
D Resume of independent fish taxonomist who will verify fish vouchers (if applicable)
CL
CL
147
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 148 of 224
This documentation may be submitted electronically via e-mail to forde.kendra@epa.gov with a cc: to
tarquinio.ellen@epa.gov and lehmann.sarah@epa.gov. Questions concerning this request can be
submitted forde.kendra@epa.gov (202-566-0417) or tarquinio.ellen@epa.gov (202-566-2267).
cc.
O
O
tc
cc.
cc.
O
cc.
O
CO
s
CO
X
Q
CL
CL
148
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 149 of 224
Lab Signature Form - Biology Labs
I certify that the lab,
located in , will abide by the following standards in
performing biology data analysis and reporting for the 2013-2014 National Rivers and
Streams Assessment (NRSA).
This applies to the biological indicator.
9.) Utilize procedures identified in the 2013-2014 NRSA Lab Operations Manual (or
equivalent). If using equivalent procedures, please provide procedures manual
and obtain approval from EPA.
10.) Read and abide by the 2013-2014 NRSA Quality Assurance Project Plan (QAPP)
and related Standard Operating Procedures (SOPs).
11.) Have an organized IT tracking system in place for recording sample tracking and
analysis data.
12.)Use taxonomic standards outlined in the 2013-2014 NRSA Lab Manual.
13.) Participate in taxonomic reconciliation exercises during the field and data
analysis season, which include conference calls and other lab reviews.
14.) Provide Quality Control (QC) data for internal QC check, including for sorting, on
a monthly basis.
15.) Provide data using the template provided on the NARS Sharefile.
16.) 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, 2014 (for samples collected in 2013) and May 1, 2015 (for
samples collected in 2014) or as otherwise negotiated with EPA. Samples results
for independent taxonomic QC described in the LOM and QAPP must be
provided to EPA prior to final datasets (fall and winter) to allow for
reconciliation to take place.
17.) Participate in a lab technical assessment or audit if requested by EPA NRSA staff
(this may be a conference call or on-site audit).
18.)Agree to utilize taxonomic nomenclature and hierarchical established for NRSA
2013-2014.
Signature Date
149
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 150 of 224
APPENDIX C: SAMPLE LABORATORY FORMS
cc.
O
u_
cc
QC
O
CD
5
l/l
u
X
Q
CL
CL
150
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Benthic Macroinvertebrate: Sorting Bench Sheet
Laboratory Operations Manual
Page 151 of 224
Laboratory Information Sample Information
Project ID
Station Name
Station Location
Station Number
Sample ID
Site ID
Date Collected
Field Crew ID
Sorter Name
Date Sorting lnitiated_
Proficiency:
Proportion of Sample Sorted:
(e.g,A5)
Each grid
QuarterW
(if any)
Each quarter
Sorter's Results
# Organisms
Sorting QC Officer
(if QC check)
Sorting has unique has unique
Order identifier identifier
Per Grid
Cumulative
#Additional
Organisms
Cumulative
(include sorter#)
10
11
12
13
14
15
16
Is cumulative number between 500 and 600? Y / N (N permissible only if entire sample sorted)
If QC check, provide PSE:
Sorting QC Officer Initials,
151
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 152 of 224
Benthic Macroinvertebrates: Taxonomy Bench Sheet (optional)
Laboratory Information Sample Information
Project ID
Station Name
Station Location
Station Number
Sample ID
Site ID
Date Collected
Field Crew ID
Taxonomist Name
Date 1st Organism Identified in Sample:_
QC Check? Y / N
Counts of Organisms in the
Taxon:
(Use # in Taxon
Uniqueldentifier (seetargettaxon Distinct
from WQX) in Table 3) (Y/N)
Data
Cumulative Qualifier
Number of (Codes
Organisms in Table
in Sample 4)
Is cumulative number of organisms > 500? If not, unless the sample was completed sorted, obtain
more sorted sample.
Comments:
152
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Enterrococci (EPA Method 1606) Laboratory Bench Sheet
Site ID Sample ID Date_
Laboratory Operations Manual
Page 153 of 224
Purified DNA Extracts
Vol. SAE
Batch Sample QA/QC Sample Vol Buffer Added Color of 25X Dilution
Sample # ID# Qual Code (ml) Filtered (ul) Filter Needed? Comments
153
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Fish Voucher: Taxonomy Bench Sheet (optional)
Laboratory Operations Manual
Page 154 of 224
Laboratory Information Sample Information
Labname
QC Taxonomist
Project ID
Internal Sample ID
Sample ID
Site ID
Visit Number
Date Collected
Taxonomist Name:
Date 1st Specimen Identified in Sample:
Condi
Tag New tion Data Comment Including Citation
No. Taxon? Common or Scientific Name Code Flag if Appropriate
Additional Comments
154
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 155 of 224
APPENDIX D: OTHER PERTINENT ATTACHMENTS
u
<
cc.
LJJ
CL
Q
X
Q
CL
CL
155
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Table D.O.I Fish identification: standard common and scientific names
Laboratory Operations Manual
Page 156 of 224
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
Perciformes
Percopsiformes
Perciformes
Cypriniformes
Clupeiformes
Cypriniformes
Acipenseriformes
Esociformes
Clupeiformes
Lepisosteiformes
Cypriniformes
Cypriniformes
Cyprinodontiformes
Cyprinodontiformes
Siluriformes
Perciformes
Petromyzontiformes
Anguilliformes
Clupeiformes
Cyprinodontiformes
Cypriniformes
Perciformes
Salmoniformes
Salmoniformes
Salmoniformes
Petromyzontiformes
Perciformes
Cypriniformes
Perciformes
Perciformes
Perciformes
Cypriniformes
Cyprinodontiformes
Perciformes
Synbranchiformes
Perciformes
Beloniformes
Salmoniformes
Cichlidae
Amblyopsidae
Percidae
Catostomidae
Clupeidae
Cyprinidae
Acipenseridae
Umbridae
Clupeidae
Lepisosteidae
Cyprinidae
Cyprinidae
Cyprinodontidae
Poeciliidae
Loricariidae
Percidae
Petromyzontidae
Anguillidae
Clupeidae
Poeciliidae
Cyprinidae
Percidae
Salmonidae
Salmonidae
Salmonidae
Petromyzontidae
Percidae
Cyprinidae
Percidae
Percidae
Gobiidae
Cyprinidae
Goodeidae
Percidae
Synbranchidae
Sciaenidae
Belonidae
Salmonidae
Hemichromis letourneuxi
Speoplatyrhinus poulsoni
Etheostoma ramseyi
Hypentelium etowanum
Alosa alabamae
Cyprinella callistia
Scaphirhynchus suttkusi
Dallia pectoralis
Alosa pseudoharengus
Atractosteus spatula
Cyprinella xaenura
Gila alvordensis
Cyprinodon nevadensis
Poecilia formosa
Pterygoplichthys pardalis
Percina antesella
Lampetra appendix
Anguilla rostrata
Alosa sapidissima
Gambusia amistadensis
Pteronotropis grandipinnis
Percina gymnocephala
Salvelinus a 1 pin us
Coregonus autumnalis
Thymallus arcticus
Lampetra camtschatica
Etheostoma cragini
Notropis girardi
Etheostoma euzonum
Etheostoma sagitta
Cleveland/a ios
Gila orcuttii
Empetrichthys merriami
Etheostoma cinereum
Monopterus albus
Micropogonias undulatus
Strongylura marina
Salmo salar
african jewelfish
alabama cavefish
alabama darter
alabama hog sucker
alabama shad
alabama shiner
alabama sturgeon
alaska blackfish
alewife
alligator gar
altamaha shiner
alvord chub
amargosa pupfish
amazon molly
amazon sailfin catfish
amber darter
american brook lamprey
american eel
american shad
amistad gambusia
apalachee shiner
appalachia darter
arctic char
arctic Cisco
arctic grayling
arctic lamprey
arkansas darter
arkansas river shiner
arkansas saddled darter
arrow darter
arrow goby
arroyo chub
ash meadows poolfish
ashy darter
asian swamp eel
atlantic croaker
atlantic needlefish
atlantic salmon
156
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 157 of 224
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
Myliobatiformes
Acipenseriformes
Clupeiformes
Gadiformes
Perciformes
Perciformes
Perciformes
Perciformes
Cyprinodontiformes
Perciformes
Scorpaeniformes
Perciformes
Cyprinodontiformes
Perciformes
Cypriniformes
Cypriniformes
Perciformes
Perciformes
Perciformes
Cyprinodontiformes
Clupeiformes
Pleuronectiformes
Perciformes
Cyprinodontiformes
Cyprinodontiformes
Scorpaeniformes
Salmoniformes
Cypriniformes
Cypriniformes
Salmoniformes
Cyprinodontiformes
Cypriniformes
Cypriniformes
Cypriniformes
Cypriniformes
Cypriniformes
Cypriniformes
Cypriniformes
Perciformes
Dasyatidae
Acipenseridae
Clupeidae
Gadidae
Percidae
Sciaenidae
Cichlidae
Percidae
Fundulidae
Elassomatidae
Cottidae
Centrarchidae
Fundulidae
Percidae
Cyprinidae
Cyprinidae
Centrarchidae
Percidae
Percidae
Fundulidae
Engraulidae
Paralichthyidae
Percidae
Fundulidae
Fundulidae
Cottidae
Salmonidae
Cyprinidae
Cyprinidae
Salmonidae
Poeciliidae
Cyprinidae
Catostomidae
Cyprinidae
Cyprinidae
Catostomidae
Cyprinidae
Cyprinidae
Eleotridae
Dasyatis sabina
Acipenser oxyrinchus
Opisthonema oglinum
Microgadus tomcod
Etheostoma zonifer
Bairdiella icistia
Heros severus
Etheostoma zonale
Fundulus diaphanus
Elassoma zonatum
Cottus carolinae
Enneacanthus obesus
Fundulus cingulatus
Etheostoma zonistium
Luxilus zonistius
Cyprinella leedsi
Lepomis symmetricus
Etheostoma obeyense
Etheostoma forbesi
Fundulus julisia
Anchoa mitchilli
Citharichthys spilopterus
Etheostoma rubrum
Fundulus pulvereus
Fundulus nottii
Cottus extensus
Prosopium abyssicola
Cyprinella formosa
Notropis rupestris
Coregonus laurettae
Gambusia gaigei
Hybopsis amblops
Moxostoma ariommum
Notropis boops
Hypophthalmichthys nobilis
Ictiobus cyprinellus
Nocomis platyrhynchus
Notropis dorsal is
Gobiomorus dormitor
atlantic stingray
atlantic sturgeon
atlantic thread herring
atlantic tomcod
backwater darter
bairdiella
banded cichlid
banded darter
banded killifish
banded pygmy sunfish
banded sculpin
banded sunfish
banded topminnow
bandfin darter
bandfin shiner
bannerfin shiner
bantam sunfish
barcheek darter
barrens darter
barrens topminnow
bay anchovy
bay whiff
bayou darter
bayou killifish
bayou topminnow
bear lake sculpin
bear lake whitefish
beautiful shiner
bedrock shiner
bering Cisco
big bend gambusia
bigeye chub
bigeye jumprock
bigeye shiner
bighead carp
bigmouth buffalo
bigmouth chub
bigmouth shiner
bigmouth sleeper
157
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 158 of 224
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
Perciformes
Cypriniformes
Perciformes
Cypriniformes
Siluriformes
Perciformes
Siluriformes
Cypriniformes
Scorpaeniformes
Perciformes
Perciformes
Cypriniformes
Perciformes
Salmoniformes
Perciformes
Cypriniformes
Cypriniformes
Cypriniformes
Cypriniformes
Perciformes
Perciformes
Cypriniformes
Cyprinodontiformes
Cyprinodontiformes
Cypriniformes
Cypriniformes
Cypriniformes
Cypriniformes
Cypriniformes
Perciformes
Salmoniformes
Perciformes
Perciformes
Cypriniformes
Cyprinodontiformes
Perciformes
Siluriformes
Cypriniformes
Scorpaeniformes
Percidae
Cyprinidae
Cichlidae
Catostomidae
Ictaluridae
Centrarchidae
Ictaluridae
Catostomidae
Cottidae
Percidae
Centrarchidae
Cyprinidae
Cichlidae
Salmonidae
Percidae
Catostomidae
Cyprinidae
Cyprinidae
Cyprinidae
Percidae
Percidae
Cyprinidae
Fundulidae
Fundulidae
Catostomidae
Cyprinidae
Catostomidae
Cyprinidae
Cyprinidae
Percidae
Salmonidae
Percidae
Gobiidae
Cyprinidae
Poeciliidae
Percidae
Ictaluridae
Cyprinidae
Cottidae
Percina macrolepida
Rhodeus sericeus
Cichlasoma bimaculatum
Ictiobus niger
Ameiurus melas
Pomoxis nigromaculatus
Noturus funebris
Moxostoma duquesnei
Cottus baileyi
Percina nigrofasciata
Enneacanthus chaetodon
Notropis heterodon
Sarotherodon melanotheron
Coregonus nigripinnis
Etheostoma nigripinne
Thoburnia atripinnis
Notropis melanostomus
Notropis heterolepis
Phoxinus cumberlandensis
Percina maculata
Etheostoma duryi
Notropis atrocaudalis
Fundulus olivaceus
Fundulus notatus
Moxostoma poecilurum
Cyprinella venusta
Moxostoma cervinum
Lythrurus atrapiculus
Luxilus zonatus
Etheostoma blennius
Coregonus hoyi
Etheostoma sanguifluum
Ctenogobius fasciatus
Erimystax insignis
Gambusia senilis
Percina burtoni
Ictalurus furcatus
Gila coerulea
Cottus caeruleomentum
bigscale logperch
bitterling
black acara
black buffalo
black bullhead
black crappie
black madtom
black redhorse
black sculpin
blackbanded darter
blackbanded sunfish
blackchin shiner
blackchin tilapia
blackfin Cisco
blackfin darter
blackfin sucker
blackmouth shiner
blacknose shiner
blackside dace
blackside darter
blackside snubnose darter
blackspot shiner
blackspotted topminnow
blackstripe topminnow
blacktail redhorse
blacktail shiner
blacktip jumprock
blacktip shiner
bleeding shiner
blenny darter
bloater
bloodfin darter
blotchcheek goby
blotched chub
blotched gambusia
blotchside logperch
blue catfish
blue chub
blue ridge sculpin
158
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 159 of 224
LINE NO.
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
ORDER
Cypriniformes
Cypriniformes
Perciformes
Clupeiformes
Perciformes
Perciformes
Cyprinodontiformes
Cypriniformes
Perciformes
Cypriniformes
Cypriniformes
Cypriniformes
Cypriniformes
Perciformes
Perciformes
Perciformes
Cypriniformes
Cypriniformes
Perciformes
Cypriniformes
Cypriniformes
Salmoniformes
Salmoniformes
Cypriniformes
Cypriniformes
Perciformes
Amiiformes
Cypriniformes
Cypriniformes
Cypriniformes
Perciformes
Siluriformes
Salmoniformes
Cypriniformes
Cyprinodontiformes
Perciformes
Perciformes
Atheriniformes
Gasterosteiformes
FAMILY
Cyprinidae
Catostomidae
Cichlidae
Clupeidae
Elassomatidae
Percidae
Fundulidae
Cyprinidae
Centrarchidae
Cyprinidae
Cyprinidae
Catostomidae
Cyprinidae
Percidae
Centrarchidae
Percidae
Cyprinidae
Cyprinidae
Percidae
Cyprinidae
Cyprinidae
Salmonidae
Salmonidae
Cyprinidae
Cyprinidae
Percidae
Amiidae
Cyprinidae
Catostomidae
Cyprinidae
Percidae
Ictaluridae
Salmonidae
Cyprinidae
Fundulidae
Percidae
Percidae
Atherinopsidae
Gasterosteidae
SCIENTIFIC NAME
Cyprinella caerulea
Cycleptus elongatus
Oreochromis aureus
Alosa aestivalis
Elassoma okatie
Etheostoma camurum
Lucania goodei
Campostoma pauciradii
Lepomis macrochirus
Nocomis leptocephalus
Pteronotropis hubbsi
Catostomus discobolus
Pteronotropis welaka
Etheostoma jessiae
Enneacanthus gloriosus
Percina cymatotaenia
Cyprinella callitaenia
Cyprinella camura
Etheostoma chlorosoma
Pimephales notatus
Notropis simus
Prosopium gemmifer
Prosopium spilonotus
Gila elegans
Gila boraxobius
Etheostoma wapiti
Amia co/i/o
Hybognathus hankinsoni
Catostomus columbianus
Notropis bifrenatus
Etheostoma lynceum
Noturus miurus
Coregonus nasus
Pteronotropis euryzonus
Fundulus euryzonus
Percina palmaris
Etheostoma burri
Labidesthes sicculus
Culaea inconstans
COMMON NAME
blue shiner
blue sucker
blue tilapia
blueback herring
bluebarred pygmy sunfish
bluebreast darter
bluefin killifish
bluefin stoneroller
bluegill
bluehead chub
bluehead shiner
bluehead sucker
bluenose shiner
blueside darter
bluespotted sunfish
bluestripe darter
bluestripe shiner
bluntface shiner
bluntnose darter
bluntnose minnow
bluntnose shiner
bonneville Cisco
bonneville whitefish
bonytail
borax lake chub
boulder darter
bowfin
brassy minnow
bridgelip sucker
bridle shiner
brighteye darter
brindled madtom
broad whitefish
broadstripe shiner
broadstripe topminnow
bronze darter
brook darter
brook silverside
brook stickleback
159
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 160 of 224
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
Salmoniformes
Siluriformes
Perciformes
Siluriformes
Siluriformes
Salmoniformes
Perciformes
Cypriniformes
Carcharhiniformes
Salmoniformes
Cypriniformes
Perciformes
Gadiformes
Cypriniformes
Cypriniformes
Perciformes
Siluriformes
Cypriniformes
Cypriniformes
Perciformes
Cypriniformes
Cypriniformes
Cypriniformes
Perciformes
Siluriformes
Perciformes
Esociformes
Cypriniformes
Esociformes
Perciformes
Siluriformes
Perciformes
Cypriniformes
Siluriformes
Perciformes
Perciformes
Cypriniformes
Petromyzontiformes
Perciformes
Salmonidae
Ictaluridae
Percidae
Callichthyidae
Ictaluridae
Salmonidae
Percidae
Cyprinidae
Carcharhinidae
Salmonidae
Cyprinidae
Channidae
Gadidae
Cyprinidae
Cyprinidae
Cichlidae
Ictaluridae
Cyprinidae
Cyprinidae
Percidae
Cyprinidae
Cyprinidae
Cyprinidae
Percidae
Ictaluridae
Elassomatidae
Umbridae
Cyprinidae
Esocidae
Percidae
Ictaluridae
Percidae
Cyprinidae
Ictaluridae
Percidae
Percidae
Cyprinidae
Petromyzontidae
Percidae
Sal veli n us fan final is
Ameiurus nebulosus
Etheostoma edwini
Hoplosternum littorale
Noturus phaeus
Salmo trutta
Etheostoma bison
Nocomis raneyi
Carcharhinus leucas
Salvelinus confluentus
Pimephales vigilax
Channa marulius
Lota lota
Macrhybopsis marconis
Notropis asperifrons
Cichla ocellaris
Noturus taylori
Notropis cahabae
Hesperoleucus symmetricus
Etheostoma osburni
Notropis mekistocholas
Luxilus cardinalis
Notropis percobromus
Etheostoma collis
Noturus furiosus
Elassoma boehlkei
Umbra limi
Campostoma anomalum
Esox niger
Percina nevisense
Ictalurus punctatus
Percina copelandi
Notropis wickliffi
Noturus flavater
Etheostoma scoff/
Etheostoma etnieri
Lythrurus roseipinnis
Ichthyomyzon castaneus
Etheostoma cervus
brook trout
brown bullhead
brown darter
brown hoplo
brown madtom
brown trout
buffalo darter
bull chub
bull shark
bull trout
bullhead minnow
bullseye snakehead
burbot
burrhead chub
burrhead shiner
butterfly peacock bass
caddo madtom
cahaba shiner
California roach
candy darter
cape fear shiner
cardinal shiner
carmine shiner
Carolina darter
Carolina madtom
Carolina pygmy sunfish
central mudminnow
central stoneroller
chain pickerel
chainback darter
channel catfish
channel darter
channel shiner
checkered madtom
