National Rivers and Streams Assessment 2018/19 Laboratory Operations Manual Version 1.1


rtED STAJ
United States Environmental Protection Agency
Office of Water
Washington, DC
EPA 841-B-17-004
National Rivers and Streams Assessment
2018/19
Laboratory Operations
Manual
Version 1,1 June 2018

-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page ii of 185
NOTICE
The intention of the National Rivers and Streams Assessment 2018-2019 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 2018-19: Quality Assurance Project Plan EPA-841-B-17-001
National Rivers and Streams Assessment 2018-19: Site Evaluation Guidelines EPA-841-B-17-002
National Rivers and Streams Assessment 2018-19: Non-Wadeable Field Operations Manual EPA-841-B-
17-003a
National Rivers and Streams Assessment 2018-19: Wadeable Field Operations Manual EPA-841-B-17-
003b
National Rivers and Streams Assessment 2018-19: Laboratory Operations Manual EPA-841-B-17-004
Addendum to the National Rivers and Streams Assessment 2018-19: 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 2018-2019. 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. 2017. National Rivers and Streams Assessment 2018-19: Laboratory Operations Manual. EPA-
841-B-17-004. U.S. Environmental Protection Agency, Office of Water, Washington, DC.

-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page iii of 185
TABLE OF CONTENTS
NOTICE	ii
TABLE OF CONTENTS	iii
LIST OF TABLES	vi
LIST OF FIGURES	vii
LIST OF ACRONYMS	vii
1.0 INTRODUCTION	9
2.0 GENERAL LABORATORY GUIDELINES	10
2.1	Responsibility and Personnel Qualifications	10
2.2	Roles and Contact Information	10
2.3	Sample T racking	10
2.4	Reporting	11
3.0 ALGAL TOXIN: CYLINDROSPERMOPSIN IMMUNOASSAY PROCEDURE	12
3.1	Definitions and Required Personnel Qualifications	12
3.1.1	Definitions	12
3.1.2	Personnel Qualifications	13
3.2	Precautions	13
3.3	Equipment/Materials	14
3.4	Sample Receipt	14
3.5	Procedure	15
3.5.1	Sample Preparation	15
3.5.2	Kit Preparation	16
3.5.3	Insertion of Contents into Wells	17
3.5.4	Dilutions (if needed)	21
3.6	Pertinent QA/QC Procedures	22
3.6.1	QC Samples	22
3.6.2	Summary of QA/QC Requirements	23
4.0 ALGAL TOXIN: MICROCYSTIN IMMUNOASSAY PROCEDURE	25
4.1	Summary of the Procedure	25
4.2	Health and Safety Warnings	25
4.3	Definitions and Required Resources (Personnel, Laboratories, and Equipment)	25
4.3.1 Definitions	25
4.4	General Requirements for Laboratories	27
4.4.1	Expertise	27
4.4.2	Quality assurance and quality control requirements	27
4.4.3	Personnel	27
4.4.4	Equipment/Materials	27
4.5	Sample Receipt	28
4.6	Procedure	29
CO
4.6.1	Sample Preparation	29	\—
4.6.2	Kit Preparation	29	llj
4.6.3	Insertion of Contents into Wells	30	2
4.6.4	Dilutions (if needed)	35	u
4.7	Quality Measures	36	q
4.7.1	Assist an ce Visits	36	u-j
4.7.2	QC Samples	36	^
iii

-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page iv of 185
4.7.3 Summary ofQA/QC Requirements	36
4.8	Sample and Record Retention	38
4.9	References	38
5.0 BENTHIC MACROINVERTEBRATES	39
5.1	Introduction	39
5.2	Summary of Method	39
5.3	Health and Safety Warnings	39
5.4	Definitions and Required Resources (Laboratory, Personnel, and Equipment)	40
5.4.1	Definitions	40
5.4.2	Laboratory	41
5.4.3	Personnel	42
5.4.4	Equipment/Materials	43
5.4.4.1	Sample Preparation (Subsampling) and Sorting Equipment/Materials	43
5.4.4.2	Taxonomy Identification Equipment/Materials	44
5.5	Sample Receipt	44
5.6	Subsampling	45
5.7	Sorting	47
5.8	Taxonomy Identification	50
5.9	Data Entry	55
5.10	Sample and Record Retention	55
5.11	External Taxonomic Quality Control	55
5.12	Quality Assurance/Quality Control (QA/QC)	57
5.13	References	60
6.0 FECAL INDICATOR: ENTEROCOCCI	61
6.1	Scope & Application	61
6.2	Summary of Method	61
6.3	Definitions of Method	61
6.4	Interferences	62
6.5	Health & Safety Warnings	62
6.6	Personnel Qualifications	63
6.7	Equipment and Supplies	63
6.8	Reagents & Standards	63
6.9	Preparations Prior to DNA Extraction & Analysis	63
6.10	Procedures for Processing & qPCR Analysis of Sample Concentrates	65
6.10.1	Sample Processing (DNA Extraction)	65
6.10.2	Sample Analysis by Enterococcus qPCR	66
6.10.2.1 Preparation of qPCR assay mix	66
6.10.3	Sample analysis sequence for SmartCycler	68
6.11	Storage & Timing of Processing/Analysis of Filter Concentrates	68
6.12	Chain of Custody	68
6.13	Quality Assurance/Quality Control (QA/QC) Procedures	68
6.14	Method Performance	69
6.15	Record Keeping & Data Management	69
6.16	Waste Management & Pollution Prevention	69	^
6.17	Literature Cited	70	z
6.18	Tables, Diagrams, Flowcharts, Checklists, and Validation Data	70	h
6.18.1	Enterococcus qPCR Analysis Decision Tree (ADT)	73	o
6.18.2	"Modified" MagNA Pure LC DNA Purification Kit III Protocol	73	^
O
7.0 FISH VOUCHER SPECIMENS	76	l±j
	I
CO
7.1 Summary of Procedures	76	<
iv

-------
2018/19 National Rivers & Streams Assessment	Laboratory Operations Manual
Version 1.1, June 2018	Page v of 185
7.2	Health and Safety Warnings	76
7.3	Definitions and Required Resources (Personnel, Taxonomy Laboratories, and Equipment)	77
7.3.1	Definitions	77
7.3.2	General Requirements for Taxonomists and Taxonomy Laboratories	78
7.3.3	Personnel	78
7.3.4	Equipment/Materials	79
7.4	Sample Receipt	79
7.5	QC Identification	81
7.6	Assistance Visits	85
7.7	Sample and Record Retention	85
7.8	Summary of QC Requirements for Fish Voucher Specimens	85
7.9	References	87
8.0 FISH TISSUE FILLET (Whole Fish Collection)	89
9.0 FISH TISSUE PLUG	90
10.0 DIATOMS	92
10.1	Summary of Procedure	92
10.2	Health and Safety Warnings	92
10.3	Required Equipment	92
10.3.1	Laboratory	92
10.3.2	Equipment/Materials	93
10.3.2.1	Subsampling Equipment/Materials	93
10.3.2.2	Diatoms	94
10.3.2.2.1	Preparation of Diatom Slides	94
10.3.2.2.2	Analysis of Diatoms	94
10.4	Sample Receipt	94
10.5	Sample Preparation	96
10.6	Diatom Cleaning and Mounting	96
10.7	Analysis of Diatoms	102
10.7.1	Creation of Pre-Count Regional Voucher Flora and Develop Diatom Analyst Schedule	102
10.7.2	Diatom Sample Count	106
10.7.3	Internal Quality Control	108
10.8	Data Entry	109
10.9	Sample and Record Retention	109
11.0 PERIPHYTON BIOMASS	110
12.0 PERIPHYTON META-GENOMICS (Research Effort)	112
13.0 WATER CHEMISTRY and CHLOROPHYLL A	113
13.1	Analytical Parameters	
13.2	Sample Processing and Preservation	
13.2.1	Water Chemistry Samples	
13.2.2	Chlorophyll-a Samples	
13.3	Performance-based Methods	
13.4	Pertinent QA/QC Procedures	
13.4.1	Laboratory Performance Requirements	
13.4.2	Laboratory Quality Control Samples	
13.4.3	Data Reporting, Review, and Management
13.5	Literature Cited	
APPENDIX A: CONTACT INFORMATION	126	,1,
	I
APPENDIX B: LABORATORY REMOTE EVALUATION FORMS	127	<
.113
.114
.115
.116
.116
.118
.118
.118
.123
.125
O
u
V

-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page vi of 185
APPENDIX C: SAMPLE LABORATORY FORMS	134
Benthic Macroinvertebrate: Sorting Bench Sheet	135
Benthic Macroinvertebrates: Taxonomy Bench Sheet (optional)	136
Enterrococci (EPA Method 1606) Laboratory Bench Sheet	137
Fish Voucher: Taxonomy Bench Sheet (optional)	138
APPENDIX D: OTHER PERTINENT ATTACHMENTS	139
APPENDIX E: REPORTING TEMPLATES	167
APPENDIX F: EXAMPLE SOP FOR ASH FREE DRY MASS ANALYSIS OF PERIPHYTON BIOMASS	168
APPENDIX G: EXAMPLE SOPS FOR MERCURY IN FISH TISSUE PLUG ANALYSIS	185
LIST OF TABLES
Table 2.1 Contact information	10
Table 3.1 Cylindrospermopsin: required data elements- data submission	19
Table 3.2 Cylindrospermopsin: quality control- sample analysis	23
Table 4.1 Microcystis required data elements-data submission	33
Table 4.2 Microcystis quality control-sample analysis	36
Table 5.1 Benthic macroinvertebrate: required data elements-login	45
Table 5.2 Benthic macroinvertebrate: list of taxa that are not to be sorted/counted	48
Table 5.3 Benthic macroinvertebrate: required data elements-sorting	49
Table 5.4 Benthic macroinvertebrate: target level of taxonomic identification - benthics commonly found in
FRESHWATER	52
Table 5.5 Benthic macroinvertebrate: target Level of taxonomic identification - chironomidae	53
Table 5.6 Benthic macroinvertebrate: required data elements - taxonomic identification	54
Table 5.7 Benthic macroinvertebrate: measurement data quality objectives	58
Table 5.8 Benthic macroinvertebrate: quality control-laboratory	59
Table 6.1 Enterococci: PCR assay mix composition (according to draft EPA method 1606)	70
Table 6.2 Enterococci: batch calibrator & enterococcus standards PCR run - 7 samples	70
Table 6.3 Enterococci: sub batch test sample PCR run-26 samples & 1 method blank	70
Table 6.4 Enterococci: laboratory methods	71
Table 6.5 Enterococci: parameter measurement data quality objectives	72
Table 6.6 Enterococci: laboratory QC procedures - enterococci DNA sequences	72
Table 7.1 Fish voucher: required data elements-login	80
Table 7.2 Fish voucher: required data elements - data submission worksheet	84
Table 7.3 Fish voucher: measurement data quality objectives	86
Table 7.4 Fish Voucher: quality control-taxonomic identification	87
Table 7.5 Fish voucher: data validation	87
Table 9.1 Fish tissue plug: measurement data quality objectives	
Table 9.2 Fish tissue plug: quality control	
Table 10.1 Required data elements for diatom sample login	
Table 10.2 Required data elements for diatom subsampling	
Table 10.3 Required data elements for diatom cleaning & mounting	
Table 10.4 Diatom sample analysis required data elements	
Table 11.1 Quality assurance measures for AFDM	
Table 11.2 Data field requirements from labs for AFDM	
Table 13.1 Water chemistry: NRSA 2018/19 parameters	
Table 13.2 Water chemistry: acid preservatives added for various analytes	
Table 13.3 Water chemistry: NRSA 2018/19 analytical methods (Central Laboratory, EPAORD-Corvallis)
...90
...90
...95
...96
.101
.108
.110
.111
.113
.116
.117
CO
<
o
I—
LD
vi

-------
2018/19 National Rivers & Streams Assessment	Laboratory Operations Manual
Version 1.1, June 2018	Page vii of 185
Table 13.4 Water chemistry & chlorophyll-a: laboratory method performance requirements	119
Table 13.5 Water chemistry: quality control-laboratory samples	121
Table 13.6 Water chemistry: quality control-data validation	123
Table 13.7 Water chemistry: data reporting criteria	123
Table 13.8 Water chemistry: constants for converting major ion concentration from mg/Lto heq/L	124
Table 13.9 Water chemistry: factors to calculate equivalent conductivities of major ions	125
Table D.0.1 Fish identification: standard common and scientific names	140
LIST OF FIGURES
Figure 3.1 Cylindrospermopsin: sample template	17
Figure 4.1 Microcystis sample template	31
Figure 6.1 Enterococci: qPCR analysis decision tree (ADT)	73
Figure 7.1 Fish voucher: example QC fish taxonomy bench sheet (optional)	88
Figure 10.1 Example of light micrograph image after rotation, histogram, and scale bar processing	104
Figure 10.2 Example plate of a regional voucher flora collected before diatom sampling counting has begun	105
Figure 13.1 Water chemistry: sample processing procedures	115
LIST OF ACRONYMS
A
absorbance
EMAP
Environmental Monitoring and Assessment
ADT
analysis decision tree

Program
AFDM
ash-free dry mass
ENT
enterococci
ANC
acid neutralizing capacity
EPA
Environmental Protection Agency
ANS
Academy of Natural Sciences
ETOH
ethyl alcohol
AQM
absolute quantitation method
FOM
Field Operations Manual
ASTM
American Society for Testing and Materials
GEQ
genomic equivalent
Avg
Average
GIS
geographic information system
BHI
brain heart infusion
GPS
global positioning device
Ca
Calcium
HCI
hydrogen chloride
CCE
calibrator cell equivalents
HDPE
high density polyethylene
CEQ
cell equivalent
hno3
nitric acid
Chl-a
chlorophyll-a
HQ
headquarters
C02
carbon dioxide
HRP
antibody-Horseradish Peroxidase
Ct
threshold cycle
H2S
hydrogen sulfide
CPR
cardiopulmonary resuscitation
H2S04
sulphuric acid
cv
curriculum vitae
IBD
ionic balance difference
CV
coefficent of variation
ID
Identification
DCF
dilution/concentration factor
IM
information management
Dl
de-ionized
IPC
internal positive control
DIC
differential interference contrast
ISBN
International Standard Book Number
DL
detection limit
ISO
International Organization for
DNA
Deoxyribo-nucleic Acid

Standardization
DO
dissolved oxygen
IT IS
Integrated Taxonomic Information System
DOC
dissolved organic carbon

(IT IS)
DTH
depositional targeted habitat
K
potassium
DW
distilled water
KC
kit control
ELISA
enzyme-linked Immunosorbent assay
LFB
lab fortified blanks


LFM
lab fortified matrices

-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
LIMS
Laboratory Information Management

System
LOM
Lab Operations Manual
LRL
lower reporting limit
Mg
magnesium
MDL
method detection limit
MPCA
Minnesota Pollution Control Agency
MSDS
Materials Safety Data Sheet
N
nitrogen
Na
sodium
NABS
North American Benthological Society
NALMS
North American Lakes Management Society
NARS
National Aquatic Resource Surveys
NAWQA National Water Quality Assessment

Program
NC
negative control
ND
nondetect
NELAC
National Environmental Laboratory

Accreditation Conference
NELAP
National Environmental Laboratory

Accreditation Program
nh4
ammonium
NIST
National Institute of Standards
N02
nitrite
N03
nitrate
NRSA
National Rivers and Streams Assessment
NTL
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
Laboratory Operations Manual
Page viii of 185
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
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
S0-S5	standards provided with microcystin kit
SFS	Society of Freshwater Science
SEG	Site Evaluation Guidelines
Si02	silica
S04	sulphate
SOPs	Standard Operating Procedures
SPC	sample processing control
S-R	Sedgewick-Rafter count
SRM	standard reference material
SS	salmon sperm
TMB	tetramethylbenzidine
TN	total nitrogen
TOC	total organic carbon
TP	total phosphorus
TRANS	transect
TSN	taxonomic serial number
TSS	total suspended solids
TVS	total volatile solids
UNK	unknown
USGS	United States Geological Survey
WSA	Wadeable Streams Assessment
WQX	Water Quality Exchange

-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 9 of 185
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, the NRSA 2008/09,
and NRSA 2013/2014
•	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.
EPA used an unequal probability to create two separate designs to address the dual objectives of (1)
estimating current status and (2) estimating change in status for all flowing waters. There are 825 new
unique sites selected to estimate current satus and 983 unique sites that are resamples from the 2004
Wadeable Streams Assessment, the NRSA 2008/09, and/or NRSA 2013/2014. 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.

-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 10 of 185
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 2018-2019
sample that involves post-processing. The coordinator will be the labs main point of contact in regard to
sample tracking and data submission.
Table 2.1 Contact information
EPA HQ NRSA Project Lead
Richard Mitchell, OW
mitchell.richardPepa.gov
202-566-0644
EPA HQ NRSA Project QA
Lead
Sarah Lehmann, OW
lehmann.sarah(® eoa.gov
202-566-1379
EPA HQ NRSA Laboratory
Review Manager
Kendra Forde, OW
kendra.forde@epa.gov
202-564-0417
Information Management
Center Coordinator
Marlys Cappaert, SRA
International Inc.
cappaert. marlvs(®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 5
system, where they can acquire tracking numbers and information on samples that have been shipped g
to them by field crews (either by overnight shipment for perishable samples or batch shipments for 5
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 q
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 O
CO
Coordinator, listed above, to ensure access is granted. <
I
When the samples arrive from the field crews, laboratories should also receive tracking forms in the ^
shipment (refer to the NRSA 2018-2019 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 S
10

-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 11 of 185
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, 2020 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: 2018_11_01)
• Lab name (ex: MyLab)
Combined, the file name would look as follows: WaterChemistry_2018_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 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.

-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 12 of 185
3.0 ALGAL TOXIN: CYLINDROSPERMOPSIN IMMUNOASSAY PROCEDURE
This chapter describes an immunoassay procedure that measures concentrations of total
cylindrospermopsin in water samples. In applying the procedure, the laboratory uses Abraxis'
Cylindrospermopsin Test Kits ("kits"). Each kit is an enzyme-linked immunosorbent assay (ELISA) for the
determination of cylindrospermopsin in water samples.
Frozen cylindrospermopsin samples will be shipped on dry ice from the field crews to the contract
batching laboratory. The contract batching laboratory will freeze samples and send the batched samples
to the analysis laboratory in coolers on dry ice. Samples will arrive in the analysis laboratory frozen and
they can be held in a freezer for several weeks. Cylindrospermopsin analysis laboratories will need to
process samples in accordance with the time frame outlined in contractual agreements.
The procedure is an adaption of the instructions provided by Abraxis for determining total
cylindrospermopsin concentrations using its ELISA kits. For freshwater samples, the procedure's
reporting range is 0.1 ng/Lto 2.0 ng/L, although, theoretically, the procedure can detect, not quantify,
cylindrospermopsin concentrations as low as 0.05 ng/L. For samples with concentrations higher than 2.0
Hg/L of cylindrospermopsin, the procedure includes the necessary dilution steps.
3.1 Definitions and Required Personnel Qualifications
This section provides definitions and required resources for using the procedure.
3.1.1 Definitions
The following terms are used throughout the procedure:
Absorbance (A) is a measure of the amount of light that is absorbed in a sample. A standard statistical
curve is used to convert the absorbance value to the concentration value of cylindrospermopsin.
Q
Calibration Range is the assay range for which analysis results can be reported with confidence. For
undiluted samples, it ranges from the reporting limit of 0.1 ng/L to a maximum value of 2.0 ng/L. Values §
outside the range are handled as follows. If the value is: ^
<
• < 0.05 ng/L, then the laboratory reports the result as being non-detected ("<0.05 ng/L"). $
<
• Between 0.05 ng/L and the reporting limit of 0.1 ng/L (i.e., >0.05 ng/L and <0.1 ng/L), the O
laboratory should record the value, but assign a Quality Control (QC) code to the value (i.e., ^
DATA_FLAG=J). i
• >2.0 ng/L, the laboratory must dilute and reanalyze the sample.
l/l
CL
O
QC
LU
Q_
LD
Equation 3.1 Standard deviation O
Q
Coefficient of Variation (CV): The precision for a sample is reported in terms of the percent CV of its
absorbance values. To calculate the %CV, first calculate 5 (standard deviation) as follows:
5 =
n
hZ(A> ~Ay
1/2
>
U
n
i=1
where n is the number of replicate samples, A„ is the absorbance measured for the replicate. Per q
Section 3.5.3, samples are evaluated in duplicate (i=l or 2); controls are either evaluated in duplicate or
triplicate (i=l, 2, 3). A is the average absorbance of the replicates. Then, calculate %CV as: ^
12

-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 13 of 185
Equation 3.2 Percent (%) coefficient of variation
S
%CV = - x 100
A
Dark or Dimly Lit: Away from sunlight, but under incandescent lighting is acceptable.
Detection Limit is the minimum concentration at which the analyte can be detected with confidence
(0.05 ng/L). In other words, the outcome can be reported with confidence that it is greater than zero
(i.e., present in the sample). The detection limit is less than the reporting limit of 0.1 ng/L, at which the
measured value of the analyte can be reported with confidence. Also see "Sample-Specific Detection
Limit" below.
Duplicates are defined as two aliquots of the same sample which are analyzed separately using identical
procedures. The results are used to evaluate the precision of the laboratory analyses. Per Section 3.5.3,
controls are evaluated in duplicate or triplicate (i.e., three aliquots).
Relative Standard Deviation (RSD) is the same as the coefficient of variation (%CV). Because many of
the plate reader software programs provide the CV in their outputs, the procedure presents the quality
control requirement in terms of %CV instead of RSD.
Reporting Limit: For undiluted freshwater samples, the reporting limit is 0.1 ng/L. A reporting limit is the
point at which the measured value of the analyte can be reported with confidence.
Standard Deviation (S) shows variation from the average
Sample-Specific Detection Limit: Most samples will have a sample-specific detection limit equal to the
method's detection limit of 0.05 ng/L. For diluted samples, the sample-specific detection limit will be the
product of the method's detection limit of 0.05 ng/L and the dilution factor. Typical values for the
dilution factor will be 10 or 100.
Laboratory Technician: This procedure may be used by any laboratory technician who is familiar with
the NRSA QAPP, and this procedure in the NRSA 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 USEPA staff person who is responsible for selecting and managing the
"QC contractor." To eliminate the appearance of any inherent bias, the QC contractor must be
dedicated to QA/QC functions, and thus, must not be a primary laboratory or a field sampling contractor
for the 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.
3.2 Precautions
The laboratory must require its staff to abide by appropriate health and safety precautions, because the
kit substrate solution contains tetramethylbenzidine (TMB) and the stop solution contains diluted
sulfuric acid. In addition to the laboratory's usual requirements such as a Chemical Hygiene Plan, the
laboratory must adhere to the following health and safety procedures:
1. Laboratory facilities must properly store and dispose of solutions of weak acid.
2. Laboratory personnel must wear proper personal protection clothing and equipment (e.g.,
laboratory coat, protective eyewear, gloves).
3.1.2 Personnel Qualifications

-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 14 of 185
3. When working with potential hazardous chemicals (e.g., weak acid), laboratory personnel must
avoid inhalation, skin contact, eye contact, or ingestion. Laboratory personnel must avoid
contacting skin and mucous membranes with the TMB and stopping solution. If skin contact
occurs, remove clothing immediately. Wash and rinse the affected skin areas thoroughly with
large amounts of water.
3.3 Equipment/Materials
The procedures require the following equipment and information:
• Abraxis Cylindrospermospin ELISA (Microtiter) Test Kit, Product # 522011 (see items in Section
3.5.2).
Adhesive Sealing Film (Parafilm) for Micro Plates: Used to cover plates during incubation.
Data Template - See Figure 3.1.
Distilled or Deionized Water: For diluting samples when necessary.
ELISA evaluation software.
2 glass scintillation, LC, vials (20 mL).
Multichannel Pipette & Tips: An 8-channel pipette is used for this method. Familiarity of the use
of the multichannel pipette is necessary to achieve reliable results. Practice with water if you
have never used this before.
Norm-ject syringes (or equivalent).
Paper Towels: For blotting the microtiter plates dry after washing.
Permanent Marker (Sharpie Fine Point): For labeling samples, bottles, plates and covers.
Plate Reader (such as Metertech, Model M965 AccuReader): Complete with Metertech PC Mate
software for operation of machine. This machine reads the microtiter plates.
Project Quality Control Samples.
Reagent Reservoirs (Costar Cat Number 4870): Plain plastic reservoir for reagents that
accommodate the use of a multi-channel pipette.
Timer: For measuring incubation times.
QC
3
Q
LU
U
Test tubes: For dilutions, if needed. O
EC
CL
>~
Vortex Genie: For mixing dilutions.
Whatman Glass fiber syringe filter (25mm, GF 0.45 pim filter). <
-z.
3
3.4 Sample Receipt ^
Because USEPA initiates tracking procedures designed to recover any missing shipment, the laboratory z
personnel responsible for tracking samples must start the following login steps within 24 clock hours of
receiving a delivery. ^
QC
1. Report receipt of samples to the NARS IM Team by completing and emailing the sample tracking a.
spreadsheet with the sample login and sample condition information. (See Section 2.2 Roles and §
Q
Contact Information of the manual for contact information).
Inspect each sample THE SAME DAY THEY ARE RECEIVED: >
a. Verify that the sample IDs in the shipment match those recorded on the: 2
i. Chain of custody forms when the batching laboratory sends the samples to the q
cylindrospermopsin laboratory; or
ii. Sample tracking form if the field crew sends the shipment directly to the state. 53
14

-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 15 of 185
b. For each sample, record the date received and lab comment (including Condition Code
as described below) in the sample tracking spreadsheet with the appropriate Site ID/
Sample ID for the NARS IM Team.
i. OK: Sample is in good condition
ii. C: Sample container was cracked
iii. L: Sample container is leaking
iv. ML: Sample label is missing
v. NF: Sample is not frozen
c. If any sample is damaged or missing, contact the USEPA HQ Laboratory Review Coordinator
to discuss whether the sample can be analyzed. (See contact information in Chapter 2 of
the Manual).
3. Store samples in the freezer until sample preparation begins.
4. Maintain the sample tracking forms with the samples.
3.5 Procedure
The following sections describe the sample, kit preparation and analysis.
3.5.1 Sample Preparation
For each frozen sample (500 mL per sample), the laboratory technician runs it through a freeze-thaw
cycle three times to lyse the cells as follows: ^
o
5. All cycles: Keep the samples in dark or dimly lit areas (i.e., away from sunlight, but under y
incandescent lighting is acceptable). §
CL
6. First freeze-thaw cycle: >
a. Start with a frozen 500 ml sample. $
<
b. Thaw the sample to room temperature (approximately 25° C). Swirl the sample to check for O
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. ?
CO
7. Second freeze-thaw cycle: q
a. Freeze the vial.
c. Thaw the sample vial contents to room temperature. §
b. Keep the large sample bottle (from the 500 mL initial sample) frozen for future use. ^
c. Thaw the sample vi.
8. Third freeze-thaw cycle
a. Freeze the vial.
b. Thaw the vial contents to room temperature.
Filter the vial contents through a new, syringe
glass scintillation vial. Norm-ject syringes and Whatman Glass fiber syringe filters (25mm, GF
a. Freeze the vial. ^
z
c. Filter the vial contents through a new, syringe filter (0.45 pim) into a new, labeled 20 mL o
15

-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 16 of 185
0.45 nm filter) or other similar alternative are acceptable. One new syringe and filter should
be used per sample.
3.5.2 Kit Preparation
The technician prepares the kits using the following instructions:
1. Check the expiration date on the kit box and verify that it has not expired. If the kit has expired,
discard and select a kit that is still within its marked shelf life. (Instead of discarding the kit,
consider keeping it for training activities.)
2. Verify that each kit contains all the required contents:
• Microtiter plate
• Standards (7) referenced in this procedure as follows with the associated concentration:
o SO:0 ng/L
o SI: 0.05 ng/L
o S2: 0.1 Hg/L,
o S3: 0.25 ng/L
o S4: 0.5 ng/L
o S5: 1.0 ng/L
o S6: 2.0 ng/L
• Kit Control (KC): 0.75 ng/L
• Sample Diluent (distilled or deionized water)
• Cylindrospermospin-HRP conjugate Soultion (vortex before use)
• Antibody solution (rabbit anti-Cylindrospermopsin)
• Wash Solution 5X Concentrate
• Substrate (Color) Solution
• Stop Solution
3. If any bottles are missing or damaged, discard the kit. This step is important because Abraxis has
calibrated the standards and reagents separately for each kit.
4. Adjust the microtiter plate, samples, standards, and the reagents to room temperature.
5. Remove 12 microtiter plate strips (each for 8 wells) from the foil bag for each kit. The plates
contain 12 strips of 8 wells. If running less than a whole plate, remove unneeded strips from the
strip holder and store in the foil bag, ziplocked closed, and store in the refrigerator (4-8 °C).
6. Prepare a negative control (NC) using distilled or deionized water
7. The standards, controls, antibody solution, enzyme conjugate, color solution, and stop solutions
are ready to use and do not require any further dilutions.
8. Dilute the wash solution with distilled or deionized water. (The wash solution is a 5X
concentrated solution.) In a 1L container, dilute the 5X solution 1:5 (i.e., 100 mL of the 5X wash
solution plus 400 mL of distilled or deionized water). Mix thoroughly. Set aside the diluted
solution to wash the microtiter wells later.
9. Handle the stop solution containing diluted H2S04 (sulfuric acid) with care.

-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 17 of 185
3.5.3 Insertion of Contents into Wells
This section describes the steps for placing the different solutions into the 96 wells. Because of the
potential for cross contamination using a shaker table, the following steps specify manual shaking of the
kits instead mechanized shaking.
1.	While preparing the samples and kit, turn the plate reader on so it can warm up. The plate
reader needs a minimum of 30 minutes to warm up.
2.	Turn on the computer so that it can control and access the plate reader.
3.	Print the template (Figure 3.1) to use as reference when loading the standards, controls, and
samples as described in the next step. Templates contain rows, labeled with a marking pen, of
strips of 8 wells that snap into the blank frame. (If the laboratory wishes to use a different
template, provide a copy to the USEPA HQ Laboratory Review Coordinator for approval prior to
first use. (See Section 2.2 of the manual for contact information.)
4.	Using the lOO-piL pipette, add 50 piL, each, of the standards, controls, and samples to the
appropriate wells in the plate. Place all seven standards (0.00, 0.05, 0.10, 0.25, 0.50, 1.0 and 2.0
Hg/L), the kit control (0.75 piL), and negative control, in pairs (duplicate), starting in the well in
the upper left-hand corner of the kit as shown in Figure 3.1. Verify that the software displays the
same template or make any necessary corrections.
10 11 12
A
SO
S4
NC
U4
U8
U12
U16
U20
U24
U28
U32
U36
B
SO
S4
NC
U4
U8
U12
U16
U20
U24
U28
U32
U36
C
SI
S5
U1
U5
U9
U13
U17
U21
U25
U29
U33
U37
D
SI
S5
U1
U5
U9
U13
U17
U21
U25
U29
U33
U37
E
S2
S6
U2
U6
U10
U14
U18
U22
U26
U30
U34
U38
F
S2
S6
U2
U6
U10
U14
U18
U22
U26
U30
U34
U38
G
S3
KC
U3
U7
Ull
U15
U19
U23
U27
U31
U35
U39
H
S3
KC
U3
U7
Ull
U15
U19
U23
U27
U31
U35
U39
Figure 3.1 Cylindrospermopsin: sample template
Key:
S0-S6 = Standards;
KC = Control supplied with Kit (i.e., Kit Control);
NC= Negative Control (Laboratory Reagent Blank);
U = Unknown ("REGULAR" sample collected by the field crew);
5.	Add 50 piL of the conjugate solution to each well using the multi-channel pipettor and a reagent
reservoir. Add 50 piL of the cylindrospermopsin antibody solution to each well using the multi-
channel pipettor and a reagent reservoir. Use dedicated reagent reservoirs for each reagent to
avoid contamination from one reagent to another.
6.	Place the sealing Parafilm over the wells.
7.	Manually mix the contents by moving the strip holder in a rapid circular motion on the benchtop
for 30 seconds. Be careful not to spill the contents.