Cherokee darter
cherry darter
cherryfin shiner
chestnut lamprey
chickasaw darter
160
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 161 of 224
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
232
233
Cypriniformes
Cypriniformes
Salmoniformes
Cypriniformes
Perciformes
Perciformes
Cypriniformes
Salmoniformes
Salmoniformes
Cypriniformes
Cyprinodontiformes
Cypriniformes
Perciformes
Osteoglossiformes
Perciformes
Perciformes
Cypriniformes
Scorpaeniformes
Salmoniformes
Perciformes
Cypriniformes
Scorpaeniformes
Cyprinodontiformes
Cypriniformes
Cypriniformes
Cypriniformes
Perciformes
Perciformes
Cyprinodontiformes
Perciformes
Perciformes
Cypriniformes
Perciformes
Perciformes
Cypriniformes
Cypriniformes
Perciformes
Cypriniformes
Perciformes
Cyprinidae
Cyprinidae
Salmonidae
Cyprinidae
Percidae
Percidae
Cyprinidae
Salmonidae
Salmonidae
Cyprinidae
Poeciliidae
Cyprinidae
Gobiidae
Notopteridae
Percidae
Percidae
Cyprinidae
Cottidae
Salmonidae
Percidae
Cyprinidae
Cottidae
Cyprinodontidae
Cyprinidae
Cyprinidae
Cyprinidae
Centropomidae
Percidae
Cyprinodontidae
Cichlidae
Percidae
Cyprinidae
Percidae
Percidae
Cyprinidae
Catostomidae
Percidae
Cyprinidae
Gobiidae
Gila nigrescens
Notropis chihuahua
Oncorhynchus tshawytscha
Acrocheilus alutaceus
Etheostoma davisoni
Etheostoma hopkinsi
Notropis potter/
Oncorhynchus keta
Coregonus artedi
Hybopsis winchelli
Gambusia heterochir
Pogonichthys ciscoides
Microgobius gulosus
Chitala ornata
Percina brevicauda
Etheostoma colorosum
Notropis petersoni
Cottus aleuticus
Oncorhynchus kisutch
Etheostoma ditrema
Ptychocheilus lucius
Cottus hubbsi
Cyprinodon elegans
Notropis amoenus
Cyprinus carpio
Luxilus cornutus
Centropomus undecimalis
Percina jenkinsi
Cyprinodon eximius
Cichlasoma nigrofasciatum
Etheostoma coosae
Notropis xaenocephalus
Etheostoma aquali
Etheostoma basilare
Semotilus atromaculatus
Erimyzon oblongus
Etheostoma collettei
Luxilus cerasinus
Lophogobius cyprinoides
chihuahua chub
chihuahua shiner
chinook salmon
chiselmouth
choctawhatchee darter
Christmas darter
chub shiner
chum salmon
Cisco
clear chub
clear creek gambusia
clear lake splittail
clown goby
clown knifefish
coal darter
coastal darter
coastal shiner
coastrange sculpin
coho salmon
coldwater darter
Colorado pikeminnow
Columbia sculpin
comanche springs pupfish
comely shiner
common carp
common shiner
common snook
conasauga logperch
conchos pupfish
convict cichlid
coosa darter
coosa shiner
coppercheek darter
corrugated darter
creek chub
creek chubsucker
Creole darter
crescent shiner
crested goby
161
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 162 of 224
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
234
235
236
237
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267
268
269
270
271
272
Perciformes
Perciformes
Perciformes
Cypriniformes
Perciformes
Perciformes
Cypriniformes
Salmoniformes
Perciformes
Cypriniformes
Perciformes
Salmoniformes
Scorpaeniformes
Salmoniformes
Cypriniformes
Cyprinodontiformes
Cypriniformes
Cyprinodontiformes
Cypriniformes
Cypriniformes
Perciformes
Salmoniformes
Perciformes
Cypriniformes
Cypriniformes
Perciformes
Cypriniformes
Cyprinodontiformes
Esociformes
Perciformes
Cypriniformes
Perciformes
Siluriformes
Perciformes
Cypriniformes
Gasterosteiformes
Perciformes
Salmoniformes
Perciformes
Belontiidae
Percidae
Percidae
Catostomidae
Percidae
Percidae
Cyprinidae
Salmonidae
Percidae
Cyprinidae
Gobiidae
Salmonidae
Cottidae
Osmeridae
Cyprinidae
Cyprinodontidae
Catostomidae
Cyprinodontidae
Cyprinidae
Cyprinidae
Centrarchidae
Salmonidae
Percidae
Cyprinidae
Cyprinidae
Percidae
Cyprinidae
Poeciliidae
Umbridae
Percidae
Cyprinidae
Percidae
Ictaluridae
Percidae
Cyprinidae
Gasterosteidae
Percidae
Osmeridae
Elassomatidae
Trichopsis vittata
Etheostoma corona
Crystal/arid asprella
Chasmistes cujus
Etheostoma susanae
Etheostoma uniporum
Exoglossum maxillingua
Oncorhynchus clarkii
Etheostoma proeliare
Hybognathus hayi
Ctenogobius boleosoma
Coregonus johannae
Myoxocephalus thompsonii
Hypomesus transpacificus
Eremichthys acros
Cyprinodon macularius
Catostomus clarkii
Cyprinodon diabolis
Dionda diaboli
Semotilus thoreauianus
Lepomis marginatus
Salvelinus malma
Percina sciera
Notropis cummingsae
Luxilus pilsbryi
Etheostoma percnurum
Rhinichthys atratulus
Gambusia holbrooki
Umbra pygmaea
Ammocrypta pellucida
Hybognathus regius
Etheostoma pseudovulatum
Noturus elegans
Etheostoma baileyi
Notropis atherinoides
Gasterosteus aculeatus
Etheostoma etowahae
Thaleichthys pacificus
Elassoma evergladei
croakinggourami
crown darter
crystal darter
cui-ui
Cumberland darter
current darter
cutlip minnow
cutthroat trout
cypress darter
cypress minnow
darter goby
deepwater Cisco
deepwater sculpin
delta smelt
desert dace
desert pupfish
desert sucker
devils hole pupfish
devils river minnow
dixie chub
dollar sunfish
dolly varden
dusky darter
dusky shiner
duskystripe shiner
duskytail darter
eastern blacknose dace
eastern mosquitofish
eastern mudminnow
eastern sand darter
eastern silvery minnow
egg-mimic darter
elegant madtom
emerald darter
emerald shiner
espinocho
etowah darter
eulachon
everglades pygmy sunfish
162
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 163 of 224
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
273
274
275
276
277
278
279
280
281
282
283
284
285
286
287
288
289
290
291
292
293
294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
Cypriniformes
Perciformes
Perciformes
Cypriniformes
Cypriniformes
Cypriniformes
Cypriniformes
Perciformes
Perciformes
Cypriniformes
Cyprinodontiformes
Cypriniformes
Cypriniformes
Siluriformes
Siluriformes
Cypriniformes
Perciformes
Lepisosteiformes
Perciformes
Cypriniformes
Perciformes
Scorpaeniformes
Gasterosteiformes
Perciformes
Siluriformes
Perciformes
Siluriformes
Perciformes
Perciformes
Perciformes
Cypriniformes
Cyprinodontiformes
Cypriniformes
Cyprinodontiformes
Salmoniformes
Perciformes
Clupeiformes
Perciformes
Perciformes
Cyprinidae
Percidae
Eleotridae
Cyprinidae
Cyprinidae
Cyprinidae
Cyprinidae
Percidae
Cichlidae
Cyprinidae
Cyprinodontidae
Cyprinidae
Catostomidae
Ictaluridae
Ictaluridae
Cyprinidae
Centrarchidae
Lepisosteidae
Percidae
Cyprinidae
Percidae
Cottidae
Gasterosteidae
Percidae
Ictaluridae
Percidae
Ictaluridae
Sciaenidae
Gobiidae
Percidae
Cyprinidae
Aplocheilidae
Cyprinidae
Poeciliidae
Salmonidae
Percidae
Clupeidae
Percidae
Percidae
Semotilus corpora/is
Etheostoma flabellare
Dormitator maculatus
Pimephales promelas
Phenacobius crassilabrum
Cyprinella pyrrhomelas
Phoxinus neogaeus
Etheostoma pyrrhogaster
Cichlasoma meeki
Pteronotropis signipinnis
Jordanella floridae
Hemitremia flammed
Catostomus latipinnis
Ameiurus platycephalus
Pylodictis olivaris
Platygobio gracilis
Centrarchus macropterus
Lepisosteus platyrhincus
Ammocrypta bifascia
Notropis edwardraneyi
Etheostoma fonticola
Myoxocephalus quadricornis
Apeltes quadracus
Percina stictogaster
Noturus munitus
Percina lenticula
Noturus nocturnus
Aplodinotus grunniens
Ctenogobius shufeldti
Etheostoma crossopterum
Notropis buchanani
Rivulus hartii
Gila intermedia
Poeciliopsis occidentalis
Oncorhynchus gilae
Percina evides
Dorosoma cepedianum
Etheostoma vitreum
Etheostoma denoncourti
fallfish
fantail darter
fat sleeper
fathead minnow
fatlips minnow
fieryblack shiner
finescale dace
firebelly darter
firemouth cichlid
flagfin shiner
flagfish
flame chub
flannelmouth sucker
flat bullhead
flathead catfish
flathead chub
flier
florida gar
florida sand darter
fluvial shiner
fountain darter
fourhorn sculpin
fourspine stickleback
frecklebelly darter
frecklebelly madtom
freckled darter
freckled madtom
freshwater drum
freshwater goby
fringed darter
ghost shiner
giant rivulus
gila chub
gila topminnow
gila trout
gilt darter
gizzard shad
glassy darter
golden darter
163
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 164 of 224
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
312
313
314
315
316
317
318
319
320
321
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
Cypriniformes
Cypriniformes
Cyprinodontiformes
Hiodontiformes
Cypriniformes
Perciformes
Perciformes
Cypriniformes
Cypriniformes
Cypriniformes
Perciformes
Cypriniformes
Cypriniformes
Acipenseriformes
Perciformes
Cyprinodontiformes
Perciformes
Perciformes
Cypriniformes
Cypriniformes
Perciformes
Perciformes
Perciformes
Cyprinodontiformes
Cypriniformes
Perciformes
Perciformes
Perciformes
Cyprinodontiformes
Perciformes
Gasterosteiformes
Cyprinodontiformes
Cypriniformes
Siluriformes
Cypriniformes
Perciformes
Siluriformes
Cypriniformes
Perciformes
Catostomidae
Cyprinidae
Fundulidae
Hiodontidae
Cyprinidae
Percidae
Percidae
Cyprinidae
Cyprinidae
Catostomidae
Lutjanidae
Catostomidae
Catostomidae
Acipenseridae
Centrarchidae
Poeciliidae
Percidae
Percidae
Cyprinidae
Cyprinidae
Percidae
Percidae
Centrarchidae
Fundulidae
Cyprinidae
Percidae
Eleotridae
Percidae
Fundulidae
Percidae
Syngnathidae
Poeciliidae
Cyprinidae
Ariidae
Catostomidae
Percidae
Ictaluridae
Cyprinidae
Percidae
Moxostoma erythrurum
Notemigonus crysoleucas
Fundulus chrysotus
Hiodon alosoides
Carassius auratus
Percina aurolineata
Etheostoma parvipinne
Ctenopharyngodon idella
Erimystax x-punctatus
Moxostoma congestion
Lutjanus griseus
Moxostoma lachneri
Moxostoma valenciennesi
Acipenser medirostris
Lepomis cyanellus
Xiphophorus hellerii
Etheostoma Jordan/
Etheostoma chlorobranchium
Cyprinella chloristia
Notropis chlorocephalus
Etheostoma blennioides
Etheostoma lepidum
Micropterus treculii
Fundulus parvipinnis
Dionda nigrotaeniata
Etheostoma oophylax
Guavina guavina
Etheostoma swaini
Fundulus grandis
Percina suttkusi
Syngnathus scovelli
Poecilia reticulata
Mylopharodon conocephalus
Ariopsis felis
Moxostoma lacerum
Etheostoma histrio
Ictalurus lupus
Gila nigra
Etheostoma lawrencei
golden redhorse
golden shiner
golden topminnow
gold eye
goldfish
goldline darter
goldstripe darter
grass carp
gravel chub
gray redhorse
gray snapper
greater jumprock
greater redhorse
green sturgeon
green sunfish
green swordtail
greenbreast darter
greenfin darter
greenfin shiner
greenhead shiner
greenside darter
greenthroat darter
guadalupe bass
guadalupe cardinalfish
guadalupe roundnose minnow
guardian darter
guavina
gulf darter
gulfkillifish
gulf logperch
gulf pipefish
guppy
hardhead
hardhead catfish
harelip sucker
harlequin darter
headwater catfish
headwater chub
headwater darter
l/l
u
<
cc.
LJJ
CL
Q
X
Q
CL
CL
164
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 165 of 224
LINE NO.
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
389
ORDER
Clupeiformes
Cypriniformes
Cypriniformes
Cypriniformes
Perciformes
Cypriniformes
Cypriniformes
Cypriniformes
Pleuronectiformes
Perciformes
Cypriniformes
Cypriniformes
Salmoniformes
Cypriniformes
Salmoniformes
Atheriniformes
Perciformes
Perciformes
Cypriniformes
Perciformes
Perciformes
Perciformes
Cypriniformes
Perciformes
Cypriniformes
Perciformes
Petromyzontiformes
Cypriniformes
Salmoniformes
Scorpaeniformes
Petromyzontiformes
Cypriniformes
Cypriniformes
Elopiformes
Cypriniformes
Cypriniformes
Cypriniformes
Acipenseriformes
Salmoniformes
FAMILY
Clupeidae
Cyprinidae
Catostomidae
Cyprinidae
Percidae
Cyprinidae
Cyprinidae
Cyprinidae
Achiridae
Percidae
Cyprinidae
Cyprinidae
Salmonidae
Cyprinidae
Salmonidae
Atherinopsidae
Percidae
Gerreidae
Cyprinidae
Cichlidae
Cichlidae
Percidae
Catostomidae
Percidae
Cyprinidae
Percidae
Petromyzontidae
Cyprinidae
Salmonidae
Cottidae
Petromyzontidae
Catostomidae
Catostomidae
Elopidae
Cyprinidae
Cyprinidae
Catostomidae
Acipenseridae
Salmonidae
SCIENTIFIC NAME
Alosa mediocris
Hybopsis hypsinotus
Carpiodes velifer
Notropis altipinnis
Etheostoma kantuckeense
Notropis micropteryx
Notropis hypsilepis
Lavinia exilicauda
Trinectes maculatus
Etheostoma brevirostrum
Nocomis biguttatus
Gila cypha
Coregonus pidschian
Leuciscus idus
Stenodus leucichthys
Menidia beryllina
Etheostoma exile
Diapterus auratus
Notropis chalybaeus
Cichlasoma octofasciatum
Cichlasoma managuense
Etheostoma nigrum
Chasmistes liorus
Etheostoma kanawhae
Phenacobius teretulus
Etheostoma rafinesquei
Lampetra hubbsi
Notropis ortenburgeri
Coregonus kiyi
Cottus princeps
Lampetra similis
Catostomus snyderi
Catostomus rimiculus
Elops saurus
Richardsonius egregius
Couesius plumbeus
Erimyzon sucetta
Acipenser fulvescens
Salvelinus namaycush
COMMON NAME
hickory shad
highback chub
highfin carpsucker
highfin shiner
highland rim darter
highland shiner
highscale shiner
hitch
hogchoker
holiday darter
hornyhead chub
humpback chub
humpback whitefish
ide
inconnu
inland silverside
iowa darter
irish pompano
ironcolor shiner
jack dempsey
jaguar guapote
johnny darter
June sucker
kanawha darter
kanawha minnow
kentucky darter
kern brook lamprey
kiamichi shiner
kiyi
klamath lake sculpin
klamath lamprey
klamath largescale sucker
klamath smallscale sucker
ladyfish
lahontan redside
lake chub
lake chubsucker
lake sturgeon
lake trout
165
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 166 of 224
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
390
391
392
393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
Salmoniformes
Perciformes
Cypriniformes
Cypriniformes
Perciformes
Cyprinodontiformes
Cypriniformes
Cypriniformes
Petromyzontiformes
Cypriniformes
Salmoniformes
Perciformes
Cyprinodontiformes
Siluriformes
Cypriniformes
Cyprinodontiformes
Cypriniformes
Perciformes
Cypriniformes
Cyprinodontiformes
Perciformes
Cypriniformes
Cypriniformes
Perciformes
Perciformes
Perciformes
Cypriniformes
Perciformes
Salmoniformes
Perciformes
Perciformes
Cypriniformes
Perciformes
Lepisosteiformes
Cypriniformes
Cypriniformes
Cypriniformes
Elopiformes
Scorpaeniformes
Salmonidae
Centrarchidae
Cyprinidae
Catostomidae
Eleotridae
Poeciliidae
Cyprinidae
Cyprinidae
Petromyzontidae
Cyprinidae
Salmonidae
Percidae
Poeciliidae
Ictaluridae
Cyprinidae
Cyprinodontidae
Cyprinidae
Percidae
Cyprinidae
Fundulidae
Percidae
Cyprinidae
Cyprinidae
Percidae
Percidae
Centrarchidae
Cyprinidae
Percidae
Osmeridae
Percidae
Gobiidae
Cyprinidae
Percidae
Lepisosteidae
Cyprinidae
Catostomidae
Catostomidae
Elopidae
Cottidae
Coregonus clupeaformis
Micropterus salmoides
Campostoma oligolepis
Catostomus macrocheilus
Eleotris amblyopsis
Gambusia geiseri
Rhinichthys deaconi
Phoxinus saylori
Lampetra aepyptera
lotichthys phlegethontis
Coregonus sardinella
Etheostoma microperca
Heterandria formosa
Noturus hildebrandi
Snyderichthys cope/
Cyprinodon bovinus
Rhinichthys falcatus
Percina pantherina
Hybopsis lineapunctata
Fundulus lineolatus
Etheostoma chuckwachatte
Lepidomeda vittata
Rhinichthys cobitis
Percina caprodes
Etheostoma neopterum
Lepomis megalotis
Agosia chrysogaster
Etheostoma longimanum
Spirinchus thaleichthys
Percina macrocephala
Gillichthys mirabilis
Rhinichthys cataractae
Percina nasuta
Lepisosteus osseus
Notropis longirostris
Catostomus catostomus
Deltistes luxatus
Elops affinis
Cottus bendirei
lake whitefish
largemouth bass
largescale stoneroller
largescale sucker
largescaled spinycheek
largespring gambusia
las vegas dace
laurel dace
least brook lamprey
least chub
least Cisco
least darter
least killifish
least madtom
leatherside chub
leon springs pupfish
leopard dace
leopard darter
lined chub
lined topminnow
lipstick darter
little Colorado spinedace
loach minnow
logperch
lollypop darter
longear sunfish
longfin dace
longfin darter
longfin smelt
longhead darter
longjaw mudsucker
longnose dace
longnose darter
longnose gar
longnose shiner
longnose sucker
lost river sucker
machete
malheur sculpin
166
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 167 of 224
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
466
467
Cypriniformes
Cyprinodontiformes
Cyprinodontiformes
Scorpaeniformes
Siluriformes
Scorpaeniformes
Cyprinodontiformes
Perciformes
Perciformes
Perciformes
Cyprinodontiformes
Cypriniformes
Cypriniformes
Characiformes
Perciformes
Petromyzontiformes
Cypriniformes
Cypriniformes
Atheriniformes
Cypriniformes
Perciformes
Cypriniformes
Perciformes
Cypriniformes
Hiodontiformes
Scorpaeniformes
Petromyzontiformes
Siluriformes
Mugiliformes
Cypriniformes
Cypriniformes
Cypriniformes
Salmoniformes
Perciformes
Perciformes
Perciformes
Cyprinodontiformes
Esociformes
Perciformes
Cyprinidae
Poeciliidae
Aplocheilidae
Cottidae
Ictaluridae
Cottidae
Fundulidae
Percidae
Cichlidae
Gobiidae
Poeciliidae
Catostomidae
Cyprinidae
Characidae
Cichlidae
Petromyzontidae
Cyprinidae
Cyprinidae
Atherinopsidae
Cyprinidae
Percidae
Cyprinidae
Percidae
Catostomidae
Hiodontidae
Cottidae
Petromyzontidae
Ictaluridae
Mugilidae
Cyprinidae
Cyprinidae
Catostomidae
Salmonidae
Cichlidae
Percidae
Centrarchidae
Fundulidae
Esocidae
Gobiidae
Dionda argentosa
Gambusia rhizophorae
Rivulus marmoratus
Cottus klamathensis
Noturus insignis
Cottus marginatus
Fundulus confluentus
Etheostoma sellare
Cichlasoma urophthalmus
Ctenogobius claytonii
Poecilia sphenops
Moxostoma austrinum
Campostoma ornatum
Astyanax mexicanus
Cichlasoma citrinellum
Lampetra minima
Notropis volucellus
Notropis spectrunculus
Menidia audens
Hybognathus nuchalis
Etheostoma tetrazonum
Moapa coriacea
Percina kathae
Catostomus microps
Hiodon tergisus
Cottus bairdii
Ichthyomyzon greeleyi
Noturus eleutherus
Agonostomus monticola
Phoxinus areas
Lythrurus lirus
Catostomus platyrhynchus
Prosopium Williamson!