-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 18 of 185
8. Place the plate in an area away from light for 45 minutes.
9. After 45 minutes, carefully remove the Parafilm.
10. Empty the contents of the plate into the sink, pat inverted plate dry on a stack of paper towels,
and then wash the wells of the plate four times with 250 piL of washing solution using the multi-
channel pipette. After adding the washing solution each time, empty the solution into the sink
and use the paper towels as before.
11. Add 100 piL of substrate/ color solution to all wells using the multi-channel pipettor.
12. Cover the wells with Parafilm.
13. Manually mix the contents by moving the strip holder in a rapid circular motion on the benchtop
for 30 seconds. Be careful not to spill the contents.
14. Place the strip holder in an area away from light for 30-45 minutes.
15. After 30-45 minutes, remove the Parafilm, add 100 piL of stop solution to the wells using the
multi-channel pipette and reagent reservoir in the same sequence as the substrate solution.
16. Use a microplate ELISA photometer (plate reader) to determine the absorbance at 450 nm. The
software (i.e., commercial ELISA evaluation program) calculates the absorbance and
concentration values of the samples from the calibration curve and the average values for each
pair. Use a 4-parameter standard curve fit to determine the concentrations.
17. Dispose of solution in plates in a laboratory sink. Rinse plates and sink with water to dilute the
weak acid present.
18. Perform QC evaluations of the data as follows:
a. If the following failures occur in the standards and controls, then the laboratory must
reanalyze all samples in the analytical run:
i. Standard curve with a correlation coefficient of less than 0.99 (i.e., R<0.99)
ii. Standards S0-S6 must have decreasing absorbance values. First, calculate the average
values for each standard. That is, if A, is the absorbance average for S,, then the
absorbance averages must be: A0> Ai > A2 > A3 > A4 >A5>A6
iii. The average absorbance of the standard SO less than 0.8 (i.e., A0< 0.8).
iv. Two or more negative control samples with detectable concentrations of
Cylindrospermopsin (i.e., values > 0.1 ng/L). If this occurs, then evaluate possible causes
(e.g., cross-contamination between samples), and if appropriate, modify laboratory
processes before the next analytical run.
v. Results for control samples of outside the acceptable range of 0.75 +/- 0.15 ppb. That is,
results must be between 0.60 and 0.90.
b. If either, or both, of the following failures occur for the sample, then the sample must be
reanalyzed (maximum of two analyses, consisting of the original analysis and, if necessary,
one reanalysis):
i. The concentration value registers as HIGH (exceeds the calibration range). Dilute the
sample for the reanalysis per Section 3.5.4.
ii. The %CV > 15% between the duplicate absorbance values for a sample.
19. Record the results, even if the data failed the quality control requirements in #18b, for each well
in the USEPA's data template (Table 3.1). The required entries are for the following columns:

-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 19 of 185
a.	SAM_CODE should be one of the following codes: S0-S6 for standards; KC or NC, for
controls; QC for quality control samples; REGULAR for unknown samples.
b.	CONC contains the numeric concentration value. Two special cases:
i.	Non-detected concentrations: If the sample is non-detected, then provide the sample-
specific detection limit which is 0.05 ng/L if the sample is undiluted. See Section 3.5.4
for calculating the sample-specific detection limit for a diluted sample.
ii.	If the result shows that it is "HI," this indicates that the sample value is outside of the
calibration range and must be diluted and re-run using another analytical run. Leave the
CONC column blank and record 'HI' in the DATA FLAG column.
c.	QUALITY FLAGS have codes for the following special cases:
i.	ND if the sample was non-detected;
ii.	J if the value is detected but at a level below the reporting limit of 0.1 ng/L (for
undiluted samples);
iii.	HI if the concentration value registers as HIGH (exceeds the calibration range).
iv.	QCF if there is a QC failure per step 18 above. The QCF code must be used for all failures
to facilitate data analysis.
v.	Qfor any other quality issue (describe in COMMENTS)
d.	DILUTION FACTOR is only required if the sample was diluted.
e.	AVG_CONC and CV_ABSORB are required for all duplicate runs (use all three values if the
controls are used in triplicate).
Table 3.1 Cylindrospermopsin: required data elements- data submission
FIELD
COLUMN HEADING
FORMAT
DESCRIPTION

LABORATORY ID
LABJD
Text
Name or abbreviation for QC laboratory
DATE RECEIVED
DATE_RECEIVED
MMDDYY
Date sample was received by lab
SITE ID
SITEJD
Text
NRSA site ID code as recorded on sample
label or tracking form (blank if standard or



control)
1
VISIT NUMBER
VISIT_NO
Numeric
Sequential visits to site (1 or 2) (blank if



standard or control)
1
SAMPLE ID
SAMPLEJD
Numeric
6-digit Sample ID number as recorded on
sample jar or tracking form (blank if



standard or control)
1
DATE COLLECTED
DATE_COL
MMDDYY
Date sample was collected (blank if



standard or control)
1
CONDITION CODE
CONDITION_CODE
Text
Sample condition upon arrival at the
laboratory (blank if standard or control)



Flag
Definition



Blank or N
Not a sample (blank,
standard, or
control)



OK
Sample is in good
condition



C
Sample container is
cracked
QC
3
~
LU
U
o
QC
CL
>
<
l/l
l/l
<
o
l/l
CL
o
QC
LU
CL
l/l
O
QC
~
>
U
X
o
<
19

-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 20 of 185



L
Sample or container
is leaking



ML
Sample label is
missing



NF
Sample is not frozen
CONDITION
COND COMMENT
Text
Any comment based on the condition code
COMMENT


flags

BATCH
BATCH ID
Numeric
Batch identification code; assigned by lab
IDENTIFICATION




TECHNICIAN
TECHNICIAN
Text
Name or initials of technician performing



the procedure

DATE ANALYZED
DATE ANALYZED
MMDDYY
Date when samples are inserted into the



wells

KIT EXPIRE DATE
KIT_EXPIRE_DATE
MMDDYY
Expiration date on kit box
KIT ID
KITJD
Text
Kit identification code. If one does not
exist, assign a unique code to each kit.
R2
R2
Numeric
R2 from curve fit to the average absorbance
values for the standards. Value is between



0 and 1.

SAMPLE CODE
SAM_CODE
Text
Type of solution being tested in the well



Code
Definition



KC
Kit Control



NC
Negative Control



SO, SI, S2, S3, S4,
Standard



S5, S6




QC
Quality Control



REGULAR
Sample of unknown
concentration
LOCATION
LOCATION
Text
Location of well in the kit (e.g., B5 would be
the fifth well from the left in the second



row B)

CONCENTRATION
CONC
Numeric
Concentration or sample-specific detection
limit of contents of well in ng/L. Sample-
specific detection limit should be 0.1 |jg/L if
the sample hasn't been diluted.
UNITS
UNITS
Text
The units of the concentration of the CONC



column

MDL
MDL
Numeric
Minimum detection limit of the machine in
same units as the CONC column
RL
RL
Numeric
Reporting limit in same units as the CONC



column

ABSORBANCE
ABSORBANCE
Numeric
Absorbance value

DILUTION FACTOR
DILUTION_FACTOR
Numeric
10,100, etc for number of times the
sample was diluted. If not diluted, leave



blank or record 1

CV ABSORBANCE
CV_ABSORB
Numeric
Calculated %CV of duplicate values of
absorbance for all runs. Enter %CV. Value is



between 0 and 100%.

AVERAGE
AVG ABSORB
Numeric
Calculated average of absorbance values
ABSORBANCE


for all samples and standards. Average
20

-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 21 of 185
AVERAGE
CONCENTRATION
AVG CONC
Numeric
OA FLAG (if
appropriate)
QA_FLAG
Text
LABORATORY
COMMENT
LAB COMMENT
Text
value of the original sample and its
duplicate (or replicates for KC and NC).
Calculated average of concentration values
for a sample. Substitute 0.1 |jg/L for any
result recorded as <0.1 ng/L
Data qualifier codes associated with
specific identifications of voucher samples.
These codes provide more information that
those used when reporting receipt of
samples. A technician may use alternative
or additional qualifiers if definitions are
provided as part of the submitted data
package (e.g., as a separate worksheet
page of the data submission file).
Flag
Definition
Concentration
below detection.
Unless the sample
was diluted, the
concentration will
be 0.05 ng/L
HI
Result indicated
that a high
concentration (i.e.,
outside calibration
range)> 2.0 ng/L
Concentration
above detection but
below reporting
limit. Without
dilution, these
values are between
0.05 and 0.1 ng/L
QCF
Q ~~
QC failure
Other quality
concerns, not
identified above
Explanation for data flag(s) (if needed) or
other comments.
3.5.4 Dilutions (if needed)
Dilutions if needed are prepared as follows (using clean glass tubes):
#1
1:10 dilution
a. Add 900 piL of distilled or deionized water to a clean vial. (Note: Dilutions may also be made
using the kit's diluent rather than distilled or deionized water.)
QC
LU
CL
l/l
O
QC
Q
>
U
X
o
<
21

-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 22 of 185
b. Pipette 100 piL from the sample into the vial. (To provide more accurate dilutions and less
chance of contaminating the diluent, the diluent should be added to the vial before the
sample.)
c. Mix by vortexing.
d. Multiply final concentration and Abraxis' detection limit of 0.05 ng/L by 10 to obtain the
sample-specific detection limit of .5 ng/L.
#2
1:100 dilution
a. Add 3.96 mL of distilled or deionized water to a clean, appropriately labeled glass vial.
(Note: Dilutions may also be made using the kit's diluent rather than distilled or deionized
water.)
b. Vortex the sample to mix thoroughly, then pipette 40 piL from the sample and add to the
water (or diluent) in the appropriate labeled vial. Vortex the sample again.
c. Multiply the final concentration and Abraxis' detection limit of 0.05 ng/L by 100 to obtain
the sample-specific detection limit of 50 ng/L.
• Other dilutions can be calculated in the same manner as #1 and #2 if needed.
3.6 Pertinent QA/QC Procedures
This section describes the quality assurance and quality control measures used to ensure that the data
will meet the NRSA's requirements.
3.6.1 QC Samples
The External QC Coordinator will instruct the QC contractor to provide one or two identical sets
of freshwater QC samples (labeled as performance test (PT) samples) to all participating
laboratories. Each set will contain five samples to test the expected range of concentrations in
the NRSA samples.
For the contract laboratory, the QC contractor will provide the first set to be run with the first
set of samples and a second set to be run at the midpoint of the assigned samples. If available, a
third set will be run with the final batch of samples. Because most state laboratories will have
relatively few samples that can be analyzed using a single kit, the QC contractor will send only
one set to each state laboratory.
Each laboratory will run the QC samples following the same procedures used for the other
samples. The External QC Coordinator will compare the results and assess patterns in the data
(e.g., one laboratory being consistently higher or lower than all others). Based upon the
evaluation, the External QC Coordinator may request additional information from one or more
laboratories about any deviations from the method or unique laboratory practices that might
account for differences between the laboratory and others. With this additional information, the
External QC Coordinator will determine an appropriate course of action, including no action,
flagging the data, or excluding some or all the laboratory's data.

-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 23 of 185
3.6.2 Summary of QA/QC Requirements
Table 3.2 provides a summary of the quality control requirements described in Sections 3.5.2 and 3.5.3.
For cylindrospermopsin, the precision for a sample is reported in terms of the percent coefficient of
variation (%CV) of its absorbance values. Relative Standard Deviation (RSD) is the same as the %CV.
Because many of the plate reader software programs provides the CV in their outputs, the procedure
presents the quality control requirement in terms of %CV instead of RSD. Accuracy is calculated by
comparing the average concentration of the kit control with the required range (0.75 +/- 0.15).
Table 3.2 Cylindrospermopsin: quality control- sample analysis
Quality Control
Description and Requirements
Corrective Action
Activity

-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 24 of 185

All samples are run in duplicate. If both of the
values are less than the upper calibration
range (i.e., 2.0 ng/Lfor undiluted samples),
then the requirement is met.
•	Record the results for both
duplicates using different start
dates and/or start times to
distinguish between the runs.
•	Report the data for both
duplicate results using Quality
Control Failure flag "QCF"; and
•	Re-analyze the sample in a
new analytical run. No
samples are to be run more
than twice.
If the second run passes, then the
data analyst will exclude the data
from the first run (which will have
been flagged with "QCF"). If both
runs fail, the data analyst will
determine if either value should
be used in the analysis (e.g., it
might be acceptable to use data if
the CV is just slightly over 15%).
Results Within
Calibration
Range
If a result registers as "HIGH", then
record the result with a data flag
of "HI." If one or both duplicates
register as 'HIGH,' then the sample
must be diluted and re-run. No
samples are to be run more than
twice. If samples are re-run, do
not enter concentration
information of the first run.
External Quality
Control Sample
External QC Coordinator, supported by QC
contractor, provides 1-2 sets of identical
samples to all laboratories and compares
results.
Based upon the evaluation, the
External QC Coordinator may
request additional information
from one or more laboratories
about any deviations from the
method or unique laboratory
practices that might account for
differences between the
laboratory and others. With this
additional information, the
External QC Coordinator will
determine an appropriate course
of action, including no action,
flagging the data, or excluding
some or all of the laboratory's
data.

-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 25 of 185
4.0 ALGAL TOXIN: MICROCYSTIN IMMUNOASSAY PROCEDURE
This chapter describes an immunoassay procedure that measures concentrations of total microcystins in
water samples. In applying the procedure, the laboratory uses Abraxis' Microcystins-ADDATest Kits (
"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.
4.1 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 ng/Lto 5.0 ng/L, although, theoretically, the procedure can detect, not quantify, microcystins
concentrations as low as 0.10 ng/L. For samples with higher concentrations of microcystins, the
procedure includes the necessary dilution steps.
4.2 Health and Safety Warnings
The laboratory must require its staff to abide by appropriate health and safety precautions, because the
kit substrate solution contains tetramethylbenzidine (TMB) and the stop solution contains diluted
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
LU
coat, protective eyeware, gloves). ^
3. When working with potential hazardous chemicals (e.g., weak acid), laboratory personnel must
contacting skin and mucous membranes with the TMB and stopping solution. If skin contact
Q
LU
U
avoid inhalation, skin contact, eye contact, or ingestion. Laboratory personnel must avoid O
CL
>
occurs, remove clothing immediately. Wash and rinse the affected skin areas thoroughly with $
large amounts of water. O
-z.
3
4.3 Definitions and Required Resources (Personnel, Laboratories, and ^
Equipment) —
This section provides definitions and required resources for using the procedure. p
i/i
4.3.1 Definitions b
o
QC
The following terms are used throughout the procedure: y
z
X
o
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. -d
25

-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 26 of 185
Absorbance (A) is a measure of the amount of light in a sample. A standard statistical curve is used to
convert the absorbance value to the concentration value of microcystins.
Calibration Range is the assay range for which analysis results can be reported with confidence. For
undiluted samples, it ranges from the reporting limit of 0.15 ng/L to a maximum value of 5.0 ng/L.
Values outside the range are handled as follows. If the value is:
•	< 0.10 ng/L, then the laboratory reports the result as being non-detected ("<0.10 ng/L").
•	Between 0.10 ng/L and the reporting limit of 0.15 ng/L (i.e., >0.10 ng/L and <0.15 ng/L), the
laboratory should record the value, but assign a QC code to the value (i.e., DATA_FLAG=J).
•	5.0 ng/L, the laboratory must dilute and reanalyze the sample.
Coefficient of Variation (CV): The precision for a sample is reported in terms of the %CV of its
absorbance values. To calculate the %CV, first calculate 5 (standard deviation) as follows:
n	-.1/2
s =

n
i = 1
where n is the number of replicate samples, A„ is the absorbance measured for the replicate. Samples
are evaluated in duplicate (i=l or 2); controls are either evaluated in duplicate or triplicate (i=l, 2, 3). A
is the average absorbance of the replicates. Then, calculate %CV as:
S
%CV =
x 100
A
Dark or Dimly Lit: Away from sunlight, but under incandescent lighting is acceptable.
Detection Limit is the minimum concentration at which the analyte can be detected with confidence. In
other words, the outcome can be reported with confidence that it is greater than zero (i.e., present in
the sample). The detection limit is less than the reporting limit of 0.15 ng/L at which the measured value
of the analyte can be reported with confidence. Also see "Sample-Specific Detection Limit."
Duplicates are defined as two aliquots of the same sample which are analyzed separately using identical	^
procedures. The results are used to evaluate the precision of the laboratory analyses. Controls are	^
evaluated in duplicate or triplicate (i.e., three aliquots).	u
O
NARS: National Aquatic Resource Surveys. The National Rivers and Streams Assessment (NRSA) is part of	^
the NARS program.	^
l/l
l/l
<
O
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.
•si
CO
NARS Information Management System (NARS IM): The IM system established to support all surveys,	<
i/i
>
Relative Standard Deviation (RSD) is the same as the coefficient of variation (%CV). Because many of	^
the plate reader software programs provides the CV in their outputs, the procedure presents the quality	^
control requirement in terms of %CV instead of RSD.	^
Reporting Limit: For undiluted freshwater sample, the reporting limit is 0.15 ng/L. A reporting limit is	^
the point at which the measured value of the analyte can be reported with confidence.	^
	I
Standard Deviation (S) shows variation from the average	^
26

-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 27 of 185
Sample-Specific Detection Limit: Most samples will have a sample-specific detection equal to the
method's detection limit of 0.1 ng/L. For diluted samples, the sample-specific detection limit will be the
product of the method's detection limit of 0.1 ng/L and the dilution factor. Typical values for the dilution
factor will be 10 or 100.
4.4 General Requirements for Laboratories
4.4.1 Expertise
To demonstrate its expertise, the laboratory shall provide EPA with one or more of the following:
• Memorandum that identifies the relevant services that the laboratory provided for the National
Aquatic Resource Surveys in the past five years.
• Documentation detailing the expertise of the organization, including professional certifications
for water-related analyses, membership in professional societies, and experience with analyses
that are the same or similar to the requirements of this method.
4.4.2 Quality assurance and quality control requirements
To demonstrate its expertise in quality assurance and quality control procedures, the organization shall
provide EPA with copies of the quality-related documents relevant to the procedure. Examples include
Quality Management Plans (QMP), QAPPs, and applicable Standard Operating Procedures (SOPs).
To demonstrate its ongoing commitment, the person in charge of quality issues for the organization
shall sign the NRSA QAPP Certification Page.
4.4.3 Personnel
Laboratory Technician: This procedure may be used by any laboratory technician who is familiar with
the NRSA 2018-19 QAPP, and this procedure in the NRSA 2018-19 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.
4.4.4 Equipment/Materials
The procedures require the following equipment and information:
• Abraxis ADDA Test Kit, Product #520011 (see items in Section 4.6.2)
• Adhesive Sealing Film (Parafilm) for Micro Plates (such as Rainin, non-sterile, Cat. No. 96-SP-
100): Used to cover plates during incubation.
• Data Template - See Table 4.1Figure 4.1
• 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

-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 28 of 185
o 4 mL (for dilutions)
• Multichannel Pipette & Tips: A single-channel and an 8-channel pipette are used for this
method.
• Norm-ject syringes (or equivalent)
• Paper Towels: For blotting the microtiter plates dry after washing.
• Permanent Marker (Sharpie Fine Point): For labeling samples, bottles, plates and covers.
• Plate Reader (e.g., Metertech Model M965 AccuReader; ChroMate®; or equivalent readers with
software to read the microtiter plates and measure absorbances).
• Reagent Reservoirs (e.g., Costar Cat Number 4870): Plain plastic reservoir for reagents that
accommodate the use of a multi-channel pipette.
• Test tubes: For dilutions, if needed.
• Timer: For measuring incubation times.
• Vortex Genie: For mixing dilutions.
• Whatman Glass fiber syringe filter (25mm, GF 0.45 pim filter)
4.5 Sample Receipt
Field crews hold the microcystins samples on ice while in the field and then pack the samples in ice for
delivery to a central facility ("batching laboratory") or the State's laboratory. The batching and State
laboratories freeze the samples upon receipt. Periodically, the batching laboratory ships samples to the
microcystins laboratory. The batching and microcystins laboratory may retain the frozen samples for
several months before analysis.
Because EPA initiates tracking procedures designed to recover any missing shipment, the laboratory
personnel responsible for tracking samples must start the following login steps within 24 clock hours of
receiving a delivery.
4. Report receipt of samples in the NARS IM sample tracking system (within 24 clock hours).
5. Inspect each sample THE SAME DAY THEY ARE RECEIVED: £
a. Verify that the sample IDs in the shipment match those recorded on the: q
LU
i. Chain of custody forms when the batching laboratory sends the samples to the u
QC
CL
microcystins laboratory; or
ii. Sample tracking form if the field crew sends the shipment directly to the state <
CO
laboratory. <
O
¦z.
b. For each sample, record the date received and lab comment (including Condition Code as ^
described below) in the sample tracking spreadsheet with the appropriate site ID/ sample ID ^
for the NARS IM Team. 5
I—
i. OK: Sample is in good condition ^
ii. C: Sample container was cracked
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 29 of 185
6. Store samples in the freezer until sample preparation begins.
7. Maintain the chain of custody or sample tracking forms with the samples.
4.6 Procedure
The following sections describe the sample and kit preparation and analysis.
4.6.1 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 25° C). Swirl the sample to check for
ice crystals. At this temperature, no ice crystals should be present in the sample.
c. Shake well to homogenize the sample, then transfer 10 mLto 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 pim) into a new, labeled 20 mL
glass scintillation vial. Norm-ject syringes and Whatman Glass fiber syringe filters (25mm, GF
LU
0.45 pirn filter) or other similar alternative are acceptable. One new syringe and filter should ^
be used per sample. £
u
O
4.6.2 Kit Preparation a.
>-
The technician prepares the kits using the following instructions: 5i
CO
<
1. Check the expiration date on the kit box and verify that it has not expired. If the kit has expired, O
discard and select a kit that is still within its marked shelf life. (Instead of discarding the kit, ^
consider keeping it for training activities.) ^
2. Verify that each kit contains all the required contents: 5
• Microtiter plate ^
• Standards (6) referenced in this procedure as follows with the associated concentration: u
o SO:0 ng/L ^
o SI: 0.15 ng/L |
o S2: 0.40 ng/L °
o S3: 1.0 ng/L <
o S4: 2.0 ng/L <
29
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 30 of 185
o S5: 5.0 ng/L
• Kit Control (KC): 0.75 ng/L
• Antibody solution
• Anti-Sheep-HRP Conjugate
• Wash Solution 5X Concentrate
• Color Solution
• Stop Solution
• Diluent
3. If any bottles are missing or damaged, discard the kit. This step is important because Abraxis has
calibrated the standards and reagents separately for each kit.
4. Adjust the microtiter plate, samples, standards, and the reagents to room temperature.
5. Remove 12 microtiter plate strips (each for 8 wells) from the foil bag for each kit. The plates
contain 12 strips of 8 wells. If running less than a whole plate, remove unneeded strips from the
strip holder and store in the foil bag, ziplocked closed, and place in the refrigerator.
6. Store the remaining strips in the refrigerator (4-8° C).
7. Prepare a negative control (NC) using distilled water
8. The standards, controls, antibody solution, enzyme conjugate, color solution, and stop solutions
are ready to use and do not require any further dilutions.
9. Dilute the wash solution with deionized water. (The wash solution is a 5X concentrated
solution.) In a 1L container, dilute the 5X solution 1:5 (i.e., 100 mL of the 5X wash solution plus
400 mL of deionized water). Mix thoroughly. Set aside the diluted solution to wash the
microtiter wells later.
10. Handle the stop solution containing diluted H2S04 with care.
4.6.3 Insertion of Contents into Wells l±j
QC
This section describes the steps for placing the different solutions into the 96 wells. Because of the o
LU
potential for cross contamination using a shaker table, the following steps specify manual shaking of the u
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. <
O
2. Turn on the computer so that it can control and access the plate reader. 2
3. Print the template (Figure 4.1) to use as reference when loading the standards, controls, and ^
samples as described in the next step. Templates contain rows, labeled with a marking pen, of —
strips of 8 wells that snap into the blank frame. (If the laboratory wishes to use a different P
template, provide a copy to the EPA HQ Laboratory Review Manager for approval prior to first b
O
use. (See Section 2.2 of the manual for contact information.) g
4. Using the lOO-piL pipette, add 50 piL, each, of the standards, controls, and samples to the ^
appropriate wells in the plate. Place all six standards (0.00, 0.15, 0.40, 1.00, 2.0 and 5.0 ng/L), M
the kit control (0.75 piL), and negative control, in pairs, starting in the well in the upper left-hand O
corner of the kit as shown in Figure 4.1. Verify that the software displays the same template or <
make any necessary corrections. ^
30
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 31 of 185
10 11 12
A
SO
S4
NC
U4
U8
U12
U16
U20
U24
U28
U32
U36
B
SO
S4
NC
U4
U8
U12
U16
U20
U24
U28
U32
U36
C
SI
S5
U1
U5
U9
U13
U17
U21
U25
U29
U33
U37
D
SI
S5
U1
U5
U9
U13
U17
U21
U25
U29
U33
U37
E
S2
KC
U2
U6
U10
U14
U18
U22
U26
U30
U34
U38
F
S2
KC
U2
U6
U10
U14
U18
U22
U26
U30
U34
U38
G
S3
KC
U3
U7
Ull
U15
U19
U23
U27
U31
U35
U39
H
S3
NC
U3
U7
Ull
U15
U19
U23
U27
U31
U35
U39
Figure 4.1 Microcystin: sample template
Key:
S0-S5 = Standards;
KC = Control supplied with Kit (i.e., Kit Control);
NC = Negative Control;
U = Unknown (sample collected by the field crew, marked as "REGULAR" in the
SAM_CODE column)
5. Add 50 piL of the pink antibody solution to each well using the multi-channel pipettor and a
reagent reservoir. Use dedicated reagent reservoirs for each reagent to avoid contamination
from one reagent to another.
6. Place the sealing Parafilm over the wells.
7. Manually mix the contents by moving the strip holder in a rapid circular motion on the benchtop
for 30 seconds. Be careful not to spill the contents.
8. Place the plate in an area away from light for 90 minutes.
9. After 90 minutes, carefully remove the Parafilm.
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 piL of washing solution using the
multi-channel pipette. After adding the washing solution each time, empty the solution into the
sink and use the paper towels as before.
11. Add 100 piL of 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.
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 32 of 185
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.
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 in the standards and controls, then the laboratory must
reanalyze all samples in the analytical run:
i. Standard curve with a correlation coefficient of less than 0.99 (i.e., R<0.99)
ii. Standards S0-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: A0 > Ai > A2 > A3 > A4 >A5
iii. The average absorbance of the standard SO less than 0.8 (i.e., A0 < 0.8).
iv. Two or more negative control samples with detectable concentrations of microcystins
(i.e., values > 0.1 ng/L). If this occurs, then evaluate possible causes (e.g., cross-
contamination between samples), and if appropriate, modify laboratory processes
before the next analytical run.
v. Results for control samples of outside the acceptable range of 0.75 +/- 0.185 ppb. That
is, results must be between 0.565 and 0.935.
b. If either, or both, of the following failures occur, then the sample must be reanalyzed
(maximum of two analyses, consisting of the original analysis and, if necessary, one
reanalysis):
i. The concentration value registers as HIGH (exceeds the calibration range). Dilute the
sample for the reanalysis per Section 4.6.4.
ii. The %CV > 15% between the duplicate absorbance values for a sample.
24. Record the results, even if the data failed the quality control requirements in #23b, for each well
in EPA's data template (see Table 4.1 for required data elements in the Analysis Stage). The
required entries are for the following columns:
a. SAM_CODE should be one of the following codes: S0-S5 for standards; KC, NC, QC for
controls; REGULAR for unknown sample.
b. CONC contains the numeric concentration value. Two special cases:
i. Non-detected concentrations: If the sample is non-detected, then provide the sample-
specific detection limit which is 0.1 ng/L if the sample is undiluted. See Section 4.6.4 for
calculating the sample-specific detection limit for a diluted sample.
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 33 of 185
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 QA_FLAG column.
c. QA_FLAG have codes for the following special cases:
i. ND if the sample was non-detected;
ii. J if the value is detected but at a level below the reporting limit of 0.15 ng/L (for
undiluted samples);
iii. HI if the concentration value registers as HIGH (exceeds the calibration range).
iv. QCF if there is a QC failure per step 23 above. The QCF code must be used for all failures
to facilitate data analysis.
v. Qfor any other quality issue (describe in LAB_COMMENT)
d. DILUTION FACTOR is only required if the sample was diluted.
e. AVG_CONC and CV_ABSORB are required for duplicate samples and control samples (use all
three values if the controls are used in triplicate).
Table 4.1 Microcystin: required data elements - data submission
FIELD COLUMN HEADING FORMAT DESCRIPTION
LABORATORY ID
LABJD
Text
Name or abbreviation for QC laboratory
DATE RECEIVED
DATE_RECEIVED
MMDDYY
Date sample was received by lab
SITE ID
SITEJD
Text
NRSA site ID code as recorded on sample
label or tracking form (blank if standard or
control)
VISIT NUMBER
VISIT_NO
Numeric
Sequential visits to site (1 or 2) (blank if
standard or control)
SAMPLE ID
SAMPLEJD
Numeric
6-digit Sample ID number as recorded on
sample jar or tracking form (blank if
standard or control)
DATE COLLECTED
DATE_COL
MMDDYY
Date sample was collected (blank if
standard or control)
CONDITION CODE
CONDITION_CODE
Text
Sample condition upon arrival at the
laboratory (blank if standard or control)

Flag
Definition

Blank or N
Not a sample (blank,
standard, or
control)

OK
Sample is in good
condition

C
Sample container is
cracked

L
Sample or container
is leaking

ML
Sample label is
missing

NF
Sample is not frozen
CONDITION
COMMENT
COND_COMMENT
Text
Any comment based on the condition code
flags
QC
3
~
LU
U
o
QC
CL
>
<
LT)
LT)
<
O
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 34 of 185
BATCH
BATCH ID
Numeric
| Batch identification code; assigned by lab
IDENTIFICATION

TECHNICIAN
TECHNICIAN
Text
Name or initials of technician performing
the procedure
DATE ANALYZED
DATE_ANALYZED
MMDDYY
Date when samples are inserted into the
¦ wells
KIT EXPIRE DATE
KIT_EXPIRE_DATE
MMDDYY
Expiration date on kit box
KIT ID
KITJD
Text
Kit identification code. If one does not
exist, assign a unique code to each kit.
R2
R2
Numeric
R2 from curve fit to the average absorbance
values for the standards. Value is between
: 0 and 1.
SAMPLE CODE
SAM CODE
Text
Type of solution being tested in the well

Code Definition

KC Kit Control

NC Negative Control

SO, SI, S2,S3, S4, S5 Standard

QC Quality Control

: REGULAR Sample of unknown

| > concentration
LOCATION
LOCATION
Text
Location of well in the kit (e.g., B5 would be
the fifth well from the left in the second
row B)
CONCENTRATION
CONC
Numeric
Concentration or sample-specific detection
limit of contents of well in ng/L. Sample-
; specific detection limit should be 0.1 |jg/L if
the sample hasn't been diluted.
UNITS
UNITS
Text
The units of the concentration of the CONC
column
MDL
MDL
Numeric
Minimum detection limit of the machine in
same units as the CONC column
RL
RL
Numeric
Reporting limit in same units as the CONC
column
ABSORBANCE
ABSORBANCE
Numeric
Absorbance value
DILUTION FACTOR
DILUTION_FACTOR
Numeric
10,100, etc for number of times the
sample was diluted. If not diluted, leave
blank or record 1
CV ABSORBANCE
CV_ABSORB
Numeric
Calculated %CV of duplicate values of
absorbance for all runs. Enter %CV. Value is
between 0 and 100%.
AVERAGE
AVG ABSORB
Numeric
Calculated average of absorbance values
ABSORBANCE

for all samples and standards. Average
value of the original sample and its
duplicate (or replicates for KC and NC).
AVERAGE
AVG_CONC
Numeric
Calculated average of concentration values
CONCENTRATION

for a sample. Substitute 0.15 ng/L for any
result recorded as <0.15 ng/L
OA FLAG (if
QA_FLAG
Text
Data qualifier codes associated with
appropriate)

specific identifications of voucher samples.
These codes provide more information that
; those used when reporting receipt of
34
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 35 of 185

samples. A technician may use alternative

or additional qualifiers if definitions are

provided as part of the submitted data

package (e.g., as a separate worksheet

page of the data submission file).

Flag
Definition

ND
Concentration

below detection.

Unless the sample

was diluted, the

concentration will

be 0.1 ng/L

HI
Result indicated

that a high

concentration (i.e.,

outside calibration

range)

J
Concentration

above detection but

below reporting

limit. Without

dilution, these

values are between

0.1 and 0.15 ng/L

1 QCF
QC failure

; Q
Other quality

concerns, not

identified above
LABORATORY
LAB_COMMENT
Text
Explanation for data flag(s) (if needed) or
COMMENT

| other comments.

4.6.4 Dilutions (if needed)
Dilutions if needed are prepared as follows (using clean glass tubes):
• 1:10 dilution
e. Add 900 piL of distilled water to a clean vial. (Note: Dilutions may also be made using the
kit's diluent rather than distilled water.)
f. Pipette 100 piL from the sample into the vial. (To provide more accurate dilutions and less
chance of contaminating the diluent, the diluent should be added to the vial before the
sample.)
g. Mix by vortexing.
h. Multiply final concentration and Abraxis' detection limit of 0.1 ng/L by 10 to obtain the
sample-specific detection limit of 1.0 ng/L.
QC
3
Q
LU
U
o
cc
o_
>
<
LD
LD
<
O
U~)
b
o
QC
u
• 1:100 dilution
d. 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.)
x
o
<
35
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 36 of 185
e. Vortex the sample to mix thoroughly, then pipette 40 piL from the sample and add to the
water (or diluent) in the appropriate labeled vial. Vortex.
f. Multiply the final concentration and Abraxis' detection limit of 0.1 ng/L by 100 to obtain the
sample-specific detection limit of 10 ng/L.
• Other dilutions can be calculated in the same manner as #1 and #2 if needed.
4.7 Quality Measures
This section describes the quality assurance and quality control measures used to ensure that the data
will meet NRSA's requirements.
4.7.1 Assistance Visits
Assistance visits are intended to familiarize EPA with actual procedures being implemented by different
laboratories; and to ensure a clear and consistent understanding of procedures and activities by both
EPA and the laboratories. If EPA decides to conduct an assistance visit, a qualified EPA scientist or
contractor will administer a checklist based upon the steps described in this chapter.
4.7.2 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 (labeled as performance test (PT) 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.
4.7.3 Summary of QA/QC Requirements
Table 4.2 provides a summary of the quality control requirements described in Sections 4.5 and 4.6.
Table 4.2 Microcystin: quality control - sample analysis
Quality Control
Description and Requirements
Corrective Action
Activity