Oreochromis mossambicus
Etheostoma asprigene
Acantharchus pomotis
Fundulus heteroclitus
Esox masquinongy
Gobiosoma base
manantial roundnose minnow
mangrove gambusia
mangrove rivulus
marbled sculpin
margined madtom
margined sculpin
marsh killifish
maryland darter
mayan cichlid
mexican goby
mexican molly
mexican redhorse
mexican stoneroller
mexican tetra
midas cichlid
miller lake lamprey
mimic shiner
mirror shiner
mississippi silverside
mississippi silvery minnow
missouri saddled darter
moapa dace
mobile logperch
modoc sucker
mooneye
mottled sculpin
mountain brook lamprey
mountain madtom
mountain mullet
mountain redbelly dace
mountain shiner
mountain sucker
mountain whitefish
mozambique tilapia
mud darter
mud sunfish
mummichog
muskellunge
naked goby
167
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 168 of 224
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
489
490
491
492
493
494
495
496
497
498
499
500
501
502
503
504
505
506
Perciformes
Siluriformes
Cypriniformes
Perciformes
Perciformes
Gasterosteiformes
Petromyzontiformes
Percopsiformes
Cypriniformes
Siluriformes
Esociformes
Cypriniformes
Cyprinodontiformes
Cypriniformes
Cyprinodontiformes
Cypriniformes
Cypriniformes
Cypriniformes
Petromyzontiformes
Perciformes
Perciformes
Perciformes
Esociformes
Gasterosteiformes
Perciformes
Perciformes
Siluriformes
Cypriniformes
Perciformes
Perciformes
Cypriniformes
Perciformes
Cypriniformes
Cypriniformes
Siluriformes
Perciformes
Siluriformes
Cypriniformes
Cyprinodontiformes
Percidae
Ictaluridae
Cyprinidae
Percidae
Cichlidae
Gasterosteidae
Petromyzontidae
Amblyopsidae
Catostomidae
Ictaluridae
Esocidae
Cyprinidae
Fundulidae
Cyprinidae
Fundulidae
Catostomidae
Cyprinidae
Cyprinidae
Petromyzontidae
Percidae
Elassomatidae
Percidae
Umbridae
Syngnathidae
Percidae
Percidae
Ictaluridae
Cyprinidae
Sciaenidae
Centrarchidae
Cyprinidae
Percidae
Cyprinidae
Cobitidae
Loricariidae
Cichlidae
Ictaluridae
Cyprinidae
Cyprinodontidae
Ammocrypta beanii
Noturus placid us
Notropis scabriceps
Etheostoma nianguae
Oreochromis niloticus
Pungitius pungitius
Ichthyomyzon fossor
Amblyopsis spelaea
Hypentelium nigricans
Noturus stigmosus
Esox lucius
Ptychocheilus oregonensis
Fundulus kansae
Phoxinus eos
Fundulus catenatus
Moxostoma collapsum
Dionda serena
Cyprinella callisema
Ichthyomyzon bdellium
Etheostoma okaloosae
Elassoma okefenokee
Percina squamata
Novumbra hubbsi
Microphis brachyurus
Etheostoma radiosum
Etheostoma bellum
Noturus gilberti
Notropis ammophilus
Cynoscion xanthulus
Lepomis humilis
Pteronotropis merlini
Etheostoma spectabile
Oregonichthys crameri
Misgurnus anguillicaudatus
Pterygoplichthys multiradiatus
Astronotus ocellatus
Noturus lachneri
Lythrurus snelsoni
Cyprinodon radiosus
naked sand darter
neosho madtom
new river shiner
niangua darter
nile tilapia
ninespine stickleback
northern brook lamprey
northern cavefish
northern hog sucker
northern madtom
northern pike
northern pikeminnow
northern plains killifish
northern redbelly dace
northern studfish
notchlip redhorse
nueces roundnose minnow
ocmulgee shiner
ohio lamprey
okaloosa darter
okefenokee pygmy sunfish
olive darter
Olympic mudminnow
opossum pipefish
orangebelly darter
orangefin darter
orangefin madtom
orangefin shiner
orangemouth corvina
orangespotted sunfish
orangetail shiner
orangethroat darter
Oregon chub
oriental weatherfish
Orinoco sailfin catfish
oscar
ouachita madtom
ouachita shiner
owens pupfish
168
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 169 of 224
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
507
508
509
510
511
512
513
514
515
516
517
518
519
520
521
522
523
524
525
526
527
528
529
530
531
532
533
534
535
536
537
538
539
540
541
542
543
544
545
Cypriniformes
Perciformes
Percopsiformes
Cypriniformes
Perciformes
Siluriformes
Cypriniformes
Scorpaeniformes
Cypriniformes
Petromyzontiformes
Scorpaeniformes
Acipenseriformes
Cypriniformes
Cyprinodontiformes
Scorpaeniformes
Perciformes
Cypriniformes
Cypriniformes
Acipenseriformes
Cypriniformes
Cypriniformes
Cypriniformes
Perciformes
Cyprinodontiformes
Cyprinodontiformes
Cypriniformes
Cypriniformes
Cypriniformes
Perciformes
Perciformes
Cyprinodontiformes
Perciformes
Cypriniformes
Perciformes
Salmoniformes
Percopsiformes
Scorpaeniformes
Petromyzontiformes
Cyprinodontiformes
Catostomidae
Centrarchidae
Amblyopsidae
Cyprinidae
Percidae
Ictaluridae
Cyprinidae
Cottidae
Cyprinidae
Petromyzontidae
Cottidae
Polyodontidae
Cyprinidae
Goodeidae
Cottidae
Percidae
Cyprinidae
Cyprinidae
Acipenseridae
Catostomidae
Cyprinidae
Cyprinidae
Percidae
Poeciliidae
Cyprinodontidae
Cyprinidae
Cyprinidae
Cyprinidae
Percidae
Haemulidae
Poeciliidae
Percidae
Cyprinidae
Sparidae
Salmonidae
Aphredoderidae
Cottidae
Petromyzontidae
Fundulidae
Catostomus fumeiventris
Ambloplites constellatus
Amblyopsis rosae
Erimystax harryi
Percina fulvitaenia
Noturus a/footer
Notropis nubilus
Cottus hypselurus
Notropis ozarcanus
Lampetra tridentata
Leptocottus armatus
Polyodon spathula
Lepidomeda altivelis
Empetrichthys latos
Cottus beldingii
Etheostoma pallididorsum
Notropis albizonatus
Hybopsis amnis
Scaphirhynchus albus
Moxostoma pisolabrum
Mylocheilus caurinus
Margariscus margarita
Percina aurora
Gambusia nobilis
Cyprinodon pecosensis
Macrhybopsis tetranema
Notropis perpallidus
Notropis area
Percina crassa
Orthopristis chrysoptera
Belonesox belizanus
Etheostoma mariae
Lythrurus matutinus
Lagodon rhomboides
Oncorhynchus gorbuscha
Aphredoderus sayanus
Cottus pitensis
Lampetra lethophaga
Fundulus zebrinus
owens sucker
ozark bass
ozarkcavefish
ozark chub
ozark logperch
ozark madtom
ozark minnow
ozark sculpin
ozark shiner
pacific lamprey
pacific staghorn sculpin
paddlefish
pahranagat spinedace
pahrump poolfish
paiute sculpin
paleback darter
palezone shiner
pallid shiner
pallid sturgeon
pealip redhorse
peamouth
pearl dace
pearl darter
pecos gambusia
pecos pupfish
peppered chub
peppered shiner
phantom shiner
piedmont darter
pigfish
pike killifish
pinewoods darter
pinewoods shiner
pinfish
pink salmon
pirate perch
pit sculpin
pit-klamath brook lamprey
plains killifish
169
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 170 of 224
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
546
547
548
549
550
551
552
553
554
555
556
557
558
559
560
561
562
563
564
565
566
567
568
569
570
571
572
573
574
575
576
577
578
579
580
581
582
583
584
Cypriniformes
Cyprinodontiformes
Cypriniformes
Salmoniformes
Cypriniformes
Cyprinodontiformes
Scorpaeniformes
Cypriniformes
Cypriniformes
Scorpaeniformes
Cypriniformes
Cypriniformes
Cypriniformes
Perciformes
Cyprinodontiformes
Siluriformes
Scorpaeniformes
Salmoniformes
Cypriniformes
Cyprinodontiformes
Perciformes
Cypriniformes
Salmoniformes
Salmoniformes
Cyprinodontiformes
Cypriniformes
Perciformes
Cyprinodontiformes
Cypriniformes
Cypriniformes
Perciformes
Perciformes
Perciformes
Perciformes
Perciformes
Cypriniformes
Cyprinodontiformes
Perciformes
Esociformes
Cyprinidae
Fundulidae
Cyprinidae
Osmeridae
Cyprinidae
Poeciliidae
Cottidae
Cyprinidae
Cyprinidae
Cottidae
Cyprinidae
Cyprinidae
Cyprinidae
Centrarchidae
Fundulidae
Ictaluridae
Cottidae
Salmonidae
Catostomidae
Goodeidae
Percidae
Cyprinidae
Osmeridae
Salmonidae
Fundulidae
Catostomidae
Sciaenidae
Cyprinodontidae
Cyprinidae
Cyprinidae
Percidae
Cichlidae
Centrarchidae
Centrarchidae
Centrarchidae
Cyprinidae
Fundulidae
Percidae
Esocidae
Hybognathus placitus
Fundulus sciadicus
Cyprinella lepida
Hypomesus olidus
Notropis ariommus
Poeciliopsis gracilis
Cottus girardi
Macrhybopsis australis
Lythrurus bellus
Cottus asper
Cyprinella proserpina
Opsopoeodus emiliae
Notropis anogenus
Lepomis gibbosus
Leptolucania ommata
Noturus stanauli
Cottus paulus
Prosopium coulterii
Carpiodes cyprinus
Crenichthys nevadae
Etheostoma caeruleum
Notropis chrosomus
Osmerus mordax
Oncorhynchus mykiss
Lucania parva
Xyrauchen texanus
Sciaenops ocellatus
Cyprinodon rubrofluviatilis
Notropis bairdi
Cyprinella lutrensis
Etheostoma luteovinctum
Tilapia zillii
Lepomis auritus
Lepomis microlophus
Micropterus coosae
Notropis harperi
Fundulus rubrifrons
Etheostoma whipplei
Esox americanus
plains minnow
plains topminnow
plateau shiner
pond smelt
popeye shiner
porthole livebearer
potomac sculpin
prairie chub
pretty shiner
prickly sculpin
proserpine shiner
pugnose minnow
pugnose shiner
pumpkinseed
pygmy killifish
pygmy madtom
pygmy sculpin
pygmy whitefish
quillback
railroad valley springfish
rainbow darter
rainbow shiner
rainbow smelt
rainbow trout
rainwater killifish
razorback sucker
red drum
red river pupfish
red river shiner
red shiner
redband darter
redbelly tilapia
redbreast sunfish
redear sunfish
redeye bass
redeye chub
redface topminnow
redfin darter
redfin pickerel
170
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 171 of 224
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
585
586
587
588
589
590
591
592
593
594
595
596
597
598
599
600
601
602
603
604
605
606
607
608
609
610
611
612
613
614
615
616
617
618
619
620
621
622
623
Cypriniformes
Perciformes
Cypriniformes
Cypriniformes
Cypriniformes
Cypriniformes
Perciformes
Perciformes
Perciformes
Cypriniformes
Cypriniformes
Perciformes
Scorpaeniformes
Cypriniformes
Cypriniformes
Scorpaeniformes
Cypriniformes
Perciformes
Perciformes
Cypriniformes
Cypriniformes
Cypriniformes
Cypriniformes
Cypriniformes
Perciformes
Perciformes
Petromyzontiformes
Cypriniformes
Cypriniformes
Perciformes
Perciformes
Perciformes
Cypriniformes
Perciformes
Cypriniformes
Perciformes
Perciformes
Cypriniformes
Cypriniformes
Cyprinidae
Percidae
Cyprinidae
Cyprinidae
Cyprinidae
Cyprinidae
Percidae
Centrarchidae
Cichlidae
Cyprinidae
Cyprinidae
Percidae
Cottidae
Cyprinidae
Cyprinidae
Cottidae
Cyprinidae
Cichlidae
Percidae
Cyprinidae
Cyprinidae
Catostomidae
Catostomidae
Cyprinidae
Percidae
Gobiidae
Petromyzontidae
Catostomidae
Cyprinidae
Percidae
Centrarchidae
Percidae
Catostomidae
Percidae
Catostomidae
Centrarchidae
Percidae
Cyprinidae
Cyprinidae
Lythrurus umbratilis
Etheostoma rufilineatum
Notropis chiliticus
Clinostomus elongatus
Richardsonius balteatus
Nocomis asper
Etheostoma artesiae
Lepomis miniatus
Geophagus surinamensis
Nocomis effusus
Relictus solitarius
Etheostoma chienense
Cottus perplexus
Lythrurus fumeus
Phenacobius catostomus
Cottus gulosus
Gila pandora
Cichlasoma cyanoguttatum
Etheostoma grahami
No tropis Jem ezan us
Hybognathus amarus
Catostomus plebeius
Carpiodes carpio
Nocomis micropogon
Percina shumardi
Awaous banana
Lampetra ayresii
Moxostoma carinatum
Notropis blennius
Etheostoma podostemone
Ambloplites cavifrons
Percina roanoka
Hypentelium roanokense
Percina rex
Moxostoma robustum
Ambloplites rupestris
Etheostoma rupestre
Notropis suttkusi
Lythrurus ardens
redfin shiner
redline darter
redlip shiner
redside dace
redside shiner
redspot chub
redspot darter
redspotted sunfish
redstriped eartheater
redtail chub
relict dace
relict darter
reticulate sculpin
ribbon shiner
riffle minnow
riffle sculpin
rio grande chub
rio grande cichlid
rio grande darter
rio grande shiner
rio grande silvery minnow
rio grande sucker
river carpsucker
river chub
river darter
river goby
river lamprey
river redhorse
river shiner
riverweed darter
roanoke bass
roanoke darter
roanoke hog sucker
roanoke logperch
robust redhorse
rock bass
rock darter
rocky shiner
rosefin shiner
171
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 172 of 224
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
624
625
626
627
628
629
630
631
632
633
634
635
636
637
638
639
640
641
642
643
644
645
646
647
648
649
650
651
652
653
654
655
656
657
658
659
660
661
662
Cypriniformes
Cypriniformes
Cypriniformes
Scorpaeniformes
Cypriniformes
Atheriniformes
Cypriniformes
Perciformes
Salmoniformes
Cypriniformes
Cypriniformes
Cypriniformes
Perciformes
Perciformes
Cyprinodontiformes
Cypriniformes
Cypriniformes
Cypriniformes
Perciformes
Cypriniformes
Cypriniformes
Perciformes
Perciformes
Cypriniformes
Cyprinodontiformes
Cypriniformes
Cyprinodontiformes
Cyprinodontiformes
Cyprinodontiformes
Percopsiformes
Cypriniformes
Cypriniformes
Cypriniformes
Cypriniformes
Cyprinodontiformes
Cypriniformes
Cypriniformes
Perciformes
Perciformes
Cyprinidae
Cyprinidae
Cyprinidae
Cottidae
Cyprinidae
Atherinopsidae
Cyprinidae
Gobiidae
Salmonidae
Cyprinidae
Cyprinidae
Cyprinidae
Percidae
Percidae
Fundulidae
Catostomidae
Cyprinidae
Cyprinidae
Centrarchidae
Cyprinidae
Catostomidae
Percidae
Percidae
Cyprinidae
Poeciliidae
Cyprinidae
Cyprinodontidae
Fundulidae
Poeciliidae
Percopsidae
Cyprinidae
Cyprinidae
Cyprinidae
Catostomidae
Cyprinodontidae
Cyprinidae
Cyprinidae
Percidae
Percidae
Hybopsis rubrifrons
Notropis rubellus
Clinostomus funduloides
Cottus asperrimus
Notropis baileyi
Membras martinica
Notropis semperasper
Neogobius melanostomus
Prosopium cylindraceum
Dionda episcopa
Gila robusta
Scardinius erythrophthalmus
Gymnocephalus cernuus
Etheostoma phytophilum
Fundulus escambiae
Thoburnia hamiltoni
Notropis sabinae
Orthodon microlepidotus
Archoplites interruptus
Ptychocheilus grandis
Catostomus occidentalis
Percina vigil
Etheostoma flavum
Notropis rubricroceus
Poecilia latipinna
Pteronotropis hypselopterus
Cyprinodon salinus
Fundulus jenkinsi
Gambusia georgei
Percopsis transmontana
Notropis stramineus
Notropis scepticus
Semotilus lumbee
Catostomus santaanae
Cyprinodon arcuatus
Cyprinella zanema
Cyprinella analostana
Sander canadensis
Etheostoma fricksium
rosyface chub
rosyface shiner
rosyside dace
rough sculpin
rough shiner
rough silverside
roughhead shiner
round goby
round whitefish
roundnose minnow
roundtail chub
rudd
ruffe
rush darter
russetfin topminnow
rustyside sucker
sabine shiner
Sacramento blackfish
Sacramento perch
Sacramento pikeminnow
Sacramento sucker
saddleback darter
saffron darter
saffron shiner
sailfin molly
sailfin shiner
salt creek pupfish
saltmarsh topminnow
san marcos gambusia
sand roller
sand shiner
sandbar shiner
sandhills chub
santa ana sucker
santa cruz pupfish
santee chub
satinfin shiner
sauger
savannah darter
172
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 173 of 224
LINE NO.
663
664
665
666
667
668
669
670
671
672
673
674
675
676
677
678
679
680
681
682
683
684
685
686
687
688
689
690
691
692
693
694
695
696
697
698
699
700
701
ORDER
Perciformes
Clupeiformes
Perciformes
Cypriniformes
Siluriformes
Petromyzontiformes
Perciformes
Cyprinodontiformes
Perciformes
Cypriniformes
Perciformes
Perciformes
Scorpaeniformes
Cypriniformes
Perciformes
Perciformes
Cyprinodontiformes
Perciformes
Perciformes
Perciformes
Perciformes
Cypriniformes
Perciformes
Cyprinodontiformes
Cypriniformes
Scorpaeniformes
Salmoniformes
Salmoniformes
Lepisosteiformes
Acipenseriformes
Cypriniformes
Scorpaeniformes
Acipenseriformes
Cypriniformes
Cypriniformes
Cypriniformes
Petromyzontiformes
Perciformes
Cypriniformes
FAMILY
Percidae
Clupeidae
Percidae
Cyprinidae
Ictaluridae
Petromyzontidae
Percidae
Fundulidae
Centrarchidae
Catostomidae
Percidae
Percidae
Cottidae
Cyprinidae
Percidae
Sparidae
Cyprinodontidae
Percidae
Gobiidae
Embiotocidae
Centrarchidae
Cyprinidae
Gobiidae
Poeciliidae
Catostomidae
Cottidae
Salmonidae
Salmonidae
Lepisosteidae
Acipenseridae
Catostomidae
Cottidae
Acipenseridae
Cyprinidae
Cyprinidae
Cyprinidae
Petromyzontidae
Sciaenidae
Catostomidae
SCIENTIFIC NAME
Etheostoma serrifer
Harengula jaguana
Ammocrypta vivax
Lythrurus fasciolaris
Noturus trautmani
Petromyzon marinus
Etheostoma thalassinum
Fundulus seminolis
Ambloplites ariommus
Erimyzon ten u is
Etheostoma acuticeps
Percina oxyrhynchus
Clinocottus acuticeps
Notropis oxyrhynchus
Etheostoma tecumsehi
Archosargus probatocephalus
Cyprinodon variegatus
Percina peltata
Tridentiger bifasciatus
Cymatogaster aggregata
Micropterus cataractae
Macrhybopsis hyostoma
Tridentiger barbatus
Poecilia mexicana
Moxostoma macrolepidotum
Cottus confusus
Coregonus zenithicus
Coregonus reighardi
Lepisosteus platostomus
Acipenser brevirostrum
Chasmistes brevirostris
Cottus greenei
Scaphirhynchus platorynchus
Macrhybopsis meeki
Hypophthalmichthys molitrix
Macrhybopsis storeriana
Ichthyomyzon unicuspis
Bairdiella chrysoura
Moxostoma anisurum
COMMON NAME
sawcheek darter
scaled sardine
scaly sand darter
scarlet shiner
scioto madtom
sea lamprey
seagreen darter
seminole killifish
shadow bass
sharpfin chubsucker
sharphead darter
sharpnose darter
sharpnose sculpin
sharpnose shiner
shawnee darter
sheepshead
sheepshead minnow
shield darter
shimofuri goby
shiner perch
shoal bass
shoal chub
shokihaze goby
shortfin molly
shorthead redhorse
shorthead sculpin
shortjaw Cisco
shortnose Cisco
shortnose gar
shortnose sturgeon
shortnose sucker
shoshone sculpin
shovelnose sturgeon
sicklefin chub
silver carp
silver chub
silver lamprey
silver perch
silver redhorse
173
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 174 of 224
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
702
703
704
705
706
707
708
709
710
711
712
713
714
715
716
717
718
719
720
721
722
723
724
725
726
727
728
729
730
731
732
733
734
735
736
737
738
739
740
Cypriniformes
Cypriniformes
Cypriniformes
Cypriniformes
Cypriniformes
Clupeiformes
Cypriniformes
Perciformes
Perciformes
Perciformes
Cypriniformes
Siluriformes
Scorpaeniformes
Perciformes
Cypriniformes
Scorpaeniformes
Perciformes
Cypriniformes
Perciformes
Cypriniformes
Cypriniformes
Perciformes
Perciformes
Perciformes
Pristiformes
Siluriformes
Siluriformes
Perciformes
Cypriniformes
Perciformes
Salmoniformes
Cypriniformes
Cypriniformes
Cyprinodontiformes
Perciformes
Cypriniformes
Petromyzontiformes
Percopsiformes
Pleuronectiformes
Cyprinidae
Cyprinidae
Cyprinidae
Cyprinidae
Cyprinidae
Clupeidae
Cyprinidae
Percidae
Percidae
Gobiidae
Cyprinidae
Ictaluridae
Cottidae
Percidae
Cyprinidae
Cottidae
Percidae
Cyprinidae
Centrarchidae
Catostomidae
Catostomidae
Percidae
Centropomidae
Eleotridae
Pristidae
Ictaluridae
Ictaluridae
Percidae
Catostomidae
Percidae
Salmonidae
Cyprinidae
Catostomidae
Cyprinodontidae
Percidae
Catostomidae
Petromyzontidae
Amblyopsidae
Paralichthyidae
Notropis photogenis
Notropis shumardi
Notropis buccatus
Notropis Candidas
Notropis stilbius
Alosa chrysochloris
Notropis uranoscopus
Etheostoma smith!
Etheostoma boschungi
Ctenogobius pseudofasciatus
Erimystax cahni
Noturus exilis
Cottus tenuis
Percina phoxocephala
Pimephales tenellus
Cottus cognatus
Etheostoma gracile
Notropis buccula
Micropterus dolomieu
Ictiobus bubalus
Moxostoma breviceps
Etheostoma microlepidum
Centropomus parallel us
Eleotris perniger
Pristis pectinata
Noturus baileyi
Ameiurus brunneus
Percina tanasi
Chasmistes muriei
Etheostoma simoterum
Oncorhynchus nerka
Gila ditaenia
Catostomus insignis
Cyprinodon eremus
Etheostoma olivaceum
Cycleptus meridionalis
Ichthyomyzon gagei
Typhlichthys subterraneus
Paralichthys lethostigma
silver shiner
silverband shiner
silverjaw minnow
silverside shiner
silverstripe shiner
skipjack herring
skygazer shiner
slabrock darter
slackwater darter
slashcheek goby
slender chub
slender madtom
slender sculpin
slenderhead darter
slim minnow
slimy sculpin
slough darter
smalleye shiner
smallmouth bass
smallmouth buffalo
smallmouth redhorse
smallscale darter
smallscale fat snook
smallscaled spinycheek
smalltooth sawfish
smoky madtom
snail bullhead
snail darter
snake river sucker
snubnose darter
sockeye salmon
sonora chub
sonora sucker
sonoyta pupfish
sooty darter
southeastern blue sucker
southern brook lamprey
southern cavefish
southern flounder
174
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 175 of 224
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
741
742
743
744
745
746
747
748
749
750
751
752
753
754
755
756
757
758
759
760
761
762
763
764
765
766
767
768
769
770
771
772
773
774
775
776
777
778
779
Perciformes
Cyprinodontiformes
Cypriniformes
Siluriformes
Perciformes
Siluriformes
Cyprinodontiformes
Cypriniformes
Cypriniformes
Perciformes
Cyprinodontiformes
Siluriformes
Cypriniformes
Perciformes
Cypriniformes
Scorpaeniformes
Perciformes
Cypriniformes
Cyprinodontiformes
Cypriniformes
Perciformes
Cypriniformes
Perciformes
Siluriformes
Perciformes
Lepisosteiformes
Perciformes
Perciformes
Cypriniformes
Perciformes
Perciformes
Percopsiformes
Perciformes
Perciformes
Cypriniformes
Cyprinodontiformes
Pleuronectiformes
Cypriniformes
Perciformes
Percidae
Poeciliidae
Cyprinidae
Loricariidae
Percidae
Doradidae
Fundulidae
Cyprinidae
Cyprinidae
Percidae
Fundulidae
Ictaluridae
Cyprinidae
Percidae
Cyprinidae
Cottidae
Sciaenidae
Cyprinidae
Fundulidae
Cyprinidae
Percidae
Cyprinidae
Centrarchidae
Ictaluridae
Percidae
Lepisosteidae
Sciaenidae
Eleotridae
Catostomidae
Centrarchidae
Cichlidae
Amblyopsidae
Elassomatidae
Percidae
Cyprinidae
Fundulidae
Pleuronectidae
Cyprinidae
Percidae
Percina austroperca
Xiphophorus maculatus
Phoxinus erythrogaster
Pterygoplichthys anisitsi
Ammocrypta meridiana
Platydoras armatulus
Fundulus stellifer
Macrhybopsis aestivalis
Rhinichthys osculus
Etheostoma stigmaeum
Fundulus rathbuni
Noturus leptacanthus
Meda fulgida
Etheostoma barrenense
Pogonichthys macrolepidotus
Cottus ricei
Leiostomus xanthurus
Erimonax monachus
Fundulus luciae
Cyprinella spiloptera
Etheostoma squamiceps
Notropis hudsonius
Micropterus punctulatus
Ameiurus serracanthus
Etheostoma maculatum
Lepisosteus oculatus
Cynoscion nebulosus
Eleotris picta
Minytrema melanops
Lepomis punctatus
Tilapia marine
Forbesichthys agassizii
Elassoma alabamae
Percina uranidea
Phenacobius uranops
Fundulus dispar
Platichthys stellatus
Cyprinella whipplei
Etheostoma punctulatum
southern logperch
southern platyfish
southern redbelly dace
southern sailfin catfish
southern sand darter
southern striped raphael
southern studfish
speckled chub
speckled dace
speckled darter
speckled killifish
speckled madtom
spikedace
splendid darter
splittail
spoonhead sculpin
spot
spotfin chub
spotfin killifish
spotfin shiner
spottail darter
spottail shiner
spotted bass
spotted bullhead
spotted darter
spotted gar
spotted seatrout
spotted sleeper
spotted sucker
spotted sunfish
spotted tilapia
spring cavefish
spring pygmy sunfish
stargazing darter
stargazing minnow
starhead topminnow
starry flounder
steelcolor shiner
stippled darter
175
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 176 of 224
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
780
781
782
783
784
785
786
787
788
789
790
791
792
793
794
795
796
797
798
799
800
801
802
803
804
805
806
807
808
809
810
811
812
813
814
815
816
817
818
Cyprinodontiformes
Perciformes
Siluriformes
Perciformes
Cypriniformes
Perciformes
Perciformes
Perciformes
Perciformes
Cypriniformes
Perciformes
Mugiliformes
Cypriniformes
Perciformes
Cypriniformes
Siluriformes
Cypriniformes
Salmoniformes
Perciformes
Cypriniformes
Perciformes
Percopsiformes
Perciformes
Perciformes
Siluriformes
Cypriniformes
Cypriniformes
Perciformes
Cypriniformes
Cypriniformes
Perciformes
Elopiformes
Perciformes
Perciformes
Cypriniformes
Cypriniformes
Cypriniformes
Cypriniformes
Perciformes
Fundulidae
Percidae
Ictaluridae
Percidae
Cyprinidae
Percidae
Percidae
Moronidae
Percidae
Catostomidae
Gerreidae
Mugilidae
Cyprinidae
Percidae
Cyprinidae
Loricariidae
Cyprinidae
Osmeridae
Centrarchidae
Cyprinidae
Percidae
Amblyopsidae
Percidae
Centropomidae
Ictaluridae
Catostomidae
Cyprinidae
Percidae
Cyprinidae
Cyprinidae
Percidae
Megalopidae
Centropomidae
Percidae
Cyprinidae
Cyprinidae
Cyprinidae
Cyprinidae
Percidae
Fundulus bifax
Etheostoma derivativum
Noturus flavus
Etheostoma /rag/
Erimystax dissimilis
Etheostoma striatulum
Percina notogramma
Morone saxatilis
Etheostoma virgatum
Moxostoma rupiscartes
Eugerres plumieri
Mug/7 cephalus
Luxilus chrysocephalus
Etheostoma kennicotti
Macrhybopsis gelida
Hypostomus plecostomus
Phenacobius mirabilis
Hypomesus pretiosus
Micropterus not/us
Notropis procne
Etheostoma fusiforme
Chologaster cornuta
Etheostoma swannanoa
Centropomus ensiferus
Noturus gyrinus
Catostomus tahoensis
Notropis maculatus
Etheostoma tallapoosae
Cyprinella gibbsi
Notropis braytoni
Percina aurantiaca
Megalops atlanticus
Centropomus pectinatus
Etheostoma barbouri
Notropis telescopus
Tinea tinea
Phoxinus tennesseensis
Notropis leuciodus
Etheostoma olmstedi
stippled studfish
stone darter
stonecat
strawberry darter
streamline chub
striated darter
stripeback darter
striped bass
striped darter
striped jumprock
striped mojarra
striped mullet
striped shiner
stripetail darter
sturgeon chub
suckermouth catfish
suckermouth minnow
surf smelt
suwannee bass
swallowtail shiner
swamp darter
swampfish
swannanoa darter
swordspine snook
tadpole madtom
tahoe sucker
taillight shiner
tallapoosa darter
tallapoosa shiner
tamaulipas shiner
tangerine darter
tarpon
tarpon snook
teardrop darter
telescope shiner
tench
tennessee dace
tennessee shiner
tessellated darter
176
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 177 of 224
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
819
820
821
822
823
824
825
826
827
828
829
830
831
832
833
834
835
836
837
838
839
840
841
842
843
844
845
846
847
848
849
850
851
852
853
854
855
856
857
Perciformes
Cypriniformes
Cyprinodontiformes
Cypriniformes
Cypriniformes
Clupeiformes
Perciformes
Perciformes
Perciformes
Perciformes
Cypriniformes
Siluriformes
Cypriniformes
Scorpaeniformes
Cypriniformes
Cypriniformes
Perciformes
Percopsiformes
Perciformes
Perciformes
Cypriniformes
Perciformes
Perciformes
Perciformes
Perciformes
Cypriniformes
Cypriniformes
Cypriniformes
Cypriniformes
Cypriniformes
Scorpaeniformes
Cypriniformes
Cyprinodontiformes
Perciformes
Siluriformes
Perciformes
Perciformes
Cypriniformes
Cypriniformes
Percidae
Cyprinidae
Poeciliidae
Cyprinidae
Cyprinidae
Clupeidae
Gobiidae
Gerreidae
Percidae
Percidae
Cyprinidae
Ictaluridae
Cyprinidae
Cottidae
Catostomidae
Cyprinidae
Percidae
Percopsidae
Gobiidae
Percidae
Cyprinidae
Embiotocidae
Percidae
Percidae
Percidae
Cyprinidae
Cyprinidae
Cyprinidae
Cyprinidae
Cyprinidae
Cottidae
Catostomidae
Poeciliidae
Percidae
Loricariidae
Percidae
Gobiidae
Cyprinidae
Cyprinidae
Percina carbonaria
Notropis a ma bills
Gambusia speciosa
Cyprinella labrosa
Gila crassicauda
Dorosoma petenense
Eucyclogobius newberryi
Eucinostomus harengulus
Etheostoma tippecanoe
Etheostoma lachneri
Exoglossum laurae
Trogloglanis pattersoni
Notropis topeka
Cottus rhotheus
Thoburnia rhothoeca
Cyprinella trichroistia
Etheostoma trisella
Percopsis omiscomaycus
Proterorhinus marmoratus
Etheostoma gutselli
Gila bicolor
Hysterocarpus traskii
Etheostoma inscriptum
Etheostoma tuscumbia
Etheostoma douglasi
Rhinichthys umatilla
Oregonichthys kalawatseti
Rhinichthys evermanni
Ptychocheilus umpquae
Gila atraria
Cottus echinatus
Catostomus ardens
Xiphophorus variatus
Etheostoma variatum
Pterygoplichthys disjunctivus
Etheostoma chermocki
Gobioides broussonetii
Gila seminuda
Lepidomeda mollispinis
texas logperch
texas shiner
tex-mexgambusia
thicklip chub
thicktail chub
threadfin shad
tidewater goby
tidewater mojarra
tippecanoe darter
tombigbee darter
tonguetied minnow
toothless blindcat
topeka shiner
torrent sculpin
torrent sucker
tricolor shiner
trispot darter
trout-perch
tubenose goby
tuckasegee darter
tui chub
tule perch
turquoise darter
tuscumbia darter
tuskaloosa darter
umatilla dace
umpqua chub
umpqua dace
umpqua pikeminnow
Utah chub
Utah lake sculpin
Utah sucker
variable platyfish
variegate darter
vermiculated sailfin catfish
vermilion darter
violet goby
virgin chub
virgin spinedace
177
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 178 of 224
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
858
859
860
861
862
863
864
865
866
867
868
869
870
871
872
873
874
875
876
877
878
879
880
881
882
883
884
885
886
887
888
889
890
891
892
893
894
895
896
Cypriniformes
Perciformes
Cyprinodontiformes
Atheriniformes
Salmoniformes
Siluriformes
Perciformes
Perciformes
Perciformes
Cypriniformes
Cypriniformes
Perciformes
Cypriniformes
Perciformes
Cypriniformes
Cypriniformes
Cypriniformes
Petromyzontiformes
Cyprinodontiformes
Perciformes
Cypriniformes
Cyprinodontiformes
Perciformes
Siluriformes
Perciformes
Mugiliformes
Perciformes
Cypriniformes
Cyprinodontiformes
Cyprinodontiformes
Cypriniformes
Acipenseriformes
Cypriniformes
Cypriniformes
Cyprinodontiformes
Cypriniformes
Cypriniformes
Siluriformes
Scorpaeniformes
Catostomidae
Percidae
Fundulidae
Atherinopsidae
Osmeridae
Clariidae
Percidae
Cichlidae
Centrarchidae
Catostomidae
Cyprinidae
Percidae
Cyprinidae
Percidae
Cyprinidae
Cyprinidae
Cyprinidae
Petromyzontidae
Poeciliidae
Percidae
Cyprinidae
Fundulidae
Moronidae
Ictaluridae
Centrarchidae
Mugilidae
Moronidae
Cyprinidae
Goodeidae
Cyprinodontidae
Cyprinidae
Acipenseridae
Catostomidae
Cyprinidae
Fundulidae
Cyprinidae
Cyprinidae
Ictaluridae
Cottidae
Moxostoma pappillosum
Etheostoma perlongum
Fundulus waccamensis
Menidia extensa
Hypomesus nipponensis
Clarias batrachus
Sander vitreus
Oreochromis urolepis
Lepomis gulosus
Catostomus warnerensis
Luxilus coccogenis
Etheostoma bellator
Lythrurus alegnotus
Etheostoma nuchale
Notropis greenei
Notropis texanus
Rhinichthys obtusus
Lampetra richardsoni
Gambusia affinis
Ammocrypta clara
Hybognathus argyritis
Fundulus blairae
Morone chrysops
Ameiurus catus
Pomoxis annularis
Mugil curema
Morone americana
Lepidomeda albivallis
Crenichthys baileyi
Cyprinodon tularosa
Luxilus albeolus
Acipenser transmontanus
Catostomus commersonii
Cyprinella nivea
Fundulus albolineatus
Notropis alborus
Cyprinella galactura
Satan eurystomus
Cottus leiopomus
v-lip redhorse
waccamaw darter
waccamaw killifish
waccamaw silverside
wakasagi
walking catfish
walleye
wami tilapia
warmouth
warner sucker
warpaint shiner
warrior darter
warrior shiner
watercress darter
wedgespot shiner
weed shiner
western blacknose dace
western brook lamprey
western mosquitofish
western sand darter
western silvery minnow
western starhead topminnow
white bass
white catfish
white crappie
white mullet
white perch
white river spinedace
white river springfish
white sands pupfish
white shiner
white sturgeon
white sucker
whitefin shiner
whiteline topminnow
whitemouth shiner
whitetail shiner
widemouth blindcat
wood river sculpin
178
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 179 of 224
LINE NO.
897
898
899
900
901
902
903
904
905
906
907
908
909
910
911
912
913
914
915
916
917
918
919
920
921
922
923
924
925
926
927
928
929
930
931
932
933
934
935
ORDER
Perciformes
Cypriniformes
Siluriformes
Cypriniformes
Cypriniformes
Perciformes
Cypriniformes
Perciformes
Siluriformes
Perciformes
Perciformes
Perciformes
Perciformes
Siluriformes
Cypriniformes
Perciformes
Perciformes
Cypriniformes
Salmoniformes
Atheriniformes
Cypriniformes
Cypriniformes
Perciformes
Perciformes
Salmoniformes
Cypriniformes
Anura
Anura
Scorpaeniformes
Salmoniformes
Scorpaeniformes
Salmoniformes
Salmoniformes
Anguilliformes
Cypriniformes
Cypriniformes
Salmoniformes
Perciformes
Perciformes
FAMILY
Percidae
Cyprinidae
Ictaluridae
Cyprinidae
Catostomidae
Percidae
Cyprinidae
Moronidae
Ictaluridae
Percidae
Cichlidae
Percidae
Gobiidae
Ictaluridae
Cyprinidae
Percidae
Percidae
Catostomidae
Salmonidae
Atherinopsidae
Cyprinidae
Cyprinidae
Pomatomidae
Percidae
Salmonidae
Catostomidae
Ranidae
Ranidae
Cottidae
Salmonidae
Cottidae
Salmonidae
Salmonidae
Congridae
Cyprinidae
Cyprinidae
Salmonidae
Percidae
Percidae
SCIENTIFIC NAME
Etheostoma vulneratum
Plagopterus argentissimus
Ictalurus price/
Gila purpurea
Catostomus bernardini
Etheostoma raneyi
Notropis rafinesquei
Morone mississippiensis
Ameiurus natalis
Perca flavescens
Cichlasoma salvini
Etheostoma moorei
Acanthogobiusflavimanus
Noturus flavipinnis
Notropis lutipinnis
Etheostoma juliae
Sander lucioperca
Moxostoma cf. poecilurum
Salmo salar
Menidia menidia
Hypophthalmichthys nobilis
Mylopharyngodon piceus
Pomatomus saltatrix
Etheostoma meadiae
Oncorhynchus clarkii Utah
Moxostoma cf. lachneri
Rana catesbeiana
Rana catesbeiana
Cottus chattahoochee
Oncorhynchus tshawytscha
Cottus cf. broadband sculpin
Oncorhynchus clarkii clarkii
Oncorhynchus clarkii pleuriticus
Conger oceanicus
Macrhybopsis cf. aestivalis
Semotilus X Luxilus atromaculatus x
chrysocephalus
Oncorhynchus clarkii x mykiss
Etheostoma planasaxatile
Etheostoma orientale
COMMON NAME
wounded darter
woundfin
yaqui catfish
yaqui chub
yaqui sucker
yazoo darter
yazoo shiner
yellow bass
yellow bullhead
yellow perch
yellowbelly cichlid
yellowcheek darter
yellowfin goby
yellowfin madtom
yellowfin shiner
yoke darter
zander
apalachicola redhorse
atlantic salmon juvenile
atlantic silverside
bighead carp
black carp
bluefish
bluespar darter
bonneville cutthroat trout
brassy jumprock
bullfrog
bullfrog tadpole
chattahoochee sculpin
chinook salmon (yoy)
clinch sculpin
coastal cutthroat trout
Colorado river cutthroat trout
conger eel
coosa chub
creek chub x striped shiner
cutbow
duck darter
eastrim darter
179
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 180 of 224
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
936
937
938
939
940
941
942
943
944
945
946
947
948
949
950
951
952
953
954
955
956
957
958
959
960
961
962
963
964
965
966
967
968
969
970
971
972
973
974
Perciformes
Esociformes
Clupeiformes
Scorpaeniformes
Salmoniformes
Cypriniformes
Perciformes
Perciformes
Cypriniformes
Cypriniformes
Cypriniformes
Cypriniformes
Caudata
Salmoniformes
Anura
Salmoniformes
Caudata
Siluriformes
Perciformes
Cypriniformes
Cypriniformes
Cypriniformes
Perciformes
Anguilliformes
Anura
Anura
Scorpaeniformes
Perciformes
Cypriniformes
Gasterosteiformes
Esociformes
Salmoniformes
Anura
Perciformes
Salmoniformes
Perciformes
Perciformes
Perciformes
Anura
Centropomidae
Esocidae
Clupeidae
Cottidae
Salmonidae
Catostomidae
Centrarchidae
Eleotridae
Catostomidae
Cyprinidae
Cyprinidae
Cyprinidae
Ambystomatidae
Salmonidae
Ranidae
Salmonidae
Salamandridae
Ictaluridae
Percidae
Cyprinidae
Cyprinidae
Catostomidae
Channidae
Ophichthidae
Ascaphidae
Ascaphidae
Cottidae
Percidae
Cyprinidae
Gasterosteidae
Esocidae
Salmonidae
Bufonidae
Percidae
Salmonidae
Moronidae
Gerreidae
Percidae
Pipidae
Centropomus parallel us
Esox americanus vermiculatus
Brevoortia patronus
Cottus kanawhae
Oncorhynchus clarkii henshawi
Pantosteus lahontan
Micropterus salmoides
Eleotris amblyopsis
Catostomus cf. latipinnis
Notropis amplamala
Pteronotropis stonei
Cyprinus carpio
Dicamptodon tenebrosus
Oncorhynchus mykiss
Rana aurora
Oncorhynchus mykiss gairdneri
Taricha granulosa
Noturus fasciatus
Sander canadensis x vitreus
Notropis cf. spectrunculus
Hypophthalmichthys molitrix
Moxostoma robustum
Channa argus
Myrophis punctatus
Ascaphus truei
Ascaphus truei
Cottus tallapoosae
Etheostoma tennesseense
Cyprinella cf. zanema
Gasterosteus aculeatus
Esox lucius x masquinongy
Salmo XSalvelinus trutta x fontinalis
Bufo boreas
Etheostoma occidentale
Oncorhynchus clarkii lewisi
Morone na
Gerres cinereus
Percina burtoni
Xenopus laevis
fat snook
grass pickerel
gulf menhaden
kanawha sculpin
lahontan cutthroat trout
lahontan sucker
largemouth bass (yoy)
largescaled spinycheek sleeper
little Colorado river sucker
longjaw minnow
lowland shiner
mirror carp
pacific giant salamander
rainbow trout (steelhead)
red-legged frog
redband rainbow trout
rough-skinned newt
saddled madtom
saugeye
sawfin shiner
silver carp
smallfin redhorse
snakehead
speckled worm eel
tailed frog
tailed frog (tadpole)
tallapoosa sculpin
tennessee darter
thinlip chub
threespine stickleback
tiger muskellunge
tiger trout
western toad
westrim darter
westslope cutthroat trout
wiper
yellowfin mojarra
blotchside darter
african clawed frog
180
-------
2013-2014 National Rivers & Streams Assessment
Version 1.3, May 2014
Laboratory Operations Manual
Page 181 of 224
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
975
976
977
978
979
980
981
982
983
984
985
986
987
988
989
990
991
992
993
994
995
996
997
998
999
1000
1001
1002
1003
Salmoniformes
Anura
Anura
Caudata
Caudata
Anura
Anura
Anura
Anura
Anura
Caudata
Anura
Salmoniformes
Anura
Caudata
Petromyzontiformes
Anura
Caudata
Anura
Anura
Anura
Anura
Salmoniformes
Anura
Cypriniformes
Anura
Anura
Anura
Anura
Salmonidae
Bufonidae
Hylidae
Ambystomatidae
Salamandridae
Hylidae
Bufonidae
Hylidae
Ranidae
Ranidae
Rhyacotritonidae
Ranidae
Salmonidae
Ranidae
Ambystomatidae
Petromyzontidae
Ranidae
Ambystomatidae
Ranidae
Ranidae
Hylidae
Ranidae
Salmonidae
Bufonidae
Cyprinidae
Ranidae
Ranidae
Bufonidae
Ranidae
Oncorhynchus apache x mykiss
Bufo microscaphus
Pseudacris maculata
Dicamptodon ensatus
Taricha torosa
Pseudacris cadaverina
Bufo hemiophrys
Hyla arenicolor
Rana cascadae
Rana luteiventris
Rhyacotriton kezeri
Rana boylii
Oncorhynchus aguabonita
Rana clamitans
Dicamptodon aterrimus
Lampetra similis
Lithobates pipiens
Ambystoma macrodactylum
Rana yavapaiensis
Rana muscosa
Pseudacris regilla
Lithobates blairi
Oncorhynchus mykiss x aguabonita
Bufo punctatus
Richardsonius X Rhinichthys balteatus x osculus
Rana pretiosa
Rana sylvatica
Bufo woodhousii
Lithobates chiricahuensis
apache x rainbow trout
arizona toad
boreal chorus frog
California giant salamander
California newt
California treefrog
Canadian toad
canyon treefrog
cascade frog
Columbia spotted frog
Columbia torrent salamander
foothill yellow-legged frog
golden trout
green frog
idaho giant salamander
klamath river lamprey
leopard frog
longtoed salamander
lowland leopard frog
mountain yellow-legged frog
pacific tree frog
plains leopard frog
rainbow x golden trout
red-spotted toad
redside shiner x speckled dace
spotted frog
wood frog
woodhouse's toad
chiricahua leopard frog
l/l
u
<
cc.
LJJ
CL
Q
X
Q
CL
CL
181
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 182 of 224
APPENDIX E: REPORTING TEMPLATES
(D
cc
O
CL
LJJ
CC
X
Q
CL
CL
182
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 183 of 224
Templates will be provided on the NARS Sharefile.
(D
cc
O
CL
LJJ
CC
X
Q
CL
CL
183
-------
2013-2014 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.3, May 2014 Page 184 of 224
APPENDIX F: EXAMPLE SOPS FOR MERCURY IN FISH TISSUE PLUG
ANALYSES
184
-------
4Seurofins
Frontier Global Sciences
Document Title:
Mercury in Water by Oxidation, Purge & Trap
__a_nd_CV-AFS (EPA Method 1631, RevE)
Eurofins Document Reference:
EFGS-SOP-137-R02
Eii irofi rts
Refererifee
EFGS-SOP-137-R02
Revision
6/17/2013
Status
Final
FGS-SOP-137.02
Level 3
SOP
L.O c 81 ,;Di6c Umi nl/jG
NA
:ReWeW6a:v://.vv;-.:;MK:;-:.:--v:fr
"' ...'!' :'' ':} ''.:. -.- ..-:,.,:",-.' ,-v .':'..