Kit - Shelf Life
Is within its expiration date listed on kit box.
If kit has expired, then discard or set
aside for training activities.
Kit - Contents
All required contents must be present and in
acceptable condition. This is important because
Abraxis has calibrated the standards and reagents
separately for each kit.
If any bottles are missing or damaged,
discard the kit.
Calibration
All of the following must be met:
o Standard curve must have a correlation
coefficient of >0.99;
o Average absorbance value, A0, for SO must
be >0.80; and
If any requirement fails:
• Results from the analytical run
are not reported.
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 37 of 185
Kit Control
Negative Control
Sample
Evaluations
Results Within
Calibration Range
External Quality
Control Sample
o Standards S0-S5 must have decreasing
average absorbance values. That is, if A, is
the average of the absorbance values for
Si, then the absorbance average values
must be: A0> Ai> A2> A3> A4>A5
The average concentration value of the duplicates
(or triplicate) must be within the range of 0.75 +/-
0.185 ng/L. That is, results must be between 0.565
and 0.935.
The values for the negative control replicates must
meet the following requirements:
o All concentration values must be < 0.15
Hg/L (i.e., the reporting limit); and
o One or more concentration results must
be nondetectable (i.e., <0.10 ng/L)
All samples are run in duplicate. Each duplicate pair
must have %CV<15% between its absorbance
values.
All samples are run in duplicate. If both of the
values are less than the upper calibration range
(i.e., 5.0 ng/Lfor undiluted samples), then the
requirement is met.
External QC Coordinator, supported by QC
contractor, provides 1-2 sets of identical samples
to all laboratories and compares results.
All samples in the analytical run
are reanalyzed until calibtration
provides acceptable results.
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.
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%).
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.
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
37
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 38 of 185
QC Coordinator will determine an
appropriate course of action,
including no action, flagging the data,
or excluding some or all of the
laboratory's data.
4.8 Sample and Record Retention
The laboratory shall retain:
1. The sample materials, including vials, for a minimum of 3 years from the date the EPA publishes
the final report. During this time, the laboratory shall freeze the materials. The laboratory shall
periodically check the sample materials for degradation.
2. Original records, including laboratory notebooks and the reference library, for a minimum of 10
years from the date that EPA publishes the final report.
After the stated time periods, the laboratory shall follow its internal protocols for disposal.
4.9 References
Abraxis, "Microcystins-ADDA ELISA (Microtiter Plate)," Product 520011, R021412, Undated. Retrieved
January 2014 from
http://www.abraxiskits.com/uploads/products/docfiles/278 Microcvstin%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 PN520011FLQW.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
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 39 of 185
5.0 BENTHIC MACROINVERTEBRATES
5.1 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 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 5.1, 5.2, 5.3, and 5.4 summarize the procedure; health and safety
concerns; and required resources. Section 5.5 provides the steps for acknowledging sample receipt.
Section 5.6 and Section 5.7 provide the steps for preparing and sorting the sample. Sections 5.8 - 5.10
provide the steps for the taxonomy identification, data entry, and sample and record retention. Section
5.11 describes EPA's external review of laboratory operations. Section 5.12 identifies references used in
developing the procedure. Table 5.8 summarizes the quality control measures.
5.2 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.
During the identification step, a taxonomist identifies the picked organisms to the target taxonomic
levels for the survey. 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.
5.3 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.
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 40 of 185
5.4 Definitions and Required Resources (Laboratory, Personnel, and
Equipment)
This section provides definitions and required resources for using this procedure. Section 5.4.1 defines
the terms used throughout the procedure. Section 5.4.2 describes the expertise required for each
laboratory using the procedure. Section 5.4.3 describes the roles and responsibilities of the personnel
involved in the procedure. Section 5.4.4 identifies the equipment necessary to apply the procedure in
preparing, sorting, and identifying benthic macroinvertebrate organisms in samples.
5.4.1 Definitions
The following terms are used throughout the procedure:
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.
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.
Distinct taxa: Data analysts use the number of distinct (i.e., unique) taxa within a given sample to
evaluate the richness associated with the sample location. The distinctness attribute is assessed sample
by sample, and not across all samples. To facilitate the data analyses, the database includes an
additional variable ("flag") that is used for the first identification of a particular taxon in a sample.
Section 5.8 provides the steps used to identify which taxa are flagged.
Elutriate: Circulate water over the sample in order to wash away the lighter or finer particles of the
detritus.
Grid: Each individual square within the Caton tray
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.
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 = ——— x 100
A + B (!)
Percent disagreement in enumeration (PDE): measure of taxonomic precision comparing the number of
organisms, rii, counted in a sample by the primary taxonomist with the number of organisms, n2,
counted by the internal or external QC taxonomist.
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 41 of 185
PDE =
x 100
m +n
'2
(2)
Percent taxonomic disagreement (PTD): measure of taxonomic precision comparing the number of
agreements (positive comparisons, comppos) 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.
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.
Secondary laboratory: The laboratory selected by the External QC Coordinator. It provides an
independent identification of the benthic macroinvertebrates in the sample. The secondary laboratory
must provide QC taxonomists who did not participate in the original identifications for the sample.
Subsample: Portion of the sample obtained by random selection and division.
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
quarter) is completed.
Target taxonomic levels: Table 5.4 and Table 5.5 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 Table 5.4 and Table 5.5.
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 the TSNs. (This is not the
identifier that should be used to identify organisms with this procedure. See WQX below for appropriate
identifier.)
5.4.2 Laboratory
The procedure may be used by any laboratory that has expertise in each of the following areas:
COmPvos
PTD = 1 ^ x 100
N
(3)
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 42 of 185
1. Analytical work: To demonstrate its expertise, the laboratory shall provide EPA with one or more
of the following:
a. Memorandum that identifies the relevant services that the laboratory provided for the
National Aquatic Resource Surveys in the past five years.
b. Dated copy of relevant Accreditation or Certification (NELAC, ISO, state, etc.) for the
laboratory and/or its experts who will perform and/or oversee the analyses. The
accreditation must be for the entirety of analysis that the laboratory will be performing.
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.
5.4.3 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
a >90% sorting efficiency) for missed organisms and records the number of missed
organisms in the appropriate field of the Bench Sheet.
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 43 of 185
• Checks 1 in 10 of an experienced individual's samples, with a minimum of 1 NRSA sample. If
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 each sample and sorter. The sorter's sorting efficiency
is recorded on the Sorter Bench Sheet.
Internal Taxonomy QC Officer is an experienced taxonomist who:
• 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. 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 QCTaxonomists, are selected by the QC contractor, and have demonstrated expertise
and experience to be used as a quasi "gold standard" for taxonomic evaluations. A QC
taxonomist may work for both a primary and secondary laboratory, but must be assigned to
different samples so that the external QC review is independent of the original identification.
QC Contractor supports the External QC Coordinator and is responsible for obtaining the
External QCTaxonomists. The QC contractor must not have previously been involved with field
sampling and/or benthic macroinvertebrate analyses.
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.
5.4.4 Equipment/Materials
The procedure requires the following equipment and materials for sample preparation (subsampling),
sorting, and taxonomic identifications.
5.4.4.1 Sample Preparation (Subsampling) and Sorting Equipment/Materials
• U.S. 35 sieve (500 pim)
• Round buckets
• Standard gridded screen (370-nm mesh screen, 30 squares, each 6 cm2) with white plastic
holding tray (Caton tray, 30cm x 36cm, 4cm deep)
• 6-cm scoop
• 6-cm2 metal dividing frame ("cookie cutter")
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 44 of 185
• White plastic or enamel pan (6" x 9") for sorting
• Scissors
• Teaspoon
• Permanent ink pen (e.g Pigma Micron® pen)
• Dropper
• Fine-tipped forceps (watchmaker type, straight and curved)
• Vials with caps or stoppers
• Sample labels for vials
• 70-80% 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)
5.4.4.2 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 Micron® pen)
• Dropper
• Fine-tipped forceps (watchmaker type, straight and curved)
• Vials with caps or stoppers
• Sample labels for vials
• 70 - 80% non-denatured ethanol in plastic wash bottle
• Taxonomic Bench Sheet (provided to each participating laboratory by EPA)
• Hand tally counter
5.5 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:
a. Refill them with 70-80% ethanol if necessary.
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 45 of 185
b. Verify that the site identification and sample number on the label also appear on the chain
of custody form in the shipment.
c. Notify the NARS IM team if any jars were broken and/or there are discrepancies between
the custody form and jars.
3. After refilling the sample containers, store them at room temperature until sorting begins.
4. Maintain the chain-of-custody form with the samples; it will be needed if the samples are
transported to any other location (e.g., for taxonomic identification, external QC evaluation).
5. Verify that the login information includes the required data elements in Table 5.1. After
completing all required elements, provide the information to the data entry personnel.
Table 5.1 Benthic macroinvertebrate: required data elements - login
FIELD
FORMAT
DESCRIPTION
LAB NAME
text
name of lab
LAB ID (optional)
text
lab sample id
DATE RECEIVED
MMDDYY
date sample was received by lab
SITE ID
text
NRSA site identification code as used on sample label
VISIT NUMBER
numeric
sequential visits to site (1 or 2, if specified on label)
SAMPLE ID
numeric
sample number as used on field sheet (on sample label)
DATE COLLECTED
MMDDYY
date sample was taken
OA FLAG (if
appropriate)
text
QA/QC flag (lab may use its own flags, if defined in QA_COMMENTS
field or provided to NARS IM team)

Flag
Definition

NP
Not enough preservative used

S
Sample shipping problem (explain in QA_COMMENTS
field)

Q
Other quality concerns, not identified above
QA.COMMENTS
text
explanation for QA FLAG (if needed)
LAB COMMENTS
text
general laboratory analysis comments
5.6 Subsampling
1. Remove the lid from the sample container(s) and pull out the internal sample label (save the label-
it will need to be returned to the sample container with the archived portion of the sample that
does not get processed). Record sample collection information on a Benthic Macroinvertebrate
Laboratory 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
field team who collected the sample (e.g., Team 1). Set the bench sheet aside.
2. Carefully decant the alcohol from the sample container by pouring the fluid through a sieve (U.S. 35)
into a separate container (the alcohol is saved to preserve the archived portion of the sample that
does not get processed). Inspect the mesh of the sieve for any organisms and return organisms
found to the sample.
3. Transfer the homogenized sample material to the gridded screen portion of the grid (use more than
one subsampling device if necessary). Wash the sample thoroughly by running tap water over it to
remove any fine material. If there is more than one jar for any particular sample, empty and wash
each jar onto the Caton-type grid one at a time, making sure to spread each jar's contents evenly
across the tray. Multiple jars from the same sample should all be emptied onto the same Caton grid
(or suitable alternative subsampling tray). If the amount of leaf litter or other detrital material
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 46 of 185
exceeds that which fills they tray to the level of the wall panels (if should be spread as evenly as
possible), it can be divided among two or more trays.
a. Elutriation of a sample is acceptable for samples with heavy amounts of inorganic substrate
(e.g., sample that has 4 or 5 jars total and 2 or 3 with gravel or sand) once it has been delivered
to the lab, before subsampling has begun on that particular sample. An example of an
acceptable elutriation method is as follows:
i. Pour alcohol off of sample containers through sieve (at least 500 pim). Also deposit leaf
litter and any other organic material (leaves, sticks, algae) onto sieve.
ii. Depending on amount of inorganic material (gravel, sand, silt), pour all or a portion of this
material into a rectangular Tupperware/Rubbermaid container and cover with water.
iii. Circulate (elutriate) sample with water and allow any organisms that might be in the
gravel/sand to float to the top of the water and pour the water through the sieve.
iv. Repeat this until the water runs clear.
v. Fill the plastic container (that still has the inorganic material in it) with water one more time,
and take it to a well lit, flat surface. Inspect it here under a ring light w/ 3x magnification for
any remaining organisms. Have another sorter double check for organisms.
vi. Once you are certain there are no organisms remaining in the plastic container, wash the
water through the sieve and dump the inorganic material into a waste bucket.
vii. Repeat this process until all of the inorganic material has been elutriated and checked for
heavier organisms, such as clams, mussles, or worms.
viii. Spread the sample now in the circular sieve over the 30-grid Caton tray.
4. Place the gridded screen into the larger white tray. (Note: Some Cooperators may not use the
gridded screen and holding tray). Add enough water to spread the sample evenly throughout the
grid (the water level should be relatively close to the top of the white tray). Spread the sample
material over the bottom of the pan as evenly as possible. Move the sample into the corners of
the pan using forceps, spoon, or by hand. Vibrate or shake the pan gently to help spread the
sample.
5. Lift the screen out of the white tray to drain. Pour off or siphon excess water from the white tray
and set the screen back into the tray. Leave just enough water in the bottom of the tray so that it
barely covers the screen once it is returned to the tray to allow the sample to remain moist.
6. Use a random number generator to select at least 10% of the grids, usually 3 grids (in a 30-grid tray)
to process (select one letter and one number, e.g., A-5, F-2). Three grids are sorted from the
sample to ensure that the subsample material is representative of the overall sample. Remove all
the material using the following procedure from that grid and place the removed material into a
separate holding container, such as a white plastic or enamel pan. If two trays are being used to
hold a large sample, the same grid on the second pan will also be removed. Continue until the
material from all 3 grids is removed. The material is removed as follows:
a. Place the metal dividing frame or "cookie cutter" over the sample at the approximate
location of the grid selected for processing (based on the letters and numbers marked on
the sides of the gridded tray). Use a pair of rulers or other straight edges to facilitate lining
up the cookie cutter at the intersection if necessary.
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 47 of 185
b. Remove the material within the "cookie cutter" using the 6-cm scoop, a teaspoon, forceps,
or dropper. Depending on the consistency of what is in the sample, it might be necessary to
cut the material along the outside of the "cookie cutter" with scissors or separate it with
forceps so that only one grid's worth of sample material is used. Inspect the screen for any
remaining organisms. Use the following rules when dealing with organisms that lie on the
line between two grids:
• An organism belongs to the grid containing its head.
• 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.
• If the head of an organism lies on the line between two grids, all organisms on the top
of a grid and those on the right side of a grid belong in that grid, and are picked with
that grid.
c. Quarter the grid (if necessary, see Section 5.7, #1). Place the material from the selected
grid(s) into a separate white plastic or enamel pan. Add the necessary amount of water to
the pan to facilitate sorting.
d. Set the subsampling device aside in case more grids need to be retrieved later. Cover the
sample with aluminum foil to prevent desiccation of the sample and damage to specimens
(periodically moisten the sample with water from a spray bottle if the top layer begins to
dry). Between each subsampling operation, be careful not to disturb the subsampling
device to prevent redistribution of specimens, which could possibly change the probability
of selection.
5.7 Sorting
1. At least 10% of the tray or three grids in the case of a Caton tray (assuming 30 grids) would be
randomly selected.
a. If the number of organisms appears to exceed the target number (500 organisms) in the
collective three grids, then each grid is quartered, and a quarter is randomly selected for initial
sorting. The quarter volume of the first grid would be sorted, with the remaining two grids
(quartered) being sorted in successive order (compositing of the first three grids is not done).
b. If the number of organisms is below the target, then another fraction of each grid would be
processed until the target number of 500 and a maximum of 600 (500+20%) is reached. All
organisms from the selected fraction, or grid, must be sorted to minimize remove bias.
c. If the target is not reached when the three grids are completely picked fully processed
(including organisms recovered during QC checks), subsequent grids would be randomly
selected and each picked to completion until 500+20% organisms is reached. If the target
number of organisms is reached within the fraction of the first or second grids, sorting is
stopped for that sample, on completion of the sorting of the corresponding fraction (i.e., the
third grid quarter would not be processed).
2. Remove the macroinvertebrates from the detritus with forceps. All samples will be sorted under a
minimum of 6x (maximum of lOx) dissecting microscope. Quality control checks will also be
performed using the same power microscope. Place picked organisms in an internally labeled vial
(or larger container, if necessary) containing 70-80% denatured ethanol.
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 48 of 185
3. 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 Benthic Macroinvertebrate Laboratory Bench Sheet. Enter
the sorter's initials in the appropriate column on the bench sheet for each grid sorted.
4. Do not remove or count empty snail or bivalve shells, specimens of surface-dwelling or strict water
column2 arthropod taxa (e.g., Collembola, Veliidae, Gerridae, Notonectidae, Corixidae, Cladocera, or
Copepoda), or incidentally-collected terrestrial taxa. Also, do not count fragments such as legs,
antennae, gills, or wings. For Oligochaeta, attempt to remove and count only whole organisms and
fragments that include the head; also, do not count fragments that do not include the head. If a
sorter is unsure as to whether a specimen should be counted or not, he or she should place the
organism in the sort vial without counting it (the final count is made by the taxonomist).
Table 5.2 Benthic macroinvertebrate: list of taxa that are not to be sorted/counted
All terrestrial life history stages are to be excluded from the counts.
TAXON
Phylum
Class (or Subclass)
Order
Family
ANNELIDA
Clitellata
Branchiobdellida

ARTHROPODA
Cladocera and other
Branchiopoda

Collembola

Insecta
Ephemeroptera (adults only)

Diptera (larvae of listed families, plus all adults)
Chaoboridae

Culicidae

Coleoptera
Dytiscidae (adult)

Gyrinidae (adult)

Heteroptera
Corixidae

Gerridae

Mesoveliidae

Notonectidae

Pleidae

Neuroptera (adults)

Odonata (adults)

Plecoptera (adults)

Trichoptera (adults)