:aH4::f;:?;;o'{,:f:^;::;/VK:^Eiii;
:l^P^||;|S:;f|l:ff
Ryan Nelson
Dave Wundertich and Patrick Garcia-Strickland
-------
C-yeuroflns j
Frontier Etobaf Sciences
Document Title:
Mercury in Water by Oxidation, Purge & Trap
__and CV-AFS (EPA Method 1631, Rev E)
Eurofins Document Reference:
EFGS-SOP-137-R02
Table of Contents
1 Revision Log; , 4
2 Reference: , , . 4
3 Cross Reference: ,.,, , 5
4 Purpose: ,, , - 5
5 Scope: , . , . 5
6 Basic Principles: , , 5
7 Reference Modifications: Q
8 Definitions: , 0
9 Interferences: g
10 Safety Precautions, Pollution Prevention and Waste Handling:.. , g
11 Personnel Training and Qualifications:, 10
12 Sample Collection, Preservation, and Handling: , .10
13 Apparatus and Equipment: -)-!
14 Reagents and Standards: ., 1-j
15 Calibration: , , ..__ U
16 Procedure: ,..,,.. -jg
17 Calculations:... , _ 21
18 Statistical Information/Method Performance: ,.,,. 22
19 Quality Assurance/Quality Control: , 22
20 Corrective Action , 24
21 List of Attachments , ,.,.,_ 25
Table 1; QC Requirements for Total Mercury. , ,, 26
Appendix A: Example - Standard Operating Procedure Training Record ....27
-------
K^eurofins [
I Frontier Global Sciences
Document Title:
Mercury in Water by Oxidation, Purge & Trap
and CV-AFS (EPA Method 1631, Rev E)
Eurofins Document Reference:
EFGS-SOP-137-R02
Approvals:
Prepared by:
Date:
/I 3
Approved by:
B
~~**u<~~ P
L A^-i a /JL.J..JJ
Date:
Approved by:
Date:
-------
%'fieurofins (
Frontier Gioba' Sciences
Document Title:
Mercury in Water by Oxidation, Purge & Trap
and CV-AFS (EPA Method 1631, Rev E)
Eurofins Document Reference:
EFGS-SOP-137-R02
1 Revision Log:
. ^^flpfesfei^y^g
Cover
AH
13.1, 13.2
14.8
14.9
15.2-15.4
16.7
>?il ^ififiitiifep? ^ .::::y;^mm;-+ >;./,>;
Required change
Formatting requirement per LOM
SOP-LAB-201
Required
Required
Required
Required
Required
?;Oh(anifcS;2^;^^^^^^
Changed company name from Frontier Global Sciences to
Eurofins Frontier Global Sciences
Reformatted document to new corporate specifications.
Added hardware and software components
Updated mercury standard prep
Updated standard and reaqent documentation procedures
Updated calibration information
Added instrument maintenance and troubleshooting
2 Reference:
EPA Method 1631, Revision E: Mercury in Water by Oxidation, Purge and Trap, and
Cold Vapor Atomic Fluorescence Spectrometry, 2002.
Method 1669, "Method for Sampling Ambient Water for Determination of Metals at
EPA Ambient Criteria Levels," U.S. Environmental Protection Agency, Office of Water,
Office of Science and Technology, Engineering and Analysis Division (4303), 401 M
Street SW, Washington, DC 20460, April 1995 with January 1996 revisions.
Bloom, N.S.; and Tsalkitzis, E. Standard Operating Procedure FGS-012 Determination
of Total Mercury in Aqueous Media (Modified EPA Method 1631). Frontier
GeoSciences inc., Quality Assurance Manual 1995.
Bloom, N.S.; Ultra-Clean Sample Handling, Environmental Lab 1995, March/April, 20.
Bloom, N.S.; Horvat M., and Watras CJ. Results of the Internationai Mercury
Speciation Intercomparison Exercise. Wat Air Soil Pollut. 1995, 80, 1257.
Bloom, N.S.; Crecelius, E.A. Determination of Mercury in Seawater at Sub-nanogram
per Liter Levels.Mar.Chem.1983, 14, 49.
Bloom, N.S.; Crecelius, E.A. Distribution of Silver, Lead, Mercury, Copper, and
Cadmium in Central Puget Sound Sediments Mar. Chem 1987, 21, 377-390.
Bloom, N.S.; Fitzgerald, W.F. Determination of Volatile Mercury Species at the
Picogram Level by Low-Temperature Gas Chromatography with Cold-Vapor Atomic
Fluorescence Detection. Anal. Chem. Acta. 1988, 208,151,
Cossa, D.; Couran, P. An International Intercomparison Exercise for Total Mercury in
Seawater. App.Organomet. Chem. 1990, 4, 49,
Fitzgerald, W.F.; Gill, G.A. Sub-Nanogram Determination of Mercury by Two-Stage
Gold Amalgamation and Gas Phase Detection Applied to Atmospheric Analysis Anal
Chem. 1979, 15, 1714.
Gill, G.A.; Fitzgerald, W.F. Mercury Sampling of Open Ocean Waters at the Picogram
Level Deep Sea Res. 1985, 32, 287.
2.12 EPA Method 30.B, Determination of total vapor phase mercury emissions from coal-
fired combustion sources using carbon sorbent traps.
2.1
2.2
2.3
2.4
2.5
2.6
2.7
2.8
2.9
2.10
2.11
-------
4>*eurofins
frontier Global Sciences
Document Title:
Mercury in Water by Oxidation, Purge & Trap
and CV-AFS (EPA Method 1631, Rev E)
Eurofins Document Reference:
EFGS-SOP-137-R02
2,13 Chemical Hygiene Plan, Eurofins Frontier Global Sciences, current version.
2.14 National Environmental Laboratory Accreditation Conference, NELAC Standard
September 8, 2009.
2.15 Department of Defense Quality Systems Manual for Environmental Laboratories,
prepared by DoD Environmental Quality Workgroup, Final Version 4.2, October 2010. '
3 Cross Reference:
Document
Document Title
SOP FGS-003
Pipette Verification, Calibration and Maintenance
SOP FGS-007
Cleaning of Sampling jEquipment and Bottles
SOP FGS-008
Ultra Clean AqueousjSampie Collection
SOP FGS-012
Oxidation of Aqueous Samples for Total Mercury Analysis
SOP FGS-061
Gold Trap Construction
I nil -l-l-l-lil-lll-l-ll-l-l-l-l-lll ^^^===^^
Standard Operating Procedure Training Record
SOP FGS-094, App F
Waste Disposal Procedure for Client Sample Waste
SOP FGS-099
SOP FGS-121
Determination of Total Mercury by Flow Injection AFS (Mod 1631EJ
SOPFGS-155
Calibration of Voiumetric Dispensers
4 Purpose;
4,1
This SOP is designed to ensure that alt reproducible traceable procedures in EPA
1631 are followed in the standardization of the total mercury analyzers and in the
analysis of samples for total mercury, as well as to establish the limits wherein data will
be considered acceptable.
5 Scope:
5.'
5.2
This Standard Operating Procedure (SOP) describes a method for the determination of
total mercury (Hg) in filtered and unfiltered water by oxidation, purge and trap,
desorption, and cold vapor atomic fluorescence spectrometry (CVAFS).
This method is designed for the determination of mercury in the range of 0.5-40 ng/L
(ppt). Application may be extended to higher levels by selection of a smaller sample
size, as long as the instrument value (intensity) remains within the calibration curve.
5.3 The Control Limits are established from EPA 1631 E.
6 Basic Principles:
6.1
6.2
6.3
For analysis of aqueous samples, an aliquot of oxidized sample is neutralized with
hydroxylamine-hydrochloride {NH2OH-HCI) to destroy free halogens, and added to a
bubbler,
Stannous chloride (SnCi2) is added to the bubbler to reduce the Hg(ll) to volatile Hg(0),
and the bubblers are sealed with Keck clips. Blanked gold traps are placed at the end
of soda-lime pre-traps. The bubbler is purged with nitrogen (N2) for 20 minutes. All gas
that flows into the bubbler should only leave the system through the soda-iime pre-trap
and then the gold trap,
The gaseous mercury amalgamates to the gold traps, which are removed and
individually placed in the analytical train. The gold trap is heated, thus releasing the
mercury into the argon gas stream flowing into the instrument.
-------
Jyeurofins
Frontier Global Sciences
Document Titie:
Mercury In Water by Oxidation, Purge & Trap
and CV-AFS (EPA Method 1631, Rev E)
Eurofins Document Reference:
EFGS-SOP-137-RG2
7 Reference Modifications:
7.1 There were no significant modifications to this method,
8 Definitions;
8.1
8.2
8,3
Analytical Duplicate (AD): A representative sample (that yielded a result within the
calibration curve) is analyzed a second time during the analytical run. The second
analysis should be at the same aliquot as the original.
Analytical Run - The continuous analysis of one or more batches during the same 12
hour-shift. Each analytical day requires a minimum five-point calibration curve, ICV, at
least 3 IBLs, and CCV/CCB every ten runs. An analytical day must conclude with a
CCV/CCB.
Analytical Spike and Analytical Spike Duplicate (AS/ASD): A representative sample is
selected and spiked, with a dilution of the primary source, during the analytical run, at
a target concentration of 1-5X the ambient concentration of the sample. These QC
samples are used to indicate sample matrix effects on the anaiyte of interest. Non-
detectable samples are spiked at 1 - 5 x of the MRL/PQL
Batch: 20 client samples or less grouped for preparation. See Quality Assurance
Section for batch requirements.
Calibration Standards (CAL) - a series of standards that will be used to calibrate the
instrument, made from a primary source stock standard. A calibration blank plus at
least five different concentrations are required, beginning with one at PQL
concentration.
Certified Reference Material (CRM) - a standard of known composition that is certified
by a recognized authority and representing a sample matrix. It is used to verify the
accuracy of a method.
Continuing Calibration Blank (CCB): An instrument blank that is used to monitor the
ambient blank concentration after the Continuing Calibration Verification (CCV).
Continuing Calibration Verification (CCV): An aliquot of standard from the same source
as the calibration standard, at a value of 20ng/L (2.0ng in ~100mL bubbler water). This
standard is analyzed after every 10 analytical runs, and determines whether the
instrument is maintaining calibration.
8.9 Continuing Demonstration of Capability (CDOC)
8.10 Control Limit (CL) - the limit of the range of acceptability for the quality control
samples
8.4
8.5
8.6
8.8
8.11
8.12
Equipment Blank (EB): Reagent water processed through the sampling devices and
placed in a sample container prior to using the equipment to collect samples and used
to demonstrate that the sampling equipment is free from contamination,
Field Blanks (FB): A sample of reagent water placed in a sample container in the field
and used to demonstrate that samples have not been contaminated by sample
collection or transport activities. EPA-1631E recommends the analysis of at least one
field blank per 10 samples collected at the same site at the same time. Analyze the
blank immediately before analyzing the samples in the batch.
-------
Frontier Gtoba! Sciences
Document Title:
Mercury in Water by Oxidation, Purge & Trap
and CV-AFS (EPA Method 1631, Rev E)
Eurofins Document Reference:
EFGS-SOP-137-R02
8.13
8.14
8,16
8.17
8.18
8.19
8.22
8.23
8,24
8.25
Initial Calibration Verification (ICV): A standard that is prepared from a secondary
source stock standard with a value of 15ng/L (1.5ng in ~1QOmL bubbler). This standard
is run immediately following the calibration curve and verifies instrument calibration. It
is always followed by the IBLs.
Initial Blank Level (IBL): An instrument blank that is used to demonstrate the ambient
blank concentration of the instrument. One per bubbler is needed at the beginning of
the analytical run.
8.15 Initial Demonstration of Capability (IDOC).
Laboratory Control Sample (LCS and LCSD) or Quality Control Sample (QCS): A
sample (and duplicate) containing a known concentration of mercury that is used to
monitor complete method performance. The preferred LCS is a matrix matched
Certified Reference Material (CRM), but a blank spike meets the requirement also. In
LIMS, the LCS is always referred to as a Blank Spike (BS), whether it is matrix
matched or not
Limit of Detection (LOD) - equal to MDL and verified on a quarterly/annual basis,
depending on the preparation, by spiking within three times the established LOD and
showing a positive result on the instrument.
Limit of Quantitation (LOQ) - equal to POL and verified on a quarterly/annual basis,
depending on the preparation, by spiking within 2 times the LOQ and showing a
recovery between 70 - 130%.
LIMS: Laboratory information Management System. Computer software used for
managing samples, standards, and other laboratory functions.
8.20 May: This action, activity, or procedural step is optional.
8.21 May Not: This action, activity, or procedural step is prohibited .
Matrix Spike (MS) and Matrix Spike Duplicate (MSD): A representative sample is
selected and spiked with a dilution of the primary source at a known concentration.
The MS and MSD are run through the entire analytical process just as the samples
are. These QC samples will indicate sample matrix effects on the analyte of interest.
Method Blank (MBLK) or Preparation Blank (PB): For waters, reagent water that is
prepared and analyzed in a manner identical to that of samples. For digested solids,
preparations blanks consist of the same reagents used to digest the samples, in the
same volume or proportion and are carried through the complete sample preparation
and analytical procedure. Boiling chips are used as a blank matrix for solids.
Preparation blanks are referred to as BLK in LIMS.
Method Detection Limit (MDL): A limit derived from 40 CFR, Part 136, Appendix B.
This method produces a defined value that is the minimum concentration that can be
measured and reported with a 99% confidence that the analyte concentration is
greater than zero from a given matrix.
Method Duplicates/Method Triplicates (MD/MT): A second or third separate sample
dilution, taken from the same source sample, prepared and analyzed in the laboratory
separately. An MSD may be used as a duplicate.
-------
Jyeurofins
Document Title;
Mercury in Water by Oxidation, Purge & Trap
and CV-AFS (EPA Method 1631, Rev E)
Eurofins Document Reference:
EFGS-SOP-137-R02
8.26 Reagent water: 18 MQ minimum, reagent water starting from a pre-purified (distilled,
Reverse Osmosis, etc.) source.
8.27 Must: This action, activity, or procedural step is required.
8.28 Ongoing Precision and Recovery (OPR): A dilution of a secondary source resulting in
an instrumental concentration of 5.0 ng/L mercury.
8.29 PM: Project Manager.
8.30 Practical Quantitation Limit (PQL), Method Reporting Limit (MRL): The minimum
concentration that can be reported quantitatively. The PQL is often described as 1-10
times higher than MDL. Eurofins Frontier defines the PQL as the lowest concentration
that can achieve 70-130% recovery for 10 replicate sample preparations, tn LI MS, the
PQL is referred to as the MRL.
8.31 Primary Source: The stock standard used to make the calibration standard. Procedural
Method: A method where standards and samples are run through the analytical
procedure exactly the same. By NELAC definition, this SOP is a procedural method.
8.32 Secondary Source: The stock standard used to make the OPR standard.
8.33 Shall: This action, activity, or procedure is required,
Should: This action, activity, or procedure is suggested, but not required.
8.34
8.35
8.36
8.37
Stock Standard Solution (SSS) - a standard of analyte that is purchased from a
certified source for the preparation of working standards.
Total mercury: As defined by this method, al! bromine monochloride-oxidizable
mercury forms and species found in aqueous solutions. This includes, but is not limited
to, Hg(ll), Hg(0), strongly organo-complexed Hg(ll) compounds, adsorbed particutate
Hg{P), and several tested covatently bound organomercuriais {i.e. CH3HgCI, (CH3)2Hg,
and C6H5HgOOCCH3). The recovery of mercury bound within microbia! cells may
require additional preparation steps (i.e. UV oxidation, or oven digestion).
Travel or Trip Blank (TB): A sample of reagent water placed in a sample container in
the laboratory and used to demonstrate that samples have not been contaminated by
transport activities.
9 Interferences;
9.1
9.2
Gold and iodide are known interferences. At a mercury concentration of 2.5 ng/L and
at increasing iodide concentrations from 30 to 100 mg/L, test data have shown that
mercury recovery will be reduced from 100 to 0 percent. At iodide concentrations
greater than 3 mg/L, the sample should be pre-reduced with SnCI2 (to remove brown
color immediately prior to analysis) and additional or more concentrated SnCI2 should
be added to the bubbler containing sample. If samples containing iodide
concentrations greater than 30 mg/L are analyzed, it may be necessary to clean the
analytical system with 4N HCi after the analysis,
Water vapor has the potential to create recovery interferences. To prevent interference
from water, ensure that soda-lime pre-traps and gold traps remain dry.
-------
4yeurofins
frontier Global Sciences
Document Title:
Mercury in Water by Oxidation, Purge & Trap
and CV-AFS (EPA Method 1631, Rev E)
Eurofins Document Reference:
EFGS-SOP-137-R02
10.1
10.2
9.3 The presence of high concentrations of silver and/or gold can cause SnCI2 to
precipitate out of solution and adhere to the bubbler walls. High concentrations of
these metals can sometimes be found in the matrix spike samples from the digestion
sets that are shared with the trace metals group. When analyzing digestates where the
matrix spike samples have been spiked with silver or gold, the matrix-spiked samples
must not be used for mercury analysis. Instead, an alternate matrix spike and matrix
spike duplicate (MS/MSD) should be prepared and analyzed. If this is not possible, an
Analytical Spike/Analytical Spike Duplicate (AS/ASD) must be analyzed on the
ambient sample,
10 Safety Precautions, Pollution Prevention and Waste Handling;
Personnel will don appropriate laboratory attire according to the Chemical Hygiene
Plan. This includes, but is not limited to, laboratory coat, safety goggles and nitrite
gloves under clean gloves.
The toxicity or carcinogenicity of reagents used in this method has not been fully
established. Each chemical should be regarded as a potential health hazard and
exposure to these compounds should be as low as reasonably achievable. Chemists
should refer to the MSDS (Material Safety Data Sheets) for each chemical they are
working with.
10.2.1 Note; Use particular caution when preparing and using BrCI, as it releases
extremely irritating, corrosive fumes similar in effect to free chlorine. Always
handle this reagent in an approved fume hood
Ail personnel handling environmental samples known to contain or to have been in
contact with human waste should be immunized against known disease-causative
agents. Eurofins Frontier will reimburse the expense of Hepatitis A and B
immunizations for any laboratory staff member who desires this protection.
Hydrochloric acid: Very hazardous in case of skin contact (corrosive, irritant,
permeator), of eye contact (irritant, corrosive), of ingestion. Slightly hazardous in case
of inhalation (lung sensitizer). Non-corrosive for lungs. Liquid or spray mist may
produce tissue damage particularly on mucous membranes of eyes, mouth and
respiratory tract. Skin contact may produce burns. Inhalation of the spray mist may
produce severe irritation of respiratory tract, characterized by coughing, choking, or
shortness of breath. Severe over-exposure can result in death. Inflammation of the eye
is characterized by redness, watering, and itching. Skin inflammation is characterized
by itching, scaling, reddening, or, occasionally, blistering. For more information see
MSDS.
10.5 See Eurofins Frontier Global Sciences Chemical Hygiene Plan (CHP) for general
information regarding employee safety, waste management, and pollution prevention,
10.6 Pollution prevention information can be found in the current Eurofins Frontier Global
Sciences Chemical Hygiene Plan (CHP), which details and tracks various waste
streams and disposal procedures.
10.7 All laboratory waste is accumulated, managed, and disposed of in accordance with all
federal, state, and local laws and regulations. Any waste generated by this procedure
should be disposed of according to SOP FGS-099 "Waste Disposal Procedure for
10.3
10.4
-------
4ฃeurofins i
Frontier GSobal Sciences
Document Title:
Mercury in Water by Oxidation, Purge & Trap
and CV-AFS {EPA Method 1631, Rev E)
Eurofins Document Reference:
EFGS-SOP-137-R02
Client Sample Waste," which provides instruction on dealing with laboratory and client
waste,
11 Personnel Training and Qualifications:
11.1 An analyst must perform an initial demonstration of capability (IDOC) that includes four
replicates of a secondary source before being qualified to analyze samples without
supervision. Continuing DOC will be maintained and monitored via performance on
CRMs and other QC samples, as well as obtaining acceptable results on proficiency
testing exercises.
The analyst/laboratory technician must have read this SOP and other relevant SOPs
and have the training documented on the applicable form(s). The analysis may be
questioned on SOP by supervisor(s) and/or trainers.
Training is documented by the employee and supervisor, and is kept on file in the QA
Office. The employee must read, understand, and by signing the training document,
agree to perform the procedures as stated in all Standard Operating Procedures
(SOPs) related to this method.
Reading of the SOP must be documented on the correct form such as "Standard
Operating Procedure Training Record," Appendix F in FGS-094, the last page of this
SOP, Appendix A "Standard Operating Procedure Training Record" or a similar
document."
All employees must also, on a yearly basis, read the Quality Manual (QM), and
complete the yearly Ethics training.
All training documents including IDOCs, CDOCs, SOP reading, Initial QA orientation,
and Ethics training are stored by the Quality Assurance Manager in the employees
training file for ten years after the employee is no longer working for Eurofins Frontier
Global Sciences.
11.7 Chemical Safety Training, Compressed Gas Training, Chemical Hygiene Plan
documentation, and Shipping of Hazardous goods, are stored by the Health and
Safety Officer for ten years after the employee is no longer working for Eurofins
Frontier Global Sciences.
12 Sample Collection, Preservation, and Handling;
12.1 Aqueous samples are collected in rigorously cleaned fluoropolymer (e.g. Teflon) or
PETG bottles and caps (as described in FGS-007 "Cleaning of Sampling Equipment
and Bottles for Mercury Analysis"). Certified clean glass bottles with fluoropolymer lids
may be used if mercury is the only analyte of interest.
12.1.1 Aqueous samples are preserved upon receipt with 0.2N BrCI that has tested
low in mercury. Samples are typically preserved to 1% BrCI v/v, but may
require further oxidation due to high levels of organic matter or mercury. Refer
to FGS-012 "Oxidation of Aqueous Samples for Total Mercury Analysis" for
oxidation of aqueous samples. Samples requiring greater than 10% BrCI must
have a method blank prepared at the time of preservation. Preservation levels
should be limited to 1%, 2%, 3%, 5%, 10%, and 100%.
11.2
11.3
11.4
11.5
11.6
-------
4>" euro fins i
Frontier Global Sciences
Document Title:
Mercury in Water by Oxidation, Purge & Trap
and CV-AFS (EPA Method 1631, Rev E)
Euroftns Document Reference:
EFGS-SOP-137-R02
12.2
12.1.2 Preservation levels other than 1% are written on the LiMS label of the sample
bottle. Preservation levels are also documented in the LIMS bench sheet by
adjusting the initial and final volumes. For example, a sample preserved at 2 %
BrCI must say "2" on the LIMS label, and have an initial volume of 100mL and a
final volume of 102mL in the bench sheet,
All samples should be collected utilizing clean techniques, so as not to cross-
contaminate samples with mercury. See FGS-008 "Ultra Clean Aqueous Sample
Collection" and EPA Method 1669 for aqueous sample techniques.
13 Apparatus and Equipment:
13.1 LIMS - Element, version 5.85 or higher; Computer - Windows XP, 7 or 8
13.2
13,3
13.4
13.5
13.6
13.7
13.8
13.9
Tehran 2500 Atomic Fluorescence Spectrophotometer (AFS) or equivalent; A high
sensitivity AFS Detector (IDL<1pg) with a required wavelength of 253.7 nm and
associated software,
Flow meter/needle valve: A unit capable of controlling and measuring gas flow to the
cold vapor generator at 200-500 mL/min.
Teflon Fittings: Connections between components and columns are made using Teflon
FEP tubing and Teflon friction fit tubing connectors.