Ostracoda

BRYOZOA

NEMATODA

5. Each sample, once it is picked by the initial sorter, must be checked for missed organisms before
another sample is processed. This step is performed by an experienced, certified, laboratory QC
Officer, as detailed below. Any missed organisms found by the QC Officer will be counted and
placed into the sample vial, or other suitable sample vial, and the number of organisms missed will
be noted on the Benthic Macroinvertebrate Laboratory Bench Sheet and added to the final count of
the sample.
2Strict water column taxa are those that do not have at least one life stage that is benthic (i.e., bottom-
dwelling).
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 49 of 185
a. If the last grid (or quarter) being processed results in more than 600 organisms (i.e., > 20%
above target number), evenly redistribute all of the organisms (without detritus) in a petri dish
(or other small container, i.e., finger bowl, etc.) divided into pie slices (1-8) containing water to
cover. Randomly choose slices and count organisms that are wholly contained within the slices.
If an organism is lying between two slices, use the criteria in Section 5.6 #6 (b) to determine
which slice it belongs in. Choose slices until you reach the target number (500 +20%). As with
picking grids and quarters, the sorter must pick an entire pie slice, even if it the sample goes
over 500 organisms as long as it remains under 600 total organisms.
6. Once the QC check of the material in the pan has been completed, it is removed from the pan and
placed in a separate container with preservative (70-80% ethanol). The container should be labeled
"Sorted Residue", on both internal and external labels ("Sorted Residue" will include material from
all grids processed for each sample). Internal sample labels should be made of cotton rag paper or
an acceptable substitute, recording the same information as before.
7. After the QC check is completed, and the target number has been reached, search the entire tray for
5-10 minutes, looking for large/rare organisms (Vinson and Hawkins, 1996). Large/rare is defined as
any organism larger than Vz" long and found in less than 1/8 of the tray holding the entire sample.
Place any organisms found into a vial labeled "L/R" for "Large/Rare".
8. All material not subsampled (remaining on the grid) must be returned to the original container with
the preservative. This container should include the original sample labels and a separate label
"Unsorted Sample Remains" should be placed inside the container and on the outside. The lid
should be tightly closed and the container archived until all appropriate QC checks are completed
(subsampling and taxonomy). The decision to discard any sample portion should be done only
following joint approval of the QC officer and the Project Manager.
9. Record the sorting date each sample was completed near the top right corner of the bench sheet.
Verify that the bench sheet includes the required data elements in Table 5.3.
Table 5.3 Benthic macroinvertebrate: required data elements - sorting
FIELD FORMAT DESCRIPTION
LAB NAME
Text
name of lab
LAB ID (optional)
Text
lab sample id
DATE RECEIVED
MMDDYY
date sample was received by lab
SITE ID
Text
NRSA site identification code as used on sample label
VISIT NUMBER
numeric
sequential visits to site (1 or 2, if specified on label)
SAMPLE ID
numeric
sample number as used on field sheet (on sample label)
DATE COLLECTED
MMDDYY
date sample was taken
DATE SORTED
MMDDYY
date that the sorter started working on the sample
PROPORTION
SORTED
numeric
proportion of sample sorted based upon number of grids and
quarters selected and available
CORRECTION
FACTOR
numeric
subsampling correction factor
OA FLAG (if
appropriate)
text
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
<
QC
CO
QC
LU
>
o
QC
u
<
u
CO
49
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 50 of 185
DD Damaged Organism
IM . Immature
IN Indeterminate (explain in QA_COMMENTS field)
NP Not enough preservative used
NT Not able to meet target level for identification (may be used
with other codes, or explain in QA_COMMENTS field)
S Sample shipping problem (explain in QA_COMMENTS field)
UN Unknown. Identification is tentative. Organism has been sent
; to expert taxonomist for definitive identification.
Q Other quality concerns, not identified above
QA_COMMENTS text explanation for QA FLAG (if needed)
LAB COMMENTS text general laboratory analysis comments
5.8 Taxonomy Identification
1. On receipt of a set of sample vials from the project cooperator or contractor laboratory, remove the
chain-of-custody form from the shipping container, sign and date it to verify that the samples were
received (in the "received by" space). Compare all sample numbers on the form with those entered
on the labels of samples that actually were in the shipment. If any vials were broken, notify the
project facilitator immediately. Maintain the chain-of-custody form with the samples; it will be
needed to return the samples.
2. Empty one sample vial at a time into a small petri dish. Add 80% denatured ethanol to keep the
organisms covered. Remove the internal sample label and complete the top portion of a Benthic
Macroinvertebrate Taxonomic Bench Sheet (Appendix C; Sample Laboratory Forms, or comparable
system), using the information from the label or that provided by the project facilitator.
3. Begin by viewing the sample under the stereo dissecting microscope and removing similar
organisms to other dishes (keep covered with 80% ethanol). Organisms should be identified to the
correct taxonomic level for the project (usually genus, Table 5.4 and Table 5.5). However,
according to the laboratory manager's discretion, a taxonomist can identify any organism farther
than the target level if they are confident in the identification. Record the identifications on the
Benthic Macroinvertebrate Taxonomic Bench Sheet (under taxon). Enter the number of larvae,
pupae, and adults of each taxon under those columns on the bench sheet. Also enter the
Taxonomic Serial Number (TSN; found in ITIS). Use the following steps to compare the final taxa list
for each site to that of the ITIS website (http://www.itis.usda.gov) Record the TSN from ITIS on the
Electronic Bench Sheet (Table 5.6).
a. Copy block of taxa names to a text file.
b. Save the text file
c. Go to the ITIS taxa match screen (http://www.itis.usda.gov/taxmatch_ftp.html)
d. Follow the onscreen instructions to upload the file. Use all of the current defaults.
e. Finish with two lists, one of matches with TSNs and one with non-matches. Check the non-
matches for the following common problems.
i. Abbreviations
ii. Extra information identifiers (e.g., sp., spp.,, nr., cf., genus 1, w/ hair chaete)
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 51 of 185
VIII.
XI.
x.
VII.
VI.
v.
mi
IX.
IV.
Extra character (e.g., "?", "Acentrella Pturbida", blank space)
The word "probably" or "prob" (e.g., "Microcylloepus prob. similis")
I Ding to a lower level than is in ITIS (e.g, to species rather than genus)
Double names (e.g., Callibaetis callibaetis)
Common misspellings
Tribes/Subfamilies/Subgenus sometimes do not appear in ITIS
Species w/ incorrect Genus (Hydatopsyche betteni)
Split level taxonomy (e.g., Cricotopus/Orthocladius)
Invalid name (e.g., taxonomic change, synonym; Sphaeriidae vs. Pisiidae)
xii. Valid name, in scientific literature, but not in ITIS (e.g., appears in Merritt & Cummins
(1996) or Epler (2001), but not listed in ITIS - will not have a TSN)
4. Prepare slide mounts of Chironomidae and Oligochaeta as needed using CMCP-10 (or CMC-9, CMC-
10, or other media) and applying a coverslip. View these organisms under the compound
microscope to ensure that all necessary diagnostic characters have been observed, according to the
taxonomic key or other literature. Record the identifications on the bench sheet as above. The slides
should be labeled with the same sample number or log-in number as the alcohol specimens.
5. Prepare a list of primary and secondary technical literature used in completing the identifications.
Provide complete citations in bibliographic format, including authors' names, date of publication,
title of document, name of journal or publisher, volume and page numbers, or ISBN number, as
appropriate. These will be kept on file with the project QC officer.
6. If damaged organisms can be identified, they are counted ONLY if:
(a) the fragment includes the head, and, in the case of
• arthropods, the thorax
• oligochaetes, a sufficient number of segments
(b) the mollusk shell (bivalve or gastropod) is occupied by a specimen
(c) the specimen is the sole representative of a taxon in the sample
7. If early instar or juvenile specimens can be identified, they are counted:
(a) as the same taxon, if with confidence, they can be associated with one or more mature
specimens that have a more developed morphology.
(b) as a separate taxon, if the specimen is the sole representative of a taxon in the sample.
8. Add the number of organisms from each developmental stage and enter the total on the bench
9. Complete the bench sheet by entering totals for each developmental stage and the total number
of each taxon in the cells at the bottom of the sheet. Cross-check to be sure the totals were
summed correctly. Make a copy of the bench sheet for the project file.
sheet.
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 52 of 185
10. Create a reference collection with at least one specimen from each genus (or lowest taxonomic
level IDed). When a sample is chosen to be the source of specimen(s) to represent a name in the
master taxa list, the appropriate specimen(s) in that sample representing the concept of that
taxon to the taxonomist should be removed and placed in the reference collection. Labels will be
placed in the primary sample container indicating the placement of any specimen(s) removed for
the reference collection. Circle slide-mounted specimens with a grease pencil (or other
appropriate mark) to indicate those which belong to the reference collection. For all slides
containing reference and non-reference specimens, be sure to place a label in the sample
container that does not contain the reference collection. Each laboratory should maintain a
master list of taxa recorded. The project facilitator will coordinate any necessary inter-lab
communication and produce and integrated master taxa list for the project.
11. Carefully return the rest of the organisms to the original sample vial, fill with 70-80% denatured
ethanol, and cap tightly.
12. Re-package the samples and slide mounted specimens carefully, and sign and date the chain-of-
custody form in the next "relinquished by" space. The samples should be shipped, properly packed
in a box, by overnight carrier to the project facilitator, and receipt confirmed by the person doing
the shipping. Each taxonomist should retain a full set of bench sheet copies, and ship the original
bench sheets in an envelope to the project facilitator. Samples and bench sheets should be
shipped separately.
Table 5.4 Benthic macroinvertebrate: target level of taxonomic identification - benthics commonly found in
freshwater

Target Level of
TAXON
Identification
Phylum
Class (or
Subclass)
Order
Family
Family
Genus
ANNELIDA
(any, except
Hirudinea,
Polychaeta)

X
Hirudinea

X
Oligochaeta
(any, including
Tubificinae, except as
noted below)

X
Enchytraeidae

Lumbriculidae

Polychaeta

ARTHROPODA
Hydrachnidia

X
Insecta
(any, except as noted)

X
Coleoptera

X
Diptera (any, except as
noted)

Ceratopogoninae
subfamily

Chironomidae
see Table
4

Dolichopodidae
X

Empididae
X

<
cc
CO
cc
LU
>
o
cc
u
<
u
CO
52
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 53 of 185
TAXON

Target Level of
Identification

Ephydiridae
X

Muscidae
X

Phoridae
X

Scathophagidae
X

Sciomyzidae
X

Stratiomyidae
X

Syrphidae
X

Tabanidae
X

Ephemeroptera

Hemiptera (Heteroptera)

Lepidoptera

Megaloptera

Odonata

Plecoptera

Trichoptera

Malacostraca
(any)

Amphipoda

Decapoda

Isopoda

Mysidacea

X
COELENTERATAt

MOLLUSCA
(any, except
Hydrobiidae)

Bivalvia

Gastropoda
(any, except
Hydrobiidae)

Hydrobiidae

NEMERTEA

X
PLA TYHELMINTHES
Turbellaria
anyt

+Colelenterata need only be identified to the phylum level.
$ Turbellaria need only be identified to the class level.
Table 5.5 Benthic macroinvertebrate: target Level of taxonomic identification -chironomidae
FAMILY GENUS REQUIRED LEVEL OF IDENTIFICATION
CO
LU
CC
CO
LU
I—
CC
LU
>
O
cc
u
<
u
=C
I—
LU
CO
Chironomidae (except as noted
below)
Genus

Cricotopus
Cricotopus/Orthocladius if a clear distinction cannot be made

Orthocladius
Cricotopus/Orthocladius if a clear distinction cannot be made

Conchapelopia
Thienemannimyia genus group

Hayesomyia

Helopelopia

Meropelopia

Rheopelopia

Telopelopia

Thienemannimyia
53
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 54 of 185
Table 5.6 Benthic macroinvertebrate: required data elements -taxonomic identification
FIELD FORMAT DESCRIPTION
LAB NAME
text
name of lab
LAB ID (optional)
text
lab sample id
DATE RECEIVED
MMDDYY
date sample was received by lab
SITE ID
text
NRSA site identification code as used on sample label
VISIT NUMBER
numeric
sequential visits to site (1 or 2, if specified on label)
SAMPLE ID
numeric
sample number as used on field sheet (on sample label)
DATE COLLECTED
MMDDYY
date sample was taken
DATE TAXON
MMDDYY
date that the taxonomist started identifying organisms in the sample
ANALYST NAME
text
name of taxonomist or Internal Taxonomy QC Officer (if record
provides results of QC check)
QC VERIFICATION
text
Y if the record provides the results from the QC check
FAMILY
text
taxonomic family
SUBFAMILY
text
taxonomic subfamily
TRIBE
text
taxonomic tribe
GENUS GROUP
text
taxonomic genus group (e.g., Thienemannimyia)
GENUS
text
taxonomic genus
SPECIES
text
taxonomic species
WQX_TSN
numeric
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 TIN (OPTIONAL)
text
lab taxa ID number
TAXANAME
text
unique taxon name in WQX
ABUNDANCE LARVAE
numeric
number of individual larvae or immature bugs
ABUNDANCE PUPAE
numeric
number of individual pupae
ABUNDANCE ADULT
numeric
number of individual adults
ABUNDANCE TOTAL
numeric
total number of individuals
DISTINCT
text
distinct taxa in sample (y/n) (See description in Section 5.8)
CITATION
text
citation for reference used to identify organism, if taxon not present
in WQX database
OA FLAG (if appropriate)
text
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
NT
Not able to meet target level for identification (may be used
with other codes, or explain in QA_COMMENTS field)
S
Sample shipping problem (explain in QA_COMMENTS field)
UN
Unknown. Identification is tentative. Organism has been sent
to expert taxonomist for definitive identification.
Q
Other quality concerns, not identified above
QA.COMMENTS
text
explanation for QA FLAG (if needed)
LAB COMMENTS
text
general laboratory analysis comments
<
cc
CO
cc
LU
>
O
cc
u
<
u
CO
54
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 55 of 185
5.9 Data Entry
Table 5.1, Table 5.3 and Table 5.6 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.
5.10 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.
2. Original records, including laboratory notebooks and the reference library, for a minimum of 10
years from the date that EPA publishes the final report.
After the stated time periods, the laboratory shall follow its internal protocols for disposal.
5.11 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 QC contractor 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 QC Contractor will
randomly select three samples for the evaluation.
b. If the laboratory is responsible for processing 100 samples or more for the survey, the QC
contractor 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 contractor).
c. To the extent practicable, the External QC Coordinator and QC contractor will schedule
batch evaluations evenly throughout the project period.
2. The QC contractor coordinates and pays for delivery of the selected samples, bench sheets, and
chain-of-custody forms.
3. The primary laboratory packs the selected samples for shipping to the central holding facility
designated by the QC contractor. Although the QC contractor 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
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 56 of 185
this information must be given to the QC contractor. In the event that the reference
specimens are the only organisms representing a given taxon for a given sample, then the
primary laboratory must make these specimens available by providing a digital image and/or
one specimen from the reference library. If there is only a small number of the organisms,
the primary laboratory also shall provide the count of the organisms.
c. Shipment must contain chain-of-custody documentation.
4. 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.
5. Upon receipt, the secondary laboratory sends a confirmation to the QC contractor that the
samples have arrived. Within 24 hours, the secondary laboratory completes and returns the
chain-of-custody forms to the QC contractor.
6. The QC taxonomist:
a. Performs whole-sample re-identifications, taking care to ensure inclusion of all slide-
mounted organisms. The QC taxonomist follows the procedures in Taxonomy Identification
Section 5.8 with the following modifications:
i. All Chironomidae and Oligochaeta organisms must be mounted on slides (i.e.,
the waiver is not an option). Before and after mounting, count the number of
organisms. Compare the two numbers and attempt to resolve any discrepancies
(e.g., check for organisms remaining on the forceps).
ii. Step 5: Classifying organisms as "Distinct" is not relevant for the QC evaluations,
and thus, not required.
iii. Step 9: All organisms provided by the primary laboratory must be identified and
counted (i.e., 500 count restriction does not apply).
iv. Step 10 and 14: Internal QC taxonomist steps do not apply.
v. Step 13: Physical and digital reference libraries are not required.
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 5.9 for data entry instructions). The QC taxonomist shall
label the bench sheet and database entries with the term "QC Re-ID."
7. The External QC Coordinator will compare the taxonomic results (counts AND identifications)
i/i
generated by the primary and secondary laboratories. If either laboratory identified the £!

different than used by WQX or other acceptable references, the QC contractor will substitute
the required level or name, respectively, in the comparisons. The External QC Coordinator will
calculate the percent difference in: q
a. Enumeration as measured by PDE defined in Section 5.4.1; and u
<
b. Taxonomy as measured by PTD defined in Section 5.4.1. Table 3 of Stribling (2003) provides ^
u
examples of what would be considered agreements. ^
i—
¦z.
LU
CO
56
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 57 of 185
8. If any sample has PDE>5% and/or PTD>15%, the QC contractor, with the External QC
Coordinator's approval, will facilitate a reconciliation call with EPA and the laboratories. If the
secondary laboratory has digital photography equipment, the QC Taxonomist shall provide
digital photographs of organisms that will be discussed during the reconciliation call. The
External QC Coordinator may decide that a reconciliation call (and the digital photographs) 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
following occurs:
a. 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.
b. The QC contractor, or the secondary laboratory, will return the samples to the primary
laboratory following appropriate tracking procedures.
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
extreme 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.
5.12 Quality Assurance/Quality Control (QA/QC)
Equation 5.1 Percent sorting efficiency (PSE).
CO
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%. ^
CO
LU
I—
QC
LU
>
A
PSE = x 100
A + B
Equation 5.2 Percent disagreement in enumeration (PDE). g
Measure of taxonomic precision comparing the number of organisms, rii, counted in a sample by the
primary taxonomist with the number of organisms, n2, counted by the internal or external QC
taxonomist. PDE should be <5%.
0
QC
u
<
u
1
I—
z
LU
CO
57
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 58 of 185
PDE =
100
n,

*1 1 "2
Equation 5.3 Percent taxonomic disagreement (PTD).
Measure of taxonomic precision comparing the number of agreements (positive comparisons, compp0s)
of the primary taxonomist and internal or external QC taxonomists. In the following equation, N is the
total number of organisms in the larger of the two counts. PTD should be <15%.
PTD =
comp
\
pos
N
x 100
Table 5.7 Benthic macroinvertebrate: measurement data quality objectives
Variable or Measurement
Precision
Accuracy 1
Sort and Pick
90% a
90% a
Identification
85% b
95% c
NA = not applicable;a As measured by PSE;b As measured by (100%-PTD);c As measured by (100%-PDE)
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 59 of 185
Table 5.8 Benthic macroinvertebrate: quality control - laboratory
Check or Sample Frequency Acceptance Criteria Corrective Action
Description
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
<
cc
CO
cc
LU
>
o
cc
u
<
u
CO
59
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 60 of 185
External QC
10% of all samples
completed per
laboratory
PDE < 5%
PTD < 15%
If PDE > 5%, implement
recommended corrective actions.
If PTD > 15%, implement
recommended corrective actions.
Use of
widely/commonly
accepted
taxonomic
references by all
NRSA labs
For all
identifications
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 5.4.1 for retrieval
instructions). This
requirement demonstrates
the general acceptance of
the references by the
scientific community.
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.
Prepare reference
collection
Each new taxon
per laboratory
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
DATA VALIDATION
Taxonomic
"reasonable-ness"
checks
All data sheets
Taxa known to occur in
given rivers or streams or
geographic area
Second or third identification by
expert in that taxon
5.13 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.
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 61 of 185
6.0 FECAL INDICATOR: ENTEROCOCCI
6.1 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 < -20°C freezers will be subjected to DNA extraction (e.g. bead-beating) and
purification processes leading up to qPCR analysis. This processing can be completed up to 1 year after
cell concentration if the sample filter retentates are maintained frozen at -20 to -80°C.
6.2 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 6.4 of Section
6.18.
6.3 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
three, analyzed during the same week.
Bottle Blank: Analyte-free water is collected into a sample container, of the same lot number as the
containers used for collection of the environmental samples. Analysis of this sample is performed to
evaluate the level of contamination, if any, introduced into the environmental and control samples from
the sample container(s) from a common vendor's lot.
DNA: Deoxyribo-Nucleic Acid, double-stranded genetic molecules containing sequences of the four
nucleotide bases, adenine, thymine, guanidine, and cytosine that encode rRNA, mRNA, and tRNA
involved in protein synthesis.
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 62 of 185
Field Filter Blank: A volume of sterile PBS, free of target organisms (i.e. Enterococcus) filtered through a
sterile filter and processed in parallel with all other samples to serve as a sentinel for detection of
reagent contamination or contamination transferred between samples by processing and analysis.
Field Replicates: Samples collected from rivers and streams that are collected at the same sampling site
one right after the other with only slight temporal variation. They are not "splits" of the same sample
volume.
Filtrate: Sample liquid or buffer rinsate passing through the filter into the vacuum flask.
Laboratory Quality Samples: Mock samples created in the lab such as lab blanks, lab-fortified blanks
(LFBs), and Lab-Fortified Matrices (LFMs) used to assure lack of sample contamination and to measure
analytical recovery during performance of sample processing and analysis methods.
Performance Testing (PT) / Performance Evaluation Sample (PES): Calibrator samples (filters spiked
with E.faecalis grown in Brain Heart Infusion Broth) and Laboratory Fortified Blanks (Phosphate
Buffered Saline; PBS) spiked with Enterococcus faecalis cells from BHI Broth suspension) will be assayed
by EPA Method 1600 and Draft EPA Method 1606 to ascertain method performance. Ball-T Bioballs®
which contain a specified number of E. faecalis cells may also be acquired to determine the performance
of the Relative Quantitation Method. Purified E.faecalis DNA acquired from the American Type Culture
Collection and TIB Mol Biol Inc. is used to test the performance of the Absolute Quantitation Method.
Retentate: The sample residue retained by the filter after the sample is vacuum-filtered. The retentate
contains particulates, microbiota, and macrobiota from which the DNA is extracted into buffer by bead-
beating for subsequent qPCR analysis.
Rinsate: The volume of phosphate buffered saline (PBS) applied to a sample's filter retentate in order to
"wash" any residual fine particles, smaller than the filter's nominal pore size, through the retentate and
the filter.
Sample Processing Control (SPC): A surrogate homologue analyte (e.g. Salmon DNA) spiked into each
sample to determine the recovery of target analyte and/or detect assay inhibition caused by matrix
effects.
Standards: Known amounts or numbers of copies of Enterococcus genomic DNA analyzed by the
Enterococcus qPCR assay to generate a Standard Curve (Log Copy Number vs. Crossing Point Value) in
order to determine Enterococcus genomic copy numbers in "Unknown" test sample extracts by Absolute
Quantitation Method.
6.4 Interferences
• Low pH (acidic) water
• Humic and fulvic acid content
• Suspended solids (e.g. fecal matter) and particulates (sand, dirt)
• Excessive algal growth
6.5 Health & Safety Warnings
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
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 63 of 185
remove clothing immediately and wash/ rinse thoroughly. Wash the affected skin areas thoroughly with
large amounts of soap and water. If available consult the MSDS for prompt action, and in all cases seek
medical attention immediately. If inhalation, eye contact or ingestion occurs, consult the MSDS for
prompt action, and in all cases seek medical attention immediately.
6.6 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.
6.7 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
j^0.02 g Acid-washed glass beads (Sigma, # G-1277 or equivalent). Filled tubes are autoclaved
15-min. Liquid Cycle (Slow Exhaust) OR
• Roche MagNA Lyser Green Bead tubes (Roche Applied Science, #03-358-941-001) sterile,
siliconized 3-mm diameter ceramic beads in a siliconized 2-mL microfuge tube.
• Roche MagNA Lyser Rotor Cooling Block
• 2-mL tube racks
• Permanent marking pens (fine point and regular point) for labeling tubes
• Bench Sheets & Printouts of Computer Software Sampling Loading Screen
6.8 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
• ABI TaqMan® Universal PCR Master Mix (ABI #4304437)
• Enterococcus PCR primers and TaqMan® probe
• Sketa PCR primers and TaqMan® probe
• Bovine Serum Albumen (BSA) Sigma Cat. #B-4287)
• Roche MagNA Pure LC DNA Isolation Kit III for Fungi & Bacteria
6.9 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
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 64 of 185
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 ng/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 ng/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 ng/mL Salmon DNA Extraction Buffer
(SAE). Extraction buffer may be prepared in advance and stored at 4 ^Cfor 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 ^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 of 600 /iL per tube, generally 1 extra tube. Thus, in this example, prepare
sufficient Salmon DNA Extraction Buffer for 24 tubes plus one extra. The total volume SAE
needed per sample is 600 iiL. 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 jiL) 50 = 300 jiL
of 10 /ug/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 iiL - 300 iiL =
14,700 jiL AE buffer needed.
4. Make Dilution Series of Enterococcus faecalis purified genomic DNA for use as internal standards
in individual qPCR runs and to generate the weekly Enterococcus qPCR Standard Curve for
quantitation purposes.
5. Enterococcus faecalis DNA for Standards.
6. Frozen Reference Stock (20-nL) at 2.89 x 10s GEQs per piL.
7. Dilute 10-piL of the Frozen Reference stock 363-fold to a final volume of 3,630 piL AE buffer.
Aliquot 20-piL volumes into many 200-nL microfuge tubes and store frozen at -20 ^c. The net
concentration of Enterococcus GEQs is 8,000 / piL. Each week perform a series of 10-fold and 4-
fold dilutions from one thawed tube of the 8,000 GEQ/^L standard solution to create 800
GEQ/nL, 80 GQ/nL and 20 GEQ/^L standard solutions. The analyst performs Enterococcus qPCR
upon duplicate 5-piL volumes of each of the four standards yielding a Standard Curve of Log
GEQs ENT versus Ct value from which the assays "efficiency" is subsequently calculated in the
Relative Quantitation EXCEL Spreadsheet.
a. Make Enterococcus faecalis calibrator filter samples:
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 65 of 185
i. Assemble calibrator positive control samples by thawing tubes of E. faecalis cell stocks,
diluting their contents (lO-piL) up to 1-mL AE buffer and spotting 10-piL on sterile PC
filter previously folded and inserted into a pre-chilled Green Bead tube.
ii. Spot a sufficient number of calibrator filter samples for the entire study to insure
uniform, consistent relative quantitation of study samples. Store the calibrator filter
samples in -202C freezer and thaw individual calibrators (three per week) for extraction
with each week's batch of samples.
8. The calibrator sample filters are spotted with 104 or 10s Enterococcus faecalis 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.
6.10 Procedures for Processing & qPCR Analysis of Sample Concentrates.
6.10.1 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 6.18.1).
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-nL of SAE
Extraction Buffer instead of the usual prescribed 600-nL volume of SAE buffer. Halving the SAE buffer
volume enables the analyst to maintain an equivalent Method Detection Limit and maintain a similar
Sample Equivalence Volume (SEQ; i.e. water sample volume per extract volume) in the extract volumes
(e.g. 5-^L) of each sample filter concentrate added to the PCR reactions.
1. Pre-chill MagNA Lyser Rotor Cooling Block in -20°C freezer. Label 1.7-mL sterile microfuge tubes
with sample ID number to match them with Green Bead Tubes. Two supernatant recovery tubes
and one "5-fold" dilution tube is needed per sample and should be labeled accordingly. The
dilution tube shall be filled with 80-nL AE buffer using a micropipettor.
2. To extract sample filters, uncap green bead tube (cold) and add 0.6-mL (600-nL) SAE Buffer
(Qiagen AE Buffer spiked with Salmon DNA). Re-cap tubes tightly.
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-
min. Being careful to move filter aside, recover and transfer up to 400-nL of supernatant (sans
debris) to new tube with a P-200 or P-1000 micropipettor.
4. Spin the supernatant tubes for 5-min at 14,000 rpm at Room Temperature. Recover >350-nL
supernatant and transfer to new 1.7-mL tube. When all samples in a batch have been extracted
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 66 of 185
transfer dilute 20-piL of DNA extract (2nd supernatant) five-fold (5X) in 80-nL AE buffer (sans SS-
DNA) and store at 4°C for qPCR assays. (If supernatant, 5X and even 25X sample dilutions
possess dark pigment and exhibit severe qPCR inhibition in Sketa assays, consider extracting
replicate filters of samples using the Modified MagNA Pure LC DNA Isolation Protocol (see
Section 6.18.2).
6.10.2 Sample Analysis by Enterococcus qPCR
6.10.2.1 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 piM primer and 100 piM probe and
dissolve by extensive vortexing. Pulse centrifuge to coalesce droplets. Store stock solutions at -
20?C.
3. Prepare working stocks of Enterococcus, and Salmon DNA primer/probe mixes by adding 10 piL
of each Enterococcus or Salmon DNA primer stock and 4 piL of respective probe stock to 676 piL
of PCR grade water, and vortex. Pulse centrifuge to create pellet. Use a micropipettor with
aerosol barrier tips for all liquid transfers. Transfer aliquots of working stocks for single day use
to separate tubes and store at 4^C.
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 6.1.
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 4°C refrigerators until use in assay or return
to long term storage at -20°C. Discard disposable gloves and put on a new pair.
7. Set 32 Smart tubes in Cepheid Racks in PCR cabinet along with micro-pippetors and expose to
germicidal UV lamp for 15-min.
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.
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
tube after loading.
10. Check to make sure each Smart tube is properly labeled and identifiable by sample number or I-
core position (e.g. A4). Insert loaded Smart tubes into Smart Tube microfuge. Close lid and spin
5-sec. Pop lid to stop. Remove Smart Tubes from microfuge and insert into proper position in
SmartCycler.
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 67 of 185
Enterococcus (Ludwig) and Salmon (Sketa) qPCR assays (EPA Method 1606) will be performed upon 5-uL
aliquots of un-diluted & 5X diluted extracts of sample unknowns, calibrator, field blank, and lab blank. A
"No Template Controls" (NTC) shall be analyzed on an ongoing basis to ensure that the Master Mix PCR
reagents are not contaminated. To minimize the number of Enterococcus qPCR reactions needed to be
performed upon samples, Sketa qPCR assays will be performed upon the 5-fold diluted DNA extracts of
samples before any Enterococcus qPCR assays are run in order to screen samples for the presence and
dilution of PCR inhibitors by comparison with the undiluted and 5-fold dilution DNA extract of the
calibrator samples and unused portions of SAE buffer. Each sample's lowest dilution DNA extract not
exhibiting PCR inhibition in the Sketa qPCR assay will be re-assayed by the Enterococcus qPCR assay and
it's results will be used for quantitation of Enterococcus DNA sequences and CCEs.
Detection of reduced levels of Salmon DNA (higher instrument Ct values) is indicative of technical error
during extract dilution or excessive levels of PCR inhibitors or nuclease activity which could impact
detection of the Enterococcus DNA target sequences in the Enterococcus PCR assay. Alternatively, the
high Sketa Ct value may be indicative of the occurrence of a technical error during extract dilution. If a
test sample's Ct value is less than 3 cycles different than the blank negative control and calibrator
samples, indicating only negligible or marginal inhibition (the Sketa Assay is more sensitive to inhibitors
than the ENT Assay), an aliquot of its five-fold diluted extract is analyzed in the Enterococcus Assay. If an
abundance of PCR inhibitors or DNA nucleases are present in a sample extract which are causing a
greater increase in an extract's Ct value (> 3 cycles increase), then the extract is diluted an additional
five-fold (net 25-fold dilution) and re-assayed by both the Sketa and ENT assays. If the inhibition is not
ameliorated by the additional dilution, which should restore the Sketa Ct value to that of the 25-fold
diluted calibrator samples' extracts, the following actions are taken by the analyst. First, the analyst re-
dilutes the sample's undiluted DNA extract five-fold and re-analyzes the dilution with the Sketa PCR
assay to confirm that Ct variance is not due to a dilution error. If the Ct difference is not attributed to a
dilution error, replicate sample filters of the "inhibited" samples are subjected to DNA extraction and
purification by the MagNA Pure LC automated platform loaded with the Roche DNA Kit III (Bacteria;
Fungi) reagents (see Section 6.18.2).
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
method is not 100% conservative (no partitioning or recovery issues) like EPA Method 1606, it typically
exhibits DNA recoveries in the range of 25-50%. DNA recoveries and Enterococcus CCE concentrations
are calculated using only the Delta-Delta Ct Relative Quantitation Method. The relative DNA recoveries
are determined by comparison of the Sketa results from purified DNA eluates of each test sample with
those of the extracted lab blank and calibrator samples. The absolute DNA recovery is calculated by
comparison of the former Sketa results with those of elution buffer spiked with an amount of Salmon
DNA equivalent to the amount in the Salmon-spiked Lysis Binding Buffer added to each sample filter
lysate during the MagNA Pure LC DNA extraction process.
The "Unknown" and "Control" sample extracts whether processed using the SAE buffer or MagNA Pure
LC Kit III reagents are analyzed according to the Cepheid SmartCycler Enterococcus and Sketa qPCR
protocols described in Appendix A of the Draft EPA Method 1606 with Ct determination made by the
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 68 of 185
software using Manual Determination (equivalent of Fit Points Method of Roche LightCycler) with the
fluorescence threshold set at 8.0 units which enables uniform analysis and comparability of all samples'
qPCR results.
Example: For analyses on a single 16-position SmartCycler, calibrator samples and water samples are
analyzed in separate runs and a maximum of 6 water samples (or 2 replicates of 3 samples) are analyzed
per run, as described in Table 6.2 and Table 6.3 of Section 6.18.
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.
6.11 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.
6.12 Chain of Custody
Follow the Sample Control Procedures, Field Sampling Form/Enterococci Filtration/Sample Processing
Standard Operating Procedures.
Field Sampling forms and NRSA 2018-2019 Sample Tracking EXCEL Spreadsheet shall be consulted to
determine if a sample has been properly preserved during collection and transport prior to analysis and
that it has passed all criteria permitting its analysis. The qPCR results of samples exceeding established
criteria or whose associated field/lab blanks had positive Enterococcus qPCR detections of DNA shall be
flagged.
6.13 Quality Assurance/Quality Control (QA/QC) Procedures
The Data Quality Objectives and the Laboratory QC Procedures are listed and summarized in Table 6.5
and Table 6.6 of Section 6.18.
The number of field blanks (dilution buffer only) shipped by field crews performing the resampling of 91
re-visited rivers and streams represents a frequency of 5-10% of the total number of samples extracted
and analyzed by qPCR. All field blanks (negative controls) will be extracted and analyzed by qPCR for the
detection of Enterococcus. The blanks will be analyzed in these cases to insure that positive detections in
field samples are not due to contamination by sampling crews.
One Lab / Method Blank (LB; sterile filters) will be run per batch week in order to insure the sterility
(lack of DNA contamination) in the SAE buffer and pipette tips used to process all of the samples. The LB
sample will be processed and diluted like all other "Unknown" samples.
6.10.3 Sample analysis sequence for SmartCycler
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 69 of 185
Up to four replicate filter concentrates (retentates) derived from the field filtration of 50-mL (in some
cases 10-mL and 25-mL) sample volumes of every sample will be received by NERL and stored at -20 to -
80°C. One filter retentate of each sample (and duplicates for 10% of samples) will be extracted to obtain
DNA lysates for Enterococcus qPCR analysis. The remaining filter concentrates will be archived for
possible extraction and analysis at a later time if needed.
Enterococcus and Sketa qPCR analysis will be performed upon 5-piL volumes of the non-diluted and 5-
fold diluted (in AE buffer) extracts which will be added to 20-^L qPCR Master Mix volumes and analyzed
in the Cepheid SmartCycler qPCR instrument in accordance with draft EPA Method 1606.
Duplicate Enterococcus and Sketa qPCR assays will be performed upon 10% of the sample extracts
(diluted and un-diluted) each week (batch) to determine qPCR assay variance.
6.14 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.
6.15 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 2018-2019 database stored on a computer server in Corvallis, Oregon.
6.16 Waste Management & Pollution Prevention
During the sample processing procedures there may be hazardous waste produced. The waste must be
handled and disposed of in accordance with federal, state, and municipal regulations. All recyclable and
non-recyclable materials for disposal will be properly sorted for their respective waste streams and
placed into proper containers for janitorial staff to collect and process according to EPA guidelines.
All ethanol used shall be consumed by ignition or evaporation. Volumes of ethanol remaining at the end
of the project can be stored for later use in a flammable cabinet or disposed of through appropriate
hazardous waste disposal vendors. Reagent ethanol shall be contained in screw cap tubes along with the
filter forceps to sterilize the latter and to prevent ethanol spillage during transport between sampling
sites.
After the DNA extract is recovered from the sample filter after bead-beating in buffer and
centrifugation, the filter and bead-tube will be discarded in autoclave bags and sterilized for 30-min at
121°C/30 psi to inactivate any potential pathogens that may be associated with the samples.
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 70 of 185
6.17 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 TaqMan® 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)
6.18 Tables, Diagrams, Flowcharts, Checklists, and Validation Data
Table 6.1 Enterococci: PCR assay mix composition (according to draft EPA method 1606)