Soda-Lime pre-trap: A 10cm x 0.9cm diameter Teflon tube containing 2-3 g of reagent
grade, non-indicating 8-14 mesh soda-lime (Ca(OH)2+NaOH) aggregates, packed
between portions of silanized glass wool. This trap is purged of mercury by placing it
on the output of a clean cold vapor generator and purging it with -3-5% HCI and -600
ML of SnCI2 for approximately 20 minutes with N2 at 40 mL/min.
Cold-vapor generator (bubbler): A 150 ml, tall, flat-bottom borosilicate flask with
standard taper 24/40 neck, fitted with a sparger having a coarse glass frit which
extends to within 0.2 cm of the flask bottom.
Gold Traps: Made from 12 cm lengths of 6 mm OD quartz tubing, with a 4-way crimp
3.0 cm from one end. The tube is filled with approximately 2.5 cm of 20/40 mesh gold-
coated quartz sand, the end of which is then plugged with quartz wool. Gold-coated
sand traps are heated to 450-500ฐC (the coil should have a barely visible red glow
when the room is darkened) with a coil consisting of 75 cm of 24-gauge nichrome wire
at a potential of 10 VAC. Potential is applied and finely adjusted with an auto-
transformer. Refer to SOP FGS-061 regarding the construction of gold traps used for
total mercury analysis.
Agilent Integrator Recorder or equivalent: Any multi-range chart recorder or integrator
with 0.1-5.0 mV input and variable speeds is acceptable. Data capture software may
also be used.
Pipettes: Calibrated variable pipettes with a range of 5 uL - 10 mL Used to make
solutions and sample dilutions. Pipettes are to be calibrated weekly according to SOP
FGS-003andFGS-155.
14 Reagents and Standards:
All reagents, except those made daily, must be entered into LIMS
-------
Mercury in Water by Oxidation, Purge & Trap
and CV-AFS (EPA Method 1631, Rev E)
Document Title: _ _ _ ,
Eurofms Document Reference:
EFGS-SGP-137-R02
14.1 Reagent Water: 18-MO ultra pure deionized water starting from a pre-purified (distilled,
R.O., etc.) source is used. To remove any remaining trace metals and organics, an
activated carbon cartridge is placed between the final ion exchange bed and the 0.2-
um filter. Reagent water used in the mercury lab is checked weekly for total mercury
concentrations, and must test below 0.25ng/L.
14-2 Hydrochloric Acid (HCI): Concentrated (36-38% weight basis). Must be traee-metaf
purified and reagent grade. HCI is typically monitored through performance of the BrCI.
Sometimes it will be necessary to test the HCI directly. To do so, add 1 mL, using a
calibrated pipette, of HCI to approximately 1QQmL of purged bubbler water. Enter 1mL
as aliquot in the Excel spreadsheet. Do not prep blank correct. Analyze one replicate
per bottle. This reagent should test below 5.0 ng/L. This solution is considered stable
until the expiration date on the bottle, set by the manufacturer.
14.3 0.2N Bromine Monochloride (BrCI):
14.3.1 37.5 g of KBr is added to a 2.5-L bottle of concentrated HCI (pre-analyzed and
found to be below 0.25 ng/L Hg). The bottle is then inverted in a fume hood to
mix the acid and KBr. The solution then sits overnight allowing for the KBr to be
dissolved.
14.3.2 27.5 g of KBrO3, certified to be low in Hg, is slowly added to the acid. When all
of the KBrO3 has been added, the solution should have gone from yellow to red
to orange.
14.3.3 Loosely cap the bottle, and allow to sit for 30 minutes in a fume hood before
tightening the lid. Once capped invert bottle to make sure all of the solids goes
into solution. CAUTION: This process generates copious quantities of free
halogens (CI2, Br2, BrCI) which are released from the bottle. Add the
KBrO3 SLOWLY and in a well operating fume hood.
14.3.3,1 To test the BrCI, add 1 mL, using a calibrated pipette, of the BrCI to a
prep blank vial containing approximately 4 mL reagent water. Add 200 uL
Hydroxylamine-HCI to the vial; pour the entire contents into a bubbler
containing approximately 100 mL of purged water. Assume a 100 mL
aliquot in the Excel spreadsheet. This reagent must test below 0.20ng/L.
Do not prep blank correct. Analyze one replicate per bottle.
14.3,3.2 The expiration time for this reagent is set by default to six months in
LIMS. There is no suggested holding time in EPA method 1631E,
therefore the holding time can be extended, as long as the primary
reagent has not expired. The mercury concentration of the BrCI is
monitored through the preparation of water preparation blanks.
14,4 Hydroxylamine hvdrochlortde: dissolve 300g of NH2OH-HCI in reagent water and bring
the volume up to 1L. This solution may be purified by the addition of 1mL SnCI2
solution and purging overnight at 500mL/min with mercury-free N2. The working
reagent is a 25% solution that is made by adding one part reagent water to one part
50% hydroxylamine hydrochloride. This reagent must test below 0.25ng/L.
14.4.1 To test the Hydroxylamine-HCI (NH2OH-HCI), add 1 mL of the 50% reagent,
using a calibrated pipette, to approximately 100 mL of purged bubbler water!
2
-------
4>s< euro-fins
frontier GiobaS Sciences
Document Title;
Mercury in Water by Oxidation, Purge & Trap
andCV-AFS {EPA Method 1631, Rev E)
Eurofins Document Reference;
EFGS-SOP-137-R02
Assume a 100 ml aliquot in the Excel spreadsheet. This reagent must test
below 0.20 ng/L. Do not prep blank correct. Analyze one replicate per bottle.
14.4.2 The expiration time for this reagent is set by default to six months in LIMS.
There is no suggested holding time in EPA method 1631E; therefore the
holding time can be extended, as long as the primary reagent has not expired,
14.5 Stannous Chloride (SnCI2): Weigh out 500 g SnCI2 using a calibrated balance that also
has been verified for the day. Dissolve with three 100 ml aliquots of concentrated HCI
and transfer to a 1L I-CHEM glass bottle, which contains approximately 300 ml of
reagent water. Bring this solution up to approximately 1 L of volume and purge
overnight with mercury-free N2 at 500 ml/mm to remove all traces of mercury. Store
tightly capped. The working reagent is a 25 % solution that is made by adding one part
reagent water to one part 50 % stannous chloride.
14.5.1 To test the Stannous Chloride (SnCI2), add 1 ml of the 50% reagent, using a
calibrated pipette, to approximately 100 ml of purged bubbler water. Assume a
100 ml aliquot in the spreadsheet. This reagent must test below 0.20 ng/L. Do
not prep blank correct. Analyze one replicate per bottle.
14.5.2 The expiration time for this reagent by default is set to six months in LIMS.
There is no suggested holding time in EPA method 1631E; therefore the
holding time can be extended, as long as the primary reagent has not expired.
Argon Grade 4.7 or better {ultra high-purity grade): Argon that has been further purified
by the removal of mercury using a gold trap that is located in line between the gas
output and the analyzer gas input.
Nitrogen Grade 4.5 (standard laboratory grade): Nitrogen that can be further purified of
mercury using a gold trap that is located in line between the gas output and bubbler
14.8 Preparation of Total Mercury Standard Solutions:
14.6
14.7
14.8.1
Mercury standard solutions are prepared in ultra clean volumetric glassware
and gravimetrically calibrated pipettes. Resulting solutions must be stored in
glass or Teflon bottles and preserved to at least 2 % BrCI. All working
standards must be tested prior to use.
14.8.1.1
New working standards and standard dilutions are tested prior to use,
Three reps of the new standard are analyzed in the same run as three
reps of the current NIST 1641D standard. Analyze 200 pi of the NIST
1641D and assume 100 ml in the bubbler. The mean percent recovery of
the three standards should be ฑ5 % (95-105 %) of the true value and also
within 5 % of the average NIST 1641D recovery (e.g. If the average of
NIST 1641D recovery is 97 %, the range for the standard is 95-102 %). If
the standard does not test within this control limit, it is retested. If it still
does not meet the control limit, it is discarded and remade, unless
otherwise approved by the Quality Assurance Officer, NOTE: When
making serial dilutions to create various standard levels; the lowest
concentration may be used to test any of the higher concentration steps
(for example: if a 1Qng/mL calibration standard is created from a
1000ng/mL spiking standard, only the 10ng/mL standard requires testing.
-------
eurofins I
frontier Siobai Sciences
Document Title:
Mercury in Water by Oxidation, Purge & Trap
and CV-AFS (EPA Method 1631, Rev E)
Eurofins Document Reference:
EFGS-SOP-137-R02
14.8.2
14.8.3
If the 1Qng/mL standard passes, then both standards are considered to
be passing within the control limits.)
Total Mercury Stock Standard Solution (Stock); Certified mercury standard
purchased from High Purity Standards (1000 |jg/mL (1 000 000 ng/mL) primary
source) or Absolute Standards (100 |jg/mL (100 000 ng/mL) secondary
source), or any equivalent standard.
Total Mercury Spiking Standard Solutions (Spiking Standard): Spiking
standards are made from either the primary or secondary sources.
14.8.3.2
14.8.3.3
14.8.3.1 To make standards, use an ultra clean volumetric flask and a calibrated
pipette. Add reagent water until flask is about half full. Add 2 % 0.2N BrCI
and the specific spike volume noted below (these volumes may be
changed as long as ratio and resulting concentration remains the same).
Bring up to the mark with reagent water and mix well prior to testing.
When spiking samples, no more than 200 ML of any spiking standard is
added to the sample to minimize effects on volume, it is also
recommended that staff pipette no less than 25 uL If possible, minimize
headspace during standard storage. Expiration date is currently set at
6 months or when the stock standard expires, whichever is shorter.
100,000 ng/mL Spiking Standard: Made from the Primary Stock Standard
(High Purity, or equivalent vendor). Dilute 10 mL of the stock standard to
100 mL of reagent water containing 2 % BrCI. (Can also be made by
preserving Secondary Stock Standard to 2% BrCI).
10,000 ng/mL Spiking Standard: If made from the Primary Stock Standard
(High Purity, or equivalent vendor). Dilute 1.0 mL of the stock standard to
100 mL of reagent water containing 2 % BrCI. If made from Secondary
Stock Standard, dilute 10mL of stock standard to 100mL with reagent
water containing 2% BrCI.
14.8.3.4 1,000 ng/mL Spiking Standard: If made from the Primary Stock Standard
(High Purity, or equivalent vendor). Dilute 0.250 mL of the stock standard
to 250 mL RO water containing 2 % BrCf. If made from Secondary Stock
Standard dilute 2.5mL of stock standard to 250mL with RO water
containing 2% BrCI.
14.8.3,5 100 ng/mL Spiking Standard: Made from a stock standard or dilution of a
stock standard with a concentration of 100,000 ng/mL. Ditute 0.100 mL of
the 100,000 ng/mL dilution to 100 mL of reagent water containing 2 %
BrCI. Expiration date is currently set at 3 months or when the stock
standard expires, whichever is shorter.
14.8.4 Calibration Standard (10 ng/mL): Must be made from a dilution of the Primary
Stock Standard (High Purity, or equivalent vendor). Typically made by diluting
0.5mL of a 10,000 ng/mL Primary Spiking Standard to 500 mL of reagent water
containing 2 % BrCI. Transfer to glass or Teflon bottle. The calibration
standard is considered stable for three months or until the stock standard
expires.
-------
eurofins
Document Titie:
Mercury in Water by Oxidation, Purge & Trap
and CV-AFS (EPA Method 1631, Rev E)
Eurofins Document Reference:
EFGS-SOP-137-R02
14.8.5
14.8.6
14.8.7
14.8.8
14.8.9
Calibration Standard (1 ng/mL): Must be made from a dilution of a Primary
Stock Standard. Typically made by diluting 1.0mL of a 100 ng/mL Primary
Spiking Standard to 100mL with Reagent water containing 2% BrCL
Initial Calibration Verification (ICV): A 10 ng/mL ICV solution is prepared using
the Secondary Stock Standard (Absolute Standards, or equivalent vendor).
Use 0.100 mL (100 uL) of the Secondary Stock Standard to 1000 mL Milli-Q
containing 2 % BrCI. Transfer to one 1000 ml glass or Teflon bottle. The ICV
standard is considered stable for three months or until the stock standard
expires, it is recommended to alternate expiration date with the CAL standard.
Continuing Calibration Verification (CCV): For CCV analysis, use 200 (jL of the
10 ng/mL CAL standard {documented in LIMS as SEQ-CAL3), The True Value
is 20 ng/L.
Certified Reference Material (CRM) for Total Mercury in Water: A 1.5679 mg/L
solution (1,557 mg/kg at a density of 1.007 g/mL) is prepared by adding a 5,0
mL of CRM NIST 1641d (from ampoule) into a 1000 mL flask containing of
reagent water. This solution is diluted to 1000 mL, and an additional 10 mL of
0.2N BrCI is added, resulting in a final volume of 1010 mL, Preparing the
solution in this manner makes a 1:200 dilution of the stock CRM. This solution
is considered stable for one year, or until the stock standard expires. Results
are corrected for the additional 1 % BrCI in the analysis Excel spreadsheet and
in LIMS.
Ongoing Precision and Recovery (OPR) for "Strict" 1631 E: A 5.0 ng/L solution
is prepared by adding 100 pL of the 100 ng/mL secondary spiking standard into
2000 mL reagent water. An additional 1 % BrCI (20 mL) of BrCI is added, so
that the final volume is 2020 mL. This standard is analyzed at 100 mL at the
instrument, and preparation blank corrected exactly in the same manner as
samples
14.9 Documentation of Standards and Reagents:
14.9.1 Standards and Reagents are documented in LIMS upon receipt or creation. A
LIMS generated label is affixed to each standard and reagent that has the
name of the solution, the person who prepared or received it, the date it was
prepared or received, and the expiration date.
14.9.2 Each bottle of standard must be labeled with the following: the date of receipt
or creation, the initials (or name) of who entered the standard into LIMS, the
concentration and analyte, the expiration date and the LiMS ID. This
information must also appear on the certificate of analysis of stock standards.
14.9.3 Stock standards and CRMs are logged into LIMS upon receipt by Shipping and
Receiving (S&R) or the Quality Assurance department (QA). These do not
require testing, provided there is a Certificate of Analysis on file in QA. When
receiving a solid CRM, QA shall generate a work order in LIMS for total solids
analysis.
14.9.4 For all standards, L!MS documentation must include the following: a description
of the standard, department, expiration date of the standard (not to exceed the
expiration of the parent standard), the name of the person who made (or
-------
4fซeu refills
Frontier Global Sciences
Document Title:
Mercury in Water by Oxidation, Purge & Trap
and CV-AFS (EPA Method 1631, Rev E)
Eurofins Document Reference:
EFGS-SOP-137-R02
received) the standard or reagent, the date it was prepared (or received), final
volume, a reference date (date entered into LIMS), concentration units (ug/mL),
the vendor and vendor lot. The solvent lot is used to document the Lot Number
or LIMS ID of the BrCI that was used. In the comments section, the analyst
must enter the sequence and applicable results for documentation of standard
testing. Other notes may be entered in here as well. The correct parent
standard must be noted, as well as the amount used, Analytes are entered
individually from the list. LIMS will calculate the true value of the standard
based on the amount of the parent used and the final volume. Click the
appropriate radio button under Standard type. A Spike Mix is a standard that is
used in a bench sheet, and a Calibration standard is a standard used only in
sequences. A Reference Standard is a Certified Reference Material (CRM).
The standard must not be used until it has passed control limits and is
approved by the mercury supervisor, mercury laboratory manager, or QA for
use.
14.9.4.1 If the new standard is a calibration standard, a separate standard ID must
be created for each calibration point based on the final concentration in
the sequence (example: THg CAL1 0.10 ng orTHg CAL2 0.50 ng). These
are given the same expiration as the standard they are made from, and
will need to be generated every three months as each new working
calibration standard is made and tested.
14,9.4,2 To generate new "CAL" standards in LIMS, go to the Laboratory drop
down menu and select Standards. Open the current CAL1 standard and
click "Copy". Update the appropriate information, including the Prepared
Date, Expiration Date, Prepared By, and the Reference Date. For these
standards, which are to be used in the sequence, the final volume is
equal to the assumed aliquot in the bubbler (100 mL). Check that the
vendor lot is correct. Remove the old (expired) parent standard. Choose
the new parent standard, and enter the amount of standard added to the
bubbler for that calibration point. All depleted or expired standards are
moved into the Expired Standards Department once they are no longer
being used.
14.9.4.3 Each bottle of standard must be labeled with the following: the date of
receipt or creation, the initials (or name) of who entered the standard into
LIMS, the concentration and analyte, the expiration date and the LIMS ID.
This information must also appear on the certificate of analysis of stock
standards.
Neat reagents are logged into LIMS with a unique identifier upon receipt by
Shipping and Receiving Department and given a default expiration of 3 years,
unless otherwise noted by the manufacturer.
Working reagents are prepared by the analyst, logged into LIMS and assigned
a unique identifier. Reagents entered into LIMS must have the information
listed in section 14.9.2. In addition the parent neat reagents are added by their
unique identifier and the amount of each reagent is entered. It is not necessary
to enter anaiytes from the list for reagents. The Solvent Lot is not applicable to
working reagents. The radio button must be clicked to Reagent. If the reagent
14.9.5
14.9.6
-------
4ฃeurofins
i Frontier Giobaf Sciences,
Document Titie:
Mercury in Water by Oxidation, Purge & Trap
and CV-AFS (EPA Method 1631, Rev E)
Eurofins Document Reference:
EFGS-SOP-137-R02
requires testing, it must test clean prior to using. Ail reagents used during
analysis and prep should be added to bench sheet,
14.9.7 Depleted or expired standards and reagents are segregated and removed from
use.
15 Calibration:
15.1 The analyst should label the strip chart/integrator printout with the corresponding
dataset ID as well as print and sign their name. For strip chart printouts, the analyst
should label the baseline ratios accordingly (usually X=1 and X=20) and label with the
analysis day start time and strip chart drum speed (usually 1 mm/min). The analyst
should note the end time as well. If using an integrator, the date and time should be
checked and corrected if necessary.
15.2 The calibration sequence determines the range of sample concentrations that are
reportable. The calibration sequence starts with a 5-point curve using the total mercury
calibration standard solution. The five points are: Q.05ng (0,50 ng/L), 0.10 ng (1.00
ng/L), 0.50 ng (5.00 ng/L), 2.00 ng (20.00 ng/L), and 4.00 ng (40.0 ng/L). An ICV/OPR
and IBLs (one for every bubbler used are analyzed immediately following the standard
curve.
15.2.1
15.2.2
15.2.3
Using the 10 ng/mL calibration standard, add 5 uL, 10 uL, 50 uL, and 200 uL to
the bubblers sequentially from the left to right. Add 300 uL SnC!2 to the
bubblers and seal bubbler tops using Keck Clips.
Place blanked gold traps securely at the end of soda-lime traps (pinched
section of gold trap closest to the soda-lime trap). Purge bubblers with N2 for a
minimum of 20 minutes,
Attach individual gold traps to the analytical, train and burn in sequential order,
Peaks produced should be labelled, as well as recorded in the Excef
spreadsheet in real time.
15.3 For the second round, add 400 fjl of the 10 ng/mL mercury calibration standard to the
first bubbler. Add 50uL of the 10 ng/mL ICV(OPR) standard to the second bubbler (5.0
ng/L). The third and fourth bubblers are used for the first and second IBLs and nothing
should be added to these bubblers. To ensure that nothing is added, keep it sealed
with a Keck Clip. Add 300 uL SnCI2 to ail bubblers except the fourth and seal bubbler
tops with Keck Clips.
15.4 For the third round, use the first and second bubbler to finish the IBLs needed for
1631. The third and fourth bubbler can be used for the first portion of the batch. If the
curve does not pass or needs to be investigated any batch portions analyzed in this
round will need to be reanalyzed.
15.5 Once the instrument is calibrated and the ICV/IBLs are analyzed and judged to be in
control, the instrument is operational. The sample concentrations must fall within the
range of the calibration standards or be diluted and reanalyzed.
15.6 The purge efficiency of the bubbler system is 100 % and is independent of volume at
the volumes used in this method. Calibration of this system is typically performed using
units of mass. For purposes of working in concentration, the volume is assumed to be
100 mL.
-------
Document Title: _ ,. _ , _ ,
Mercury in Water by Oxidation, Purge & Trap E^ ' r Q "^ซ onf^
and CV-AFS (EPA Method 1631, Rev E) tr-U5-bUP-'W-KtL>
15,7 This completes the instrument calibration for total mercury analysis.
16 Procedure:
16.1 When analyzing on the Tekran 2600, follow the procedure in EFGS-121 while still
adhering to the QA/QC criteria of this method.
16.2 Pre-analysis and Organization:
16.2.1 Prior to analyzing samples it is imperative to reference LiMS for all project
specific information, such as QC requirements, suggested dilutions, project
manager information, and specifics regarding spike levels.
16.2.2 The analyst should then locate samples and check the work order in LIMS for
notes about specific project requirements.
16.2.3 The analyst should compare the sample IDs to the work order and see that the
samples are accounted for, and notify the project manager of any
discrepancies in analysis required, sample identification, etc.
16.2.4 All mercury analyses receive a unique dataset identifier. This is comprised of
the instrument type and number, the date and the calibration number for that
day. The format is as follows: THg8-091218-1, where "THg "refers to a total
mercury analysis; "8" refers to the analyzer number 8; 091218 refers to the
date (December 18, 2009 in the YYMMDD format); and "1" refers to the first
calibration of the day,
The sequence number is assigned by LIMS when the data gets imported into
LIMS. The alpha-numeric code is based on the following format: 3B02001,
where the 3 refers to the year (2013), the "B" is the month (A= January,
B=February...L=December), "02" is the day of the month (February 2nd) and'
the final 3 digits is the nth sequence created on that particular year/month/day
combination.
16.2.5 In general, the analyst should organize their samples in the order listed on the
bench sheet. The first samples analyzed should be the preparation blanks,
then the LCS if analyzing solid samples, followed by actual samples. If
possible, run total and dissolved samples side by side to facilitate verification
that total concentration is greater than dissolved concentration. See QA
section.
16.2.6 All samples specified as being High QA should be analyzed prior to any
Standard QA projects that are being analyzed on the same instrument on the
same day. However, if concentrations are known, analyze samples with low
concentrations prior to samples with high concentrations
16.3 instrument Start Up:
16.3.1 Begin blanking gold traps. To do this, attach one trap at a time to the analytical
train and burn to the instrument. Ensure the Argon, is flowing at appropriate
levels (-25-40 mL/min). The pinched portion of the gold trap should be on the
left (closest to the analytical trap). Continue to burn traps in sequential order.
-------
4>*eurofins 1
Document Title:
Mercury in Water by Oxidation, Purge & Trap
and CV-AFS {EPA Method 1631, Rev E)
Eurofins Document Reference:
EFGS-SOP-137-R02
16.3.2 Rinse out the bubbler three times with reagent water and fill with about 100 ml
of reagent water. Using a pre-purged pipette, add 3-5 ml HCI. Initially add 600
uL of SnCl2,
16.3.3 Prepare one soda-lime trap for each bubbler. To prepare soda-lime traps, hold
soda-lime between two glass wool plugs in a Teflon tube. Cap the tubes with
Teflon plugs and attach to the bubbler. Once the soda-lime traps have been
attached, the bubbler system (soda lime trap and bubbler water/acid/ SnCI2)
must purge for a minimum of 20 minutes before beginning the instrument
calibration sequence.
16.4 Analyzing Aqueous Samples;
16.4.1 All aqueous samples should be preserved with BrCI according to FGS-012 at
least 24 hours prior to analysis. In the event a sample requires further oxidation
prior to analysis, additional BrCi is added and the sample should not be
analyzed for at least 12 additional hours. In special cases where rush turn-
around-time is required and an oxidation period of less than 24 hours may be
used, a heated oven digestion procedure can be utilized.
16.4.2 While bubbling and burning the standard curve, the analyst should prepare a
minimum of three BrCI method blanks (BLK) at 1% BrCi. Add 1 mL BrCI and
200 uL hydroxyiamine hydrochloride (NH2OH-HCI) to each bubbler. The aliquot
is assumed to be 100 ml. Any sample requiring an increased amount of
reagent must be accompanied by at least one method blank that includes an
identical amount of reagent,
16.4,3 After the instrument calibration sequence, preparation blanks and the
LCS/LCSD are analyzed.
16.4.4 All known field, equipment, and trip blanks should be analyzed before any other
sample types, usually after the BLKs. Aliquots of 100 ml should be analyzed,
provided there is adequate collected sample volume. Sample aliquot sizes of
125 ml can be analyzed upon request by the project manager.
16.4.5 For all waters, select the appropriate dilution (refer to L1IV1S, historical data
etc.).
16.4.5.1 For sample aliquots of 25 uL to 10.0 ml, use calibrated pipettes to
dispense the aliquots directly into bubbler. Due to minimal amounts of
BrCI in aliquots of 10 ml or less, NH2OH-HCI is not added. It is highly
recommended that the analyst should not pipette less than 25 uL A
dilution of the sample should be made to allow a larger aliquot to be
analyzed.
16.4.5.2 For sample aliquots greater that 10 mL, gravimetrically weigh out the
selected volume (ฑ0.2 g) into a clean 125 mL Teflon bottle. Once quantity
is weighed out, neutralize BrCI with 200 uL NH2OH-HCi no more than five
minutes prior to adding the sample to bubblers. The sample should turn
from a yellowish color to a clear/cloudy solution, depending on the matrix.
16.4.6 If the material is a seawater or highly dense liquid, it may be necessary to
account for the density if the aliquot is gravimetrically determined. Density
-------
Frontier Giobai Sciences
Document Title:
Mercury in Water by Oxidation, Purge & Trap
and CV-AFS (EPA Method 1631, Rev E)
Eurofins Document Reference:
EFGS-SOP-137-R02
checks can be performed at the time of analysis to determine if further
determinations are necessary,
16.4.7 The procedure for analysis is similar to that of the caiibration. Samples to be
analyzed are pipetted or poured into the bubbler (one sample per bubbler)
along with 300[jL SnCI2. Bubbler tops are sealed with Keck Clips to ensure
nominal sample leakage. Blanked gold traps are securely placed at the end of
the soda-lime trap. Purge bubblers with N2 for a minimum of 20 minutes,
remove gold traps, and sequentially place in the analytical train. Burn individual
traps to analyzer, labeling resulting peaks with corresponding sample in real
time,
16.4.7,1 Sample IDs, aliquot volume, BrCI percentage (group ID), peak
height/peak area, and dilution factor {if applicable) associated with each
sample should be entered into the THg Waters Template Excel
spreadsheet.
16.4.7.2 While purging one set of samples, the analyst should begin preparing the
next round of water samples in the same fashion to maximize efficiency,
16,5 End of analysis close-down procedure:
16,5.1 Turn off gas flow.
16.5.2 Carryout all end of day cleaning and restocking tasks.
16,6 The analytical data is compiled into an Excel file. The data is then copied and pasted
into an Excel template that is LIMS compatible,
16.7 Maintenance and Troubleshooting
16,7,1 ISSUE: No peaks at all
16.7.1.1 Ensure that the system is powered.
16.7.2 ISSUE: Low sensitivity
16,7.2.1 Make sure that you have freshly changed soda lime in the soda lime trap,
and that it is from a good source.
16,7.2.2 Do not use old calibration standards to calibrate the system.
16.7.2.3 Ma/ce sure you are running fresh SnC/2 solution.
16.7.2.4 Make sure that your stock Hg standard has not expired and is from a
reliable source and that it is not compromised.
16.7.2,5 Check the lamp voltage
16.7.3 ISSUE: High blanks
-------
Document Title:
Mercury in Water by Oxidation, Purge & Trap
and CV-AFS (EPA Method 1631, Rev E)
Eurofins Document Reference:
EFGS-SOP-137-R02
16,7,3,1 Check reagent (including water) quality
16.7.3.2 Check for system contamination
16.7.4 ISSUE: Nonlinearity of the calibration curve
16.7.4.1 Check and investigate high blanks.
16.7,4.2 Contaminated and expired soda lime. Change soda lime,
16.7.4.3 Mate sure your calibration standards are fresh and properly prepared.
17 Calculations:
17.1 Average all instrument blanks (PHX) using the peak area values from the TekMDS
software. Subtract the average (IB) from the peak area for each standard and sample.
17.2 Calculate the calibration factor (CFX) for mercury in each of the five standards using
the mean instrument-blank-subtracted peak area and the following equation:
CFX=PAX~IB/CX
Where:
17.2.1 PAx=peak area (or peak height) for mercury in standard
17.2.2 IB= mean peak height (or peak area) for mercury in bubbler blank
17.2.3 Cx=mass in standard analyzed (ng/L)
17.2.4 CFx=Calibration Factor of each concentration
17,2.4.1 Average the five calibration factors to establish mean value: CF(Avg)
(units/ng/L).
17.3 Sample results are then corrected for the average peak area values of at least three
preparation blanks (PBs), unless otherwise requested. This result is shown as the
Initial Result on the Excel spreadsheet and in LIMS.
17.4 Total Mercury in Water:
instrument Value (ng/L) = (Peak Height - BB) / CF(Avg)
Final Result (ng/L) = [(Instrument Value x DF) - (BLK)] x (Vf A/i)
Where:
17.4,1 CF(avg} = average calibration factor for curve (in units/ng/L).
17.4.2 BB = average bubbler blank peak area or peak height (in units)
17.4.3 Vf = Final volume of sample (in mL) from bench sheet.
17.4.4 Vj = initial volume of sample analyzed in mL prior to addition of BrCI.
17.4.5 DF - Dilution Factor - takes into account any instrumental dilution of the
sample
-------
4f*eurofins
Frontier Slobat Sciences
Document Title:
Mercury in Water by Oxidation, Purge & Trap
and CV-AFS (EPA Method 1631, Rev E)
Eurofins Document Reference:
EFGS-SOP-137-R02
17.4.6 BLK = average of the preparation blanks in ng/L.
17.5 A linear regression can be used as alternate calibration. A linear regression will not
change values significantly. If linear regression is used, the correlation coefficient (R)
must be >0.995.
18 Statistical Information/Method Performance:
18.1 The Method Detection Limit (MDL) is determined according to 40 CFR Part 136
Section B. Ten replicates {9 degrees of freedom) spiked 3-10 times the expected MDL
are run. The standard deviation (s) is taken from the resulting data and the MDL is
calculated as follows: MDL=2.821*s. This value should not be interpreted as the
method reporting limit.
18.2 The Practical Quantitation Limit (PQL) is the reporting limit for this method and is
included as the lowest calibration point (2003 NELAC regulation 5.5.5.2.2,1.h.3). The
PQL is determined by running ten samples with a concentration that will produce a
recovery of 70-130 %. The PQL is referred to as the Method Reporting Limit (MRL) in
LIMS.
18.3 Using clean handling techniques and reagents tested low for Hg content, the LOD
value for Total Hg in water is typically less than 0.2 ng/L, while the PQL is 0.50 ng/L.
18.4 Current LODs, LQQs, MDLs, and PQLs are stored at: Cuprum\General and
Admin\Quality Assurance\MDLs & PQLs.
19 Quality Assurance/Quality Control:
19.1 A minimum of three preparation blanks and one LCS/LCSD (preferably NIST 1641d),
must be analyzed per preparation batch. The upper control limit for each preparation
blank is equal to the PQL.
19.2 Matrix Spikes: One Matrix Spike/Matrix Spike Duplicate (MS/MSD) must be performed
for every 10 samples. The recovery of the MS/MSD must be between 71%-125%
recovery, and the Relative Percent Difference (RPD) below 24%. If an MS/MSD is out
of control, the analyst should investigate to identify the source of the failure. The MS
and MSD may be used as duplicates. Some failures may be qualified using QA
Qualification Flow Charts (Appendix A).
19.2.1 For aqueous samples, the MS/MSD is spiked at 1 to 5 times the ambient
concentration, with 0.25 ng, in the bubbler, being the minimum spiking level.
Sample aliquots for the MS/MSD should be the same as the ambient sample
aliquot, if sufficient sample volume exists. Spikes are added to the split aliquots
for volumes of 10mL or greater. For less than 1QmL aliquots, spikes are added
directly to the bubbler. NEVER ADD SPIKE DIRECTLY TO THE ORIGINAL
SAMPLE VESSEL UNLESS OTHERWISE STATED,
19.3 Matrix Duplicates - One Matrix Duplicate (IVID) may be analyzed for every batch of 20
samples. Upon request, a Matrix Triplicate (MT) may be performed. The MSD may
serve as the M.D if necessary. The Relative Percent Difference (RPD) and the
Relative Standard Deviation (RSD) of duplicate samples must be less than 24%. Some
failures may be qualified using QA Qualification Flow Charts.
-------
v^eurofins
Frontier Global Sciences
Document Title;
Mercury in Water by Oxidation, Purge & Trap
and CV-AFS (EPA Method 1631, Rev E)
Eurofins Document Reference:
EFGS-SOP-137-R02
19.3.1 For aqueous samples, analyze the parent, duplicate and triplicate at the same
dilution,
19.4 Laboratory Control Standard (LCS) or Quality Control Sample (QCS): For every batch
of samples, at least one LCS is processed and analyzed. The recovery of the LCS
must be within 80-120% for the aqueous NiST 1641d. An LCS Duplicate (LCSD)
should accompany the LCS.
19.4.1 A Certified Reference Material (CRM) is the preferred LCS, but a Blank Spike
may serve as an LCS if an appropriate CRM does not exist. The spiking level is
based on client request, historical data, or a default of mid-curve. A duplicate
blank spike must also be prepared as an LCSD.
19,5 Ongoing Precision and Recovery (OPR): An OPR must be analyzed at the beginning
and end of each analytical batch, or at the end of each 12-hour shift. The recovery of
the OPR must be within 77-123% to be considered in control,
19.6 All calibration standards must be traceable to the original standard source. The
calibration curve must be established at the beginning of the analytical run. It must
include at least five different concentrations, with the lowest concentration equal to the
PQL. The average response factor of each calibration standard is used to calculate the
sample values. The RSD of the response factors must be less than 15% of the mean
or the calibration fails.
19.7 ICV and CCV control limit is 77-123%. The CCV is analyzed every 10 analyses, and at
the end of an analytical run. CCBs are always analyzed after the CCVs,
19.8 Field Blanks: To be compliant with EPA 1631, clients must submit a field blank for
each set of samples (samples collected from the same site at the same time, to a
maximum of 10 samples),
19.8.1 If no field blanks are submitted by the client, their data will be flagged with "FB-
1631." "Required equipment/field/filter blank not submitted by the client. The
sample has been analyzed according to 1631E, but does not meet 1631E
criteria,"
19.9 Method or Preparation Blanks (BLK): Method blanks are used to demonstrate that the
analytical system is free from contamination that could otherwise compromise sample
results. Method blanks are prepared and analyzed using sample containers, labware,
reagents, and analytical procedures identical to those used to prepare and analyze the
samples.
A minimum of three 1 % BrCI method blanks per analytical batch are required.
Any sample requiring an increased amount of reagent must be accompanied
by at least one method blank that includes an identical amount of reagent.
If the result for any 1 % BrCI method blank is found to contain >0.50 ng/L Hg
(0.25 ng/L for DOD), the system is out of control. Mercury in the analytical
system must be reduced until a method blank is free of contamination at the
0.50 ng/L level.
For method blanks containing more than 1% BrCI, the control limit is equal to
0.50 ng/L multiplied by the final preservation percentage of BrCI. For example,
for a method blank preserved to 2 % BrCI, the control limit for the blank is 0.50
19.9.1
19.9.2
19.9,3
-------
Document Title:
Mercury in Water by Oxidation, Purge & Trap
and CV-AFS (EPA Method 1631, Rev ฃ}
Eurofins Document Reference:
EFGS-SOP-137-R02
ng/L * (102/101), or 0.50 ng/L. For 3% BrCI the control limit is
(103/101 )*0.50ng/L, or0.51ng/L
19.10 Instrument Blanks (IBL): A minimum of three instrument blanks must be analyzed with
each analytical batch. To analyze an instrument blank, attach a clean gold trap to the
bubbler. Purge and analyze as previously described and determine the amount of Hg
remaining in the system.
19.10.1 An instrument blank must be performed on all bubblers used during the
analytical run (normally four, but three at a minimum).
19.10.2 If the instrument blank is found to contain more than 0,50ng/L, the system is
out of control. The problem must be investigated and remedied and the
samples run on that bubbler must be reanalyzed. If the blanks from other
bubblers contain less than 0.50 ng/L, the data associated with those bubblers
remain valid, provided that all other QC criteria are met.
19.10.2.1.1 The mean result for all instrument blanks must be <0.25ng/L with
a standard deviation of 0.10 ng/L.
19,11 The analytical day must close with a CCV/OPR/CCB,
19,12 Because the method is done in real-time, it is EFGS' position that a single non-
compliant QC sample result does not automatically invalidate a data set. Alt data
points that can be explained and rerun with a passing result can be qualified. If the
source of error cannot be corrected for a QC standard that day, none of the data can
be validated, in the event that the system becomes out of control during the analysis
day, all results bracketed between valid QC data points shall still be considered valid
(CCV, OPR, CCB, etc),
19.13 The Control Limits are established from EPA 1631E.
20 Corrective Action
20.1
20.2
20.3
20.4
The data is reviewed as in the QC section (or matrix specific QC section) for all
parameters that pass specific requirements. If the data does not meet QC
requirements it is qualified or submitted for reruns. Data may be qualified (based on
scientific peer review) by the Group Supervisor, Project Manager, Lab Manager or QA
Officer.
Control Chart data is generated through LIMS to monitor the performance of the CCV,
LCS, MS, and MSD. This is done by the QA department.
Due to the real-time nature of the CVAFS method, failures must be investigated as
they happen. If the source of the problem can be identified, and corrected, the samples
may be rerun. If source of problem cannot be isolated, see the Senior Analyst, Group
Supervisor, or Laboratory Manager for instructions.
The Senior Analyst, Group Supervisor, Laboratory Manager, or QA Officer must be
informed if QC fails. It is also advisable to always alert the Project Managers,
-------
4>*eurofins
Document Title:
Mercury in Water by Oxidation, Purge & Trap
and CV-AFS (EPA Method 1631, Rev E)
Eurofins Document Reference:
EFGS-SOP-137-R02
21 List of Attachments
Table 1: QC Requirements for Total Mercury
Appendix A: Example - Standard Operating Procedure Training Record
-------
4?eurofins
Frontier GtofcsS Sciences
Document Title:
Mercury in Water by Oxidation, Purge & Trap
and CV-AFS (EPA Method 1631, Rev E)
Euroflns Document Reference:
EFGS-SOP-137-R02
Tab!ฉ 1: QC Requirements for Total Mercury
Initial Calibration Verification (ICV)
Continuing Calibration Verification (CCV)
Ongoing Precision and Recovery (OPR)
Initial Calibration Blank (ICB)/ Continuing
Calibration Blank (CCB)
Laboratory Control Standard (LCS) or Quality
Control Standard (QCS)
Calibration Curve RSD (Referred to as "Corr.
RSD CF" in Excel spreadsheet).
Lowest Calibration Point
1% BrCi Method Blank (BLK)
Matrix Duplicate (MD) and Analytical Duplicate
(AD)
Matrix Spike and Matrix Spike Duplicate
(MS/MSD) ; Analytical Spike (AS) and
Analytical Spike Duplicate (ASD)
77-123% Recovery
77-123% Recovery
77-123% Recovery
Individually, IBL and CCB <0.50ng/L,
but the mean of all the IBLs shall be
<0.25ng/L with a standard deviation of
0.10ng/L
80-120% Recovery for NIST1641d
and 75-125% for all other CRMs,
RSD<24%
RSD of Calibration Response Factor
<15%
75-125%
Less than 0.50ng/L (0.25ng/L for DOD
projects) (individually)
< 24% RPD
71-125% Recovery
< 24% RPD
-------
eurofms i
I Frontier Global Sciences
Document Title:
Mercury in Water by Oxidation, Purge & Trap
and CV-AFS (EPA Method 1631, Rev E]
Eurofins Document Reference:
EFGS-SOP-137-R02
Appendix A: Example - Standard Operating Procedure Training Record
By signing this document, I the employee, certifies to have read, understood and agreed to follow
the test method and quality procedure as described in this procedure.
Reading of SOP FGS-137.02:
Mercury in Water by Oxidation, Purge & Trap and CV-AFS (EPA Method 1631, Rev E.
SOP name and Revision number
Employee name (print)
Employee name (sign)
Date:
Supervisor name (sign)
Date:
Initial SOP Training (leave blank if not applicable)
Initial reading of method and training
1. Read method
2. Observe the method
3. Detailed review of method and associated literature
4. Supervised practice of method with trainer
5. Unsupervised practice of the method with trainer
6. Review of work with trainer and/or peer-review
7, IDOC to determine precision and accuracy
8. Determination of blanks
Initials
Date
Supervisor
-------
%y eiirafitis |
! Frontier Gioba! Sciences
Document Title:
Digestion Of Tissues for Total Mercury Eurofins Document Reference:
Analysis Using Nitric Acid and Sulfuric EFGS-SOP-01 1 -R05
Acids (70:30)
^W^M^^W^^m'i
' : :-.:.'..: :-.:":>' .;."/. : :-"-" KV'^ ('.:.- '.''- '.' '' ::"'-'~ ': ^V^'^'-' ''.-':"'. :.v' >"
'H^st6Hciiiif/L^ci3l:p6!6u^'^ht--NUi^b^i'^
fe^i^^*;^!^^^ฎ
I rt|^ al rtfsr'i iFYliprif' TTi/i^Oi --. , ' .'.'.../.' ..: '.;
ItvV/ltt'Ol L/ULrUlllCt I L 1 VLJCS ' .. : .:;;-.. ..;:.:
toca I Jocuitieftt :;6ateg-c>rif::::.::\;>^: ;-; K
^^ffi:/;^-:;-^^;v
ApprbVed vby ;-:;.;,'.:;';:
EFGS-SOP-Q11-RQ5 .:Revisteh:v>;V;:i 5
5/20/2013 Status- ;.;;x,:;;;i Final
FGS-SOP-011.05
Level 3
SOP
NA
Ryan Nelson
Dave Wunderlich and Patrick Garcia-Strickiand
-------
eurofins
Document Title:
Digestion of Tissues for Total Mercury
Analysis Using Nitric Acid and Sulfuric
Acids (70:30)
Eurofins Document Reference:
EFGS-SOP-Q11-RQ5
Table of Contents
1 Revision Log: ,, _ _ __ 4
2 Reference: ,, , , , , .....,,,, 4
3 Cross Reference: , , __ _ 4
4 Purpose: , , , , _ 4
5 Scope: _ __.__ 4
6 Basic Principles: ,.,....., 5
7 Reference Modifications: , ,.., 5
8 Definitions: , 5
9 Interferences: , ,,,,, Q
10 Safety Precautions, Pollution Prevention and Waste Handling: Q
11 Personnel Training and Qualifications: _ 7
12 Sample Collection, Preservation, and Handling: _g
13 Apparatus and Equipment: , _ __Q
14 Reagents and Standards: , _ g
15 Procedure: _____ ^0
16 Calculations: , , _____ -JQ
17 Statistical Information/Method Performance: , _,i
-------
eurofins j
Frontier Gioba! Sciences
Document Titfe:
Digestion of Tissues for Total Mercury
Analysis Using Nitric Acid and Suifuric
Acids (70:30)
Eurofins Document Reference:
EFGS-SOP-011-RQ5
Approvals:
Prepared by:
Date:
^Itoift
Approved by: : :L-
Date:
Approved by:
Date:
-------
v^eurofins j
Document Title:
Digestion of Tissues for Total Mercury
Analysis Using Nitric Acid and Sulfurie
Acids (70:30)
Eurofins Document Referenca;
EFGS-SQP-011-R05
1 Revision Log:
liiMstewialte
Seqtibn>>-;;::s:;,:::w::;;.:,:
Cover
Ail
8.9
14.3, 14.4
17.3
18.2-18.5
18.3
lij|^^lili.i^iji^is^^jQi|tl^^l
'^pstlflcdtioti;;iv->:;-s::\;/:/;-yi:i;ff;:v;-r:Tc;w;:
Required change
Formatting requirement per LOW
SOP-LAB-201
Required
Required
Required
Required
Required
^^M^S^'^MMS^^ll^MX^^^^^nv^
.;;ihainpsr:;ew::i:^ j-y^-^-y
Changed company name from Frontier Global Sciences
Eurofins Frontier Global Sciences.
Reformatted document to new corporate specifications.
Updated spiking ievefs for the matrix spike
Updated max contamination levels of reagent acids
Replaced MDL with LOD
Updated QC limits
Incorporated QA MOC 201 1-007
;?ฃ&
:'';: .-:'
to
2 Reference;
2.1
2.2
2.3
Chemical Hygiene Plan, Eurofins Frontier Global Sciences, current version.
EPA Method 1631, Revision E: Mercury in Water by Oxidation, Purge and Trap, and
Cold Vapor Atomic Fluorescence Spectrometry, 2002.
National Environmental Laboratory Accreditation Conference, NELAC Standard
September 8, 2009,
Department of Defense Quality Systems Manual for Environmental Laboratories,
prepared by DoD Environmental Quality Workgroup, Final Version 4.2, October 2010
2.4
3 Cross Reference:
Document
SOP FGS-003
SOP FGS-008
SOP FGS-038
SOP FGS-094, App F
SOP FGS-099
SOP FGS-121
SOP FGS-155
Document Title
Pipette Verification, Calibration and Maintenance
Ultra Clean Aqueous Sample Collection
Data Review and Validation
^tandard Operating Procedure Training Record
Waste Disposal Procedure for Client Sample Waste
Determination of Total Mercury in Various Matrices by
Fluorescence Spectrometry (EPA Method 1631E)
Flow Injection Atomic
Calibration of Volumetric Dispensers
4 Purpose;
4.1 The purpose of this Standard Operating Procedure (SOP) is to describe the method for
digesting biological tissue samples prior to analysis by CV-AFS for total mercury.
5 Scope:
5.1 This method is for the preparation, of biological tissue samples for the determination of
total mercury at concentrations less than 1 ng/g. Through the analysis of smaller
digestate aliquots, contaminated tissues of up to 10,000 ng/g can be directly
measured. Using clean handling techniques and low-level reagents, the typical
detection limit for samples prepared by this method is less than 1 ng/g.
5.2 Total mercury, as defined by this method, is all HNO3/H2S04/BrCI-oxidizable mercury
forms and species found in tissue matrices. This includes, but is not limited to, Hg(ll),
Hg(O), HgS, strongly organo-complexed Hg(II) compounds, adsorbed particulate Hg,
-------
4>*eurofins j
Document Title:
Digestion of Tissues for Total Mercury
Analysis Using Nitric Acid and Su If uric
Acids (70:30)
Eurofins Document Reference:
EFGS-SOP-011-R05
and several covatently bound organo-mercurials (i.e., CH3HgCl, (CH3)2Hg, and
C6H5HgOOCCH3).
6 Basic Principles:
6.1
Samples are collected using clean sample handling protocols into commercially
available clean glass containers with Teflon-lined caps (i.e., l-Chem glass jars) or 125
ml or 250 ml HOPE jars. Freezing (< -15ฐC) preserves tissue samples until sample
preparation is performed.
6.2
6.3
A subsampie of homogenized sample is digested with 10 ml of 70:30 HNO3/H2SO4.
The digested sample is diluted up to 40 mL with 10% (v/v) BrCL
7 Reference Modifications:
7.1 No significant modifications were made to this method.
8 Definitions;
8.1 Batch - no more than 20 client samples grouped for preparation. 3 Preparation Blanks,
1 CRM or 1 LCS/LCSD (or BS/BSD) set and 1 MD are prepared per every 20 samples;
1 MS/MSD set is prepared for every 10 samples.
8.2 Celsius (C), conversion of Celsius to Fahrenheit: (C * 1.8) + 32.
8,3 Fahrenheit (F), conversion of Fahrenheit to Celsius: (F - 32) * 5/9.
8.4 Method Detection Limit (MDL) - the limit derived from an exercise as described in 40
CFR, Part 136, Appendix B. The exercise produces a defined value that is the
minimum concentration that can be measured and reported with 99% confidence that
the analyte concentration is greater than zero from a given matrix.
8.5 Certified Reference Material (CRM) - a standard of known composition that is certified
by a recognized authority and representing a sample matrix. It is used to verify the
accuracy of a method.
8.6 Laboratory Control Sample {LCS) and Laboratory Control Sample Duplicate (LCSD), is
a sample containing known concentrations of the analytes of interest that is taken
through the entire preparation and analysis process in the same manner as the
samples to monitor complete method performance. A Certified Reference Material
(CRM) is preferred as the LCS, but a blank spiked sample also meets the requirement.
8.7 Preparation Blank (BLK) - Method blanks consist of the same reagents used to digest
the samples, in the same volume or proportion, and are carried through the complete
sample preparation and analytical procedure. Teflon boiling chips are added to the
preparation blanks.
8.8 Matrix Duplicate (MD) - a representative sample is selected and digested in the same
manner. This QC sample will indicate sample homogeneity on the analytes of interest
8.9 Matrix Spike (MS) and Matrix Spike Duplicate (MSD) - a representative sample is
selected and spiked with a secondary source at two to five times the ambient
-------
Document Title:
Digestion of Tissues for Total Mercury
Analysis Using Nitric Acid and Sulfuric
Acids (70:30)
Eurofins Document Reference:
EFGS-SOP-011-R05
concentration or at two to five times the MRL, whichever is greater. These QC samples
will indicate sample matrix effects on the analytes of interest,
8.10 May: This action, activity or procedure is optional.
8.11 May Not: This action, activity or procedure is prohibited.
8.12 Shall: This action, activity or procedure is required.
8,13 Should: This action, activity or procedure is suggested, but is not required.
9 Interferences:
9.1 Due to the high levels of halogens (i.e., iodine) typically found in tissue digestates, it is
recommended that aliquots of no more than 5.0 ml_ of the digestate be analyzed.
Otherwise, soda-lime traps may be overloaded and the gold traps may lose the ability
to amalgamate and retain mercury,
9.2 The high acidity and halogen levels that are found in tissue digestates necessitate the
changing of the bubbler water after every 10 ml of digestate analyzed. Failure to do
so can lead to low recoveries that would be reflected in the analysis of QC samples.
10 Safety Precautions, Pollution Prevention and Waste Handling:
10.1 Personnel will don appropriate laboratory attire according to the Chemical Hygiene
Plan. This includes, but is not limited to, laboratory coat, safety goggles, and nitrile
gloves under clean gloves.
10.2 The toxicity or carcinogentcity of reagents used in this method has not been fully
established. Each chemical should be regarded as a potential health hazard and
exposure to these compounds should be as low as reasonably achievable. Chemists
should refer to the MSDS (Material Safety Data Sheets) for each chemical they are
working with.
10.2.1 Note: Use particular caution when preparing and using BrCi, as it releases
extremely irritating, corrosive fumes similar in effect to free chlorine. Always
handle this reagent in an approved fume hood.
10,2.2 Note: Use particular caution when preparing and using the Nitric/Sulfuric Mixture.
Always handle this reagent in an approved fume hood.
10.3 AH personnel handling environmental samples known to contain or to have been in
contact with human waste should be immunized against known disease-causative
agents. Eurofins Frontier will reimburse the expense of Hepatitis A and B
immunizations for any laboratory staff member who desires this protection.
10.4 Nitric acid (HNO3): Corrosive. Strong oxidizer. Contact with other material may cause a
fire. Causes eye and skin burns. May cause severe respiratory tract irritation with
possible burns. May cause severe digestive tract irritation with possible burns. For
more information see MSDS.
10.5
Sulfuric acid (H2SO4): Corrosive. Causes eye and skin burns. May cause severe eye
irritation with possible burns. May cause severe respiratory tract irritation with possible
burns. May cause severe digestive tract irritation with possible burns. Cancer hazard.
-------
4?eurofitis j
Document Title:
Digestion of Tissues for Total Mercury
Analysis Using Nitric Acid and Sulfuric
Acids (70:30)
Eurofins Document Reference:
EFGS-SOP-011-R05
Animal studies suggest this acid may cause fetal effects. May cause kidney damage.
May cause lung damage. May be fatal if inhaled. Hygroscopic. Strong oxidizer.
Contact with other material may cause a fire. For more information see MSDS.
10.5.1
Eyes: Get medical aid immediately. Do NOT allow victim to rub or keep eyes
closed. Extensive irrigation with water is required (at least 30 minutes).
10.5.2 Skin: Get medical aid immediately. Flush skin with soap and water for at least 15
minutes while removing contaminated clothing and shoes. Wash clothing before
reuse. Destroy contaminated shoes.
10.6 See Eurofins Frontier Global Sciences Chemical Hygiene Plan (CHP) for general
information regarding employee safety, waste management, and pollution prevention.
10.7 Pollution prevention information can be found in the current Eurofins Frontier Global
Sciences Chemical Hygiene Plan (CHP), which details and tracks various waste
streams and disposal procedures.
10.8 All laboratory waste is accumulated, managed, and disposed of in accordance with all
federal, state, and local laws and regulations. Any waste generated by this procedure
should be disposed of according to SOP FGS-099 "Waste Disposal Procedure for
Client Sample Waste," which provides instruction on dealing with laboratory and client
waste.
11 Personnel Training and Qualifications:
11,1 An analyst must perform an initial demonstration of capability (IDOC) that includes four
replicates of a secondary source before being qualified to analyze samples without
supervision. Continuing DOC will be maintained and monitored via performance on
CRMs and other QC samples, as well as obtaining acceptable results on proficiency
testing exercises.
11,2 The analyst/laboratory technician must have read this SOP and other relevant SOPs
and have the training documented on the applicable form(s). The analyst may be
questioned on SOP by supervisor(s) and/or trainers.
11.3 Training is documented by the employee and supervisor, and is kept on file in the QA
Office. The employee must read, understand, and by signing the training document,
agree to perform the procedures as stated in all Standard Operating Procedures
(SOPs) related to this method.
11.4 Reading of the SOP must be documented on the correct form such as "Standard
Operating Procedure Training Record," Appendix F in FGS-094, the last page of this
SOP, Appendix A "Standard Operating Procedure Training Record" or a similar
document"
11.5 All employees must also, on a yearly basis, read the Quality Manual (QM), and
complete the yearly Ethics training.
11,6 All training documents including IDOCs, CDOCs, SOP reading, Initial QA orientation,
and Ethics training are stored by the Quality Assurance Manager in the employees
training file for ten years after the employee is no longer working for Eurofins Frontier
Global Sciences.
-------
frontier GSofoal Sciences
Document Title:
Digestion of Tissues for Total Mercury
Analysis Using Nitric Acid and Sulfuric
Acids (70:30)
Eurofins Document Reference:
EFGS-SOP-011-RG5
11.7 Chemical Safety Training, Compressed Gas Training, Chemical Hygiene Plan
documentation, and Shipping of Hazardous goods, are stored by the Health and
Safety Officer for ten years after the employee is no longer working for Eurofins
Frontier Global Sciences.
12 Sample Collection, Preservation, and Handling:
12,1 Samples must be collected in accordance with established ultraclean sampling
techniques (see FGS-008 "Ultra Clean Aqueous Sample Collection"). Samples may
be in commercially available clean glass containers with Teflon-lined caps (i.e., l-Chem
glass jars), or 125 ml or 250 ml HOPE jars.
12.2 Tissue sample preservation - The tissue sample must be frozen in the sampling
container at less than -15ฐC or freeze-dried and stored at room temperature. The
holding time for tissue samples is 1 year.
12.3 Just prior to digestion, samples are thawed and if necessary homogenized. The
sample is well mixed to ensure the most representative sample possible.
13 Apparatus and Equipment:
13.1 LIMS ~ Element, version 5.85 or higher; Computer - Windows XP, 7 or 8
13.2 40 ml or 20 ml l-Chem Vials: Borosiiicate glass, series 300 viais with Teflon-fined
septa in lids. The size used depends on the amount of sample available. The vials are
volumetrically accurate to ฑ 0.5 ml when filled such that the meniscus is just to the
bottom of the vial neck. The person performing the preparation should verify this.
13.3 Hot plate: A hot plate with the ability to achieve and maintain a temperature of 75 ฐC.
13.4 Pipettors: All-plastic, pneumatic, fixed volume and variable pipettes in the range of 5
to 10 mL Pipettes are to be calibrated weekly according to SOP FGS-003 and
13.5
FGS-155.
Clean hood.
13.6 Analytical Balance: A laboratory analytical balance capable of weighing to ฑ 1 mg, with
documented calibration.
13.7 Calibrated thermometer: Submerged in water in a 20 ml i-Chem vial. This vial is
placed on the hotplate during the digestion process. The analysts must record the
actual digestion temperature and the serial number of the thermometer used in the
digestion logbook.
13.8 Sample Digestion Log.
13.9 Stainless steel tools for homogenization
13.10 Tissue Homogenization Log.
13.11 Disposable spatula.
13.12 Teflon boiling chips.
13.13 Teflon reflux cap to fit the 40 ml and 20 ml l-Chem vials.
-------
4? eu refills |
Document Title:
Digestion of Tissues for Total Mercury
Analysis Using Nitric Acid and Sulfuric
Acids (70:30)
Eurofins Document Reference:
EFGS-SOP-011-R05
14 Reagents and Standards:
14.1 Reagent Water: 18 MQ ultra-pure deionized water starting from a pre-purified (distilled,
R.O., etc.) source. As a final mercury and organic removal step, the activated carbon
cartridge on the 18-MQ system is placed between the final ion exchange bed and the
0.2 Mm filter.
14.2 Nitric Acid (HNOs): Trace metal purified reagent-grade HNO3 is pre-analyzed and lot
sequestered. Several brands (Baker, Fisher, Omnitrace) have been found to have lots
with acceptably low levels of trace metals. This reagent should be from a lot number
that has been previously tested to be low for the analytes of interest. This reagent shall
be entered into LIMS and the expiration date is set to the same as the manufacturer's
expiration date.
14.3 Sulfuric acid (H2SO4) - Trace metal purified reagent-grade H2SO4 is pre-analyzed to <
50 ng/L Hg and lot sequestered before purchase. This reagent shall be entered into
the LIMS and is considered stable until the expiration date on the bottle (set by the
manufacturer,
14.4 Nitric/Sulfuric Acid Mixture: Carefully add 300 ml of pre-analyzed, (ow mercury (< 50
ng/L) concentrated sulfuric acid to 700 mL of pre-analyzed, low mercury concentrated
nitric acid to a pre-marked Teflon bottle. Stir constantly. This reagent shall be entered
into the LIMS with an expiration date of six months. CAUTION: THIS MIXTURE
BECOMES VERY HOT AND EMITS CAUSTIC FUMES.
14,5
14.6
Potassium Bromide (KBr), neat: this reagent is pre-certified by the vendor to be low in
mercury and is entered into the LIMS with a five year expiration date.
Potassium Bromate (KBrO3), neat: this reagent is pre-certified by the vendor to be low
in mercury and is entered into the LIMS with a five year expiration date.
14.7 0.2N Bromine Monochloride (BrCI):
14.7.1
14.7.2
14.7.3
37.5 g of KBr is added to a 2.5 L bottle of concentrated HCl (pre-analyzed and
below 5 ng/L Hg). The bottle is inverted in a fume hood to mix the acid and KBr.
The solution sits overnight, allowing the KBr to dissolve,
27.5 g of KBrOS (certified to be low in Hg) is slowly added to the acid. As the
KBrO3 is added, the solution should go from yellow to red to orange.
CAUTION: This process generates copious quantities of free halogens {CI2,
Br2, BrCI) which are released from the bottle. Add the KBrO3 SLOWLY in a
well operating fume hood,
Loosely cap the bottle and allow to sit for 30 minutes (in a fume hood) before
tightening. Once tightly capped, invert bottle to make sure all of the solids go into
solution.
14.7.4 This reagent shall be entered into the LIMS with a six month expiration date.
14.8 10% (v/v) of 0.2N BrCI: 200 mL of 0.2N BrCI is diluted up to 2,0 L with reagent water in
a clean, empty HCl bottle. This bottle is fitted with a 10 mL repipettor. The expiration
time for this reagent is set by default to six months in the LIMS.
-------
4>*eurof!n$
Document Title:
Digestion of Tissues for Total Mercury
Analysis Using Nitric Acid and Suifuric
Acids (70:30)
Eurofins Document Reference:
EFGS-SOP-011-R05
15 Procedure;
15.1
If needed, the sample is dissected and homogenized with acid-washed stainfess steel
tools.
15.11 The process used for homogenization, number of samples, work order number,
client name, and initials of the technician are entered into the Tissue
Homogenization Log,
15.2 Weigh at least a 0.5 g aliquot (but not more than 0.65 g) for common and unknown
samples, and up to 1.0 g ฑ 0.025 g for low-level or large-grain samples. This aliquot is
placed into a 40 ml l-Chem glass vial.
15.2.1 tf limited sample is available, use 20 ml_ glass vials and drop the initial mass of
the samples to 0.25g ฑ 0.025 g.
15.2.2 It is imperative that all biological tissue samples are thoroughly homogenized.
The importance of representativeness cannot be understated.
15.2.3 Batch requirements for this digestion limit the number of samples to 20. In each
batch, there must be three method blanks (BLKs), a Blank Spike and Blank Spike
Duplicate (BS/BSD) that is preferably a Certified Reference Material (CRM) or a
Laboratory Control Spike (LCS, prepared at 8 ng/g), a Matrix Duplicate (MD), and
a Matrix Spike and Matrix Spike Duplicate (MS/MSD).
10.0 ml of 70:30 (v/v) HN03/H2SO4 solution is pipetted in and the sample is swirled.
Note: 5,0 ml of 70:30 (v/v) HA/O/H2SO4 solution is used for limited samples prepared
in 20 mL vials (15.2.1),
15.3
15.4 The vial is placed on a hot plate operating at 75ฑ5ฐC with a Teflon reflux can in place
instead of the vial's lid. An aluminum rack id often used to keep the vials from tipping
over while on the hot plate.
15.4.1 A calibrated thermometer submerged in water is placed in a 20 ml l-Chem vial.
This l-Chem vial with a calibrated thermometer is placed on the hot plate during
the digestion process. The analysts must record the actual digestion
temperature and the serial number of the thermometer used in the digestion
logbook.
15.5 After the samples start to reflux, the samples are heated at 75ฑ5ฐC for an additional 2
hours or until all organic matter is dissolved.
15.6 The samples are allowed to cool and are diluted to 40 ml {or to 20 mis for limited
sample digestions as described in 15.2.1) with a 10% (v/v) solution of 0.2N BrCI,
capped with their respective lids, and are thoroughly shaken. Sample digestates
should be allowed to settle prior to an aliquot being taken for analysis.
15.7 Analysis for total mercury is according to Eurofins Frontier SOP FGS-121.
16 Calculations:
16.1 This preparation procedure does not involve calculations.
-------
^eurofins i
Document Title:
Digestion of Tissues for Total Mercury
Analysis Using Nitric Acid and Sulfuric
Acids (70:30)
Eurofins Document Reference;
EFGS-SOP-011-R05
17 Statistical information/Method Performance:
17.1 Method Detection Limit (MDL) and Practical Quantitation Limit (PQL) studies are
based on 40 CFR 136, Appendix B. The MDL and PQL must be performed for each
analyte/rnatrix/preparation combination.
17.2 The Practical Quantitation Limit (PQL) is the reporting limit for this method and is
included as the lowest calibration point (2003 NELAC regulation 5.5.5.2.2.1.h.3). The
PQL is determined by running ten replicate samples with a concentration that wilt
produce a recovery of 70-130% for most analytes, but the recovery requirements are
analyte dependent. The PQL is referred to as the Method Reporting Limit (MRL) in
LIMS.
17.3 The current LOD value for Total Hg in tissue prepared by the Nitric and Sulfuric Acids
(70:30) Digestion is 0.16 ng/g, while the PQL is 0.8 ng/g.
17.4 Current LODs and PQLs are stored at: \Genera! and Admin\Quality Assurance\MDLs
& PQLs.
18 Quality Assurance/Quality Control:
18,1 Maximum Sample Batch Size: 20 samples.
18.2 Preparation Blanks: Minimum of three per batch. Each preparation blank must be less
than one-half the PQL for the method.
18.2.1 The preparation blanks are prepared with a similar mass of Teflon boiling chips
as the samples, with the same reagents, and put through the same preparation
process as the samples.
18,3 Certified Reference Material (CRM, representing the sample matrix when commercially
available); a Laboratory Control Spike (LCS) and Laboratory Control Spike Duplicate
(LCSD) prepared at 8 ng/g is used when a suitable CRM is not available: One per
batch in duplicate. The control limits are 77-123% recovery.
18.4 Matrix Duplicate (MD) Sample: One per batch. The control limit for the RPD is < 24%.
18.5 Matrix Spike/Matrix Spike Duplicate (MS/MSD) Samples: One set per 10 samples. The
control limits are 71-125% recoveries and an RPD of < 24%.
18.6 Follow the flow charts in SOP FGS-038 "Data Review and Validation" to determine if
any QC falling outside the established control limits can be qualified.
18.7 All of the quality control limits for the analysis method are included on the "Data
Review Checklist
18.7.1 The data review checklists are located at: \\cuprum\General and Admtn\Quality
Assurance\Data Review\Current Data Review Checklists.
19 Corrective Action:
19.1 Limiting the source of contamination/error in the preparatory stage can decrease QC
problems during analysis. Limiting such contamination/error sources may include:
cleaning all digestion tools in a 10% HCI solution, ensuring all samples are thoroughly
homogenized, changing gloves whenever appropriate, flushing repipettors at least
Revision: 5
-------
Document Title:
Digestion of Tissues for Total Mercury
Analysis Using Nitric Acid and Sulfuric
Acids (70:30)
Eurofins Document Reference:
EFGS-SOP-011-R05
three times before dispensing into vials and, in general, following ultra-clean
procedures.
19.2 A failing QC point does not necessary fail the entire dataset. If upon analysis a QC
sample is out of control, some investigation must be performed to assess if the
difficulties are related to matrix effects. The cause and method of determining the set's
failure must be documented on the checklist and in the MMO notes, and the Group
Supervisor shall be informed. See SOP FGS-038 "Data Review and Validation" for
flow charts regarding analytical issues,
19.3 Additional corrective actions are listed in the SOP for total mercury analysis (Eurofins
Frontier SOP FGS-121).
20 List of Attachments
Appendix A: Example - Standard Operating Procedure Training Record
-------
Document Title:
Digestion of Tissues for Total Mercury
Analysis Using Nitric Acid and SuSfurie
Acids (70:30)
Eurofins Document Reference:
EFGS-SOP-011-R05
Appendix A: Example - Standard Operating Procedure Training Record
By signing this document, I the employee, certifies to have read, understood and agreed to follow
the test method and quality procedure as described in this procedure.
Reading of SOP EFGS-011.05:
Digestion of Tissues for Total Mercury Analysis Using Nitric and Suifuric Acids (70:30).
SOP name and Revision number
Employee name (print)
Employee name (sign)
Date:
Supervisor name (sign)
Date:
Initial SOP Training (leave biank if not applicable)
Initial reading of method and training
1. Read method
2. Observe the method
3. Detailed review of method and associated literature
4. Supervised practice of method with trainer
5. Unsupervised practice of the method with trainer
6. Review of work with trainer and/or peer-review
7. IDOC to determine precision and accuracy
8. Determination of blanks
Initials
Date
Supervisor
------- |