Volume/Sample (multiply by #
Reagent
samples to be analyzed per day)
Sterile H O
2
1.5 nL
Bovine Serum Albumen (20 mg/mL)
2.5 nL
TaqMan® master mix
12.5 nL
Primer/probe working stock solution
3.5 nL*
Note: This will give a final concentration of 1 iuM of each primer and 80 nM of probe in the reactions. Prepare
sufficient quantity of assay mix for the number of samples to be analyzed per day including calibrators and negative
controls plus at least two extra samples. It is strongly recommended that preparation of assay mixes be performed
each day before handling of DNA samples.
Table 6.2 Enterococci: batch calibrator & enterococcus standards PCR run - 7 samples
Sample Description*
Quantity
Samples
PCR Assay Master Mix
Quantity PCR
Reactions
3 Calibrators (5- and/or 25-fold dilution)
3
Salmon DNA (Sketa)
6
3 Calibrators (5- and/or 25-fold dilution)
3
Enterococcus
6
4 Enterococcus faecalis DNA Standards
4
Enterococcus
8
No template control (reagent blank)
1
Enterococcus
1
* Diluted equivalently to the water samples

Table 6.3 Enterococci: sub batch test sample PCR run - 26 samples & 1 method blank

Sample Description*
Quantity
Samples
PCR Assay Master Mix
Quantity PCR
Reactions
Water samples, (5-fold dilution)
26
Enterococcus
26
Method blank or Sample PCR Reaction Duplicate,
(1- or 5-fold dilution)
1
Enterococcus
1
Non-diluted SAE Buffer
1
Enterococcus
1
Water samples, (1- or 5-fold dilution)
26
Salmon DNA
26
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 71 of 185
Method blank or Sample PCR Reaction Duplicate,
1
Salmon DNA
1
(1- & 5-fold dilution)

* 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 ofPCR inhibition
while 25-fold dilutions analyses may unnecessarily sacrifice sensitivity.
Table 6.4 Enterococci: laboratory methods
Variable or
Measu rement
OA
Class
Expected
Range and/
or Units
Summary of Method
References
Sample
Collection
C
NA
Sterile sample bottle submerged to collect
250-mL sample 6-12" below surface at 1-m
from shore
NRSA Field
Operations Manual
2008
Sub-sampling
N
NA
4 x 50-mL sub-samples poured in sterile 50-
mLtube after mixing by inversion 25 times.
NRSA Laboratory
Methods Manual
2008
Sub-sample
(& Buffer Blank)
Filtration
N
NA
Up to 50-mL sub-sample filtered through
sterile polycarbonate filter. Funnel rinsed
with minimal amount of buffer. Filter folded,
inserted in tube then frozen.
NRSA Laboratory
Methods Manual
2008
Preservation &
Shipment
C
-40Cto+40 C
Batches of sample tubes shipped on dry ice
to lab for analysis.
NRSA Laboratory
Methods Manual
2008
DNA Extraction
(Recovery)
C
10-141%
Bead-beating of filter in buffer containing
Extraction Control (SPC) DNA. DNA recovery
measured
EPA Draft Method
1606 Enterococcus
qPCR
Method 1606
[Enterococcus &
SPC qPCR)
C
<60 (RL) to
>100,000 ENT
CCEs/100-mL
5-nL aliquots of sample extract are analyzed
by ENT & Sketa qPCR assays along with
blanks, calibrator samples & standards. Field
and lab duplicates are analyzed at 5%
frequency. Field blanks analyzed along with
test samples.
EPA Draft Method
1606 Enterococcus
qPCR
NERLNRSA 2008
qPCR Analytical SOP
C = critical, N = non-critical quality assurance classification.
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 72 of 185
Table 6.5 Enterococci: parameter measurement data quality objectives
Expected
Variable or QA Range and/or
Measurement Class Units Summary of Method References
DNA Extraction
(Recovery)
C
10-141%
Bead-beating of filter in buffer
containing Extraction Control (SPC)
DNA. DNA recovery measured
EPA Draft Method
1606 Enterococcus
qPCR
Enterococcus & SPC
qPCR
C
<60 to
>10,000 ENT
CEQs/100-mL
5-nL aliquots of sample extract are
analyzed by ENT & Sketa qPCR assays
along with blanks, calibrator samples &
standards. Field and lab duplicates are
analyzed at 5% frequency. Field blanks
analyzed at end of testing only if
significant detections observed.
EPA Draft Method
1606 Enterococcus
qPCR; NERLNRSA
2008 2009 qPCR
Analytical SOP
(QAPP)
SPC & ENT DNA
sequence numbers
of Calibrators &
Standards by AQM
RSD =
30%
80%
95%

ENT CCEs by dCt
RQM
RSD =
55%
40%
95%

ENT CCEs by ddCt
RQM
RSD =
55%
50%
95%

C = critical, N = non-critical quality assurance classification.
*AQM = Absolute Quantitation Method; RQM = Relative Quantitation Method;
SPC = Sample Processing Control (Salmon DNA/Sketa); CCEs = Calibrator Cell Equivalents
Table 6.6 Enterococci: laboratory QC procedures - enterococci DNA sequences
Check or Sample
Description Frequency Acceptance Criteria Corrective Action
SAMPLE PROCESSING
Re-process sub-
samples
(duplicates)
10% of all
samples
completed per
laboratory
Percent Similarity >70%
If <70%, re-process additional sub-
samples
qPCR ANALYSIS
Duplicate analysis
by different
biologist within
lab
10% of all
samples
completed per
laboratory
Percent Congruence <30% RSD
If >30%, determine reason and if
cause is systemic, re-analyze all
samples in question.
Independent
analysis by
external
laboratory
None
Independent analysis TBD
Determine if independent analysis
can be funded and conducted.
u
u
o
u
o
QC
QC
O
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 73 of 185
Use single stock of
E. faecal is
calibrator
For all qPCR
calibrator
samples for
quantitation
All calibrator sample Cp (Ct)
must have an RSD <_30%
If calibrator Cp (Ct) values exceed
an RSD value of 30% a batch's
calibrator samples shall be re-
analyzed and replaced with new
calibrators to be processed and
analyzed if RSD not back within
range.
DATA PROCESSING & REVIEW
100% verification
and review of
qPCR data
All qPCR
amplification
traces, raw and
processed data
sheets
All final data will be checked
against raw data, exported
data, and calculated data
printouts before entry into
LIMS and upload to Corvallis,
OR database.
Second tier review by contractor
and third tier review by EPA.
6.18.1 Enterococcus qPCR Analysis Decision Tree (ADT)
5X & 25X Sketa
Cps Acceptable
Re-dilute Sample 5-
fold & 25-fold; Repeat
Sketa assays to
confirm dilutions and
results.
If CP >3
S.D.s or
Non-Detect
Perform Sketa qPCR
upon 5-pL aliquot of non-
diluted/diluted purified
DNA eluate to contain
<500 ng DNA
Created 10/25/07
Cp >3 S.D.:
Cp <3
S.Ds
<40 mL
If Cp value
>3 Standard
Deviations
> 40 mL
Cp value <3
Standard
Deviations
Repeat last step
if dilution has not
exceeded 9-fold
If 5X &25X
Sketa Cp still
unacceptable
Dilute eluate
3-fold and
re-assay by
Sketa qPCR.
Extract Replicate
Filter With
MagNA Pure LC
If Florescence
Cp >3
Standard
Deviations
Combine
equiv. vols
of replicate
extracts
If Florescence
Cp is <3
Standard
Deviations
Dilute sample 5-fold
more to 25-fold and
analyze for Sketa
Analyze 5-pL of 5-
fold dilution of
sample DNA by
Enterococcus qPCR
assay
Perform Sketa qPCR assay upon 5-^L of
5 fold dilution of DNA extracts
Add 300-pL SAE Buffer to
each of 2 equiv. filter
replicates; Bead-Beat
Extract Sample Filters with 600-|jL SAE
buffer and bead beating (EPA Mtd 1606)
Enter Sketa and ENT qPCR
Ct value. Sample Vol &
Dilution Factor in Calc.
Template; Calculate ENT
CCEs per 100 mL
Confirm water volume filtered for NRSA sample
filter to be processed and analyzed by qPCR
Analyze most conc. Dilution
of sample DNA with no
significant PCR inhibition
with ENT qPCR assay.
EXCEPTION: if Sketa Cp value
of 9-fold is still >3 S.D.s of mean,
DNA recovery is low or PCR
inhibition is excessive. Analyze
5-pL of the 3-fold dilution by the
Enterococcus qPCR assay.
Created 10/25/07
Updated 1/2/08
Revised 11/05/08
Figure 6.1 Enterococci: qPCR analysis decision tree (ADT)
6.18.2 "Modified" MagNA Pure LC DNA Purification Kit III Protocol
1. Pre-warm the MagNA Pure LC DNA Isolation Kit III Lysis Buffer to 65 in waterbath. Quickly
pipette 260-jiL of warm Lysis Buffer (un-amended) into each "Green Bead" tube with filter
u
u
O
u
Q
en
QC
O
<£
o
<
u
73
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 74 of 185
(preserved after filtration temporarily on ice or during long-term storage in freezer). Shake tube
5 to 10 seconds to mix buffer with beads and filter. Let stand at RT until batch of 16 samples
(including positive control LFB or LFM and negative control LB samples) have all had Lysis Buffer
and had their caps sealed tight. Leave water bath on to use during 30-minute Proteinase K
treatment period.
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-
piL of Proteinase K (dissolved in Lysis Buffer Elution Buffer). Re-cap tubes and mix lysate by
inversion. Do not vortex. Knock beads and filter down from cap into bottom of tube by tapping
tubes on bench countertop.
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-nL pipettor and sterile aerosol-proof tips to transfer approximately 150nL
lysate supernatant from tube to wells in MagNA Pure LC Sample Cartridge in pre-designated
order.
11. When all 16 sample supernatants transferred to sample cartridge put adhesive film over
cartridge to prevent contamination and evaporation. Put sample cartridge in ice water bath or
fridge to maintain 4 ^C.
12. Repeat steps 9 to 13 for second batch of 16 samples (lysates). Re-cover sample cartridge with
adhesive film for storage. Centrifuge sample cartridge opposite a balance cartridge for 75-sec (1-
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 75 of 185
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.
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 1.34nL of 9.3ng/mL Salmon DNA Stock (10
Hg/mL nominal concentration) per lmL Lysis Binding Buffer (blue soapy solution) as the Sample
Processing Control (SPC). If the amount of Salmon DNA stock to be added is less than lO-piL,
dilute the Salmon DNA stock so that a volume > 10-piL can be pipetted into the Lysis Binding
Buffer. Rinse pipette tip up and down three times in Lysis Binding Buffer.
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 lOO-piL 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 9.3ng/mL Salmon DNA Stock by diluting a small volume to
37.2pg/1000nL (1-mL). This control sample is only analyzed by the Sketa qPCR assay. The Ct
value obtained represents that value expected in Sketa qPCR assays of each MagNA Pure LC
purified sample if 100% of the Salmon DNA was recovered and detected. Vortex to mix on low
speed briefly prior qPCR analysis. Centrifuge for 1.5-min to coalesce droplets. Remove film to
aliquot sub-samples and re-place with new film cover to restore at cool temperatures.
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 76 of 185
7.0 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 7.4
describes the sample receipt and login procedures. Section 7.5 describes the identification steps.
Section 7.6 describes laboratory assistance visits. Section 7.7 identifies the sample and record retention
requirements. Section 7.9 provides a summary of the quality control requirements. The final section
provides references to aid in identifications.
7.1 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
from QC or UNK voucher samples.
7.2 Health and Safety Warnings
In addition to the laboratory's usual requirements, laboratories must adhere to the following health and
safety procedures:
1. Laboratory facilities must properly store and dispose of solutions of 10% formalin and/or 75-
95% ethanol.
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 77 of 185
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.
7.3 Definitions and Required Resources (Personnel, Taxonomy
Laboratories, and Equipment)
This section provides definitions and required resources for using the procedure. Section 7.3.1 defines
the terms used throughout the procedure. Section 7.3.2 describes the taxonomic expertise required for
each QC taxonomist using the procedure. Section 7.3.3 describes the roles and responsibilities of the
personnel involved in the procedure. Section 7.3.4 identifies the equipment necessary to apply the
procedure in identifying fish voucher specimens in samples.
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 7.1 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.
Reference Collection: A collection of voucher specimens and digital images that provide examples of
each taxon identified from the NRSA samples sent to the laboratory. A reference collection is used to
help with future identifications of unknown specimens, and to provide physical documentation of
reported identifications.
7.3.1 Definitions
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 78 of 185
Tagged lot: A group of voucher specimens believed to represent a single taxon based on field
identifications. Tagged lots are maintained separately in a voucher sample, and are associated with a
unique voucher tag number.
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.
7.3.2 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 QC taxonomists 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.
7.3.3 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
interactions, and keeps up with the pertinent literature, since systematics and taxa identifications
change over time. Taxa identifications by QC taxonomists represent the standard against which EPA
evaluates identifications by field taxonomists. It is acceptable for the QC taxonomist to also serve as a
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 79 of 185
field fish taxonomist, but not for the same sample. In other words, for any given sample, the field fish
taxonomist and QC taxonomist must be two different persons.
7.3.4 Equipment/Materials
The procedures require the following equipment and information for taxonomic identifications.
• 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
7.4 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
laboratory.
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 80 of 185
b. Verify that all jars for each sample are included in the shipment (i.e., there may be cases
where multiple jars are required to properly preserve specimens collected at a site).
c. Complete 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. 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 7.1 Fish voucher: required data elements - login
FIELD FORMAT DESCRIPTION
LAB ID
text
Name or abbreviation for QC laboratory
DATE RECEIVED
MMDDYY
Date sample was received by lab
SITE ID
text
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
Definition
OK
Sample is in good condition
C
Sample container is cracked
L
Sample or container is leaking
ML
Sample label is missing
NP
Sample is not or insufficiently preserved
S
Sample shipping problem (explain in QA_COMMENTS field)
Q
Other quality concerns, not identified above
u
LU
CL
LT)
CC
U
3
o
>
80
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 81 of 185
7.5 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 7.1 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
7.2). The steps are described below:
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 7.9 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.
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 82 of 185
5. Use the written descriptions and illustrations in determining the taxon. For example, consider if
the specimen has:
a. Diagnostic and other characters consistent with known characters for the taxon.
b. Size within known size ranges.
c. Site information data (e.g., geographic coordinates, drainage basin, stream or river name)
indicate the collecting locality that falls within the known range of the taxon.
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
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 6.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
were provided):
aspects:
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 83 of 185
a. Record the name and count for one taxon on the current line of the bench sheet or
submission worksheet
b. Record the revised name for the second taxon as a new line on the bench sheet or the
submission worksheet.
i. Record the original existing specimen tag number on the next available blank line of the
submission worksheet.
ii. Mark as New Taxon. Record the new name.
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 7.7 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 2018-
2019/Data Templates folder. Table 7.2 identifies the contents and formats used by EPA's file.
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 84 of 185
14. Save the submission worksheet file using the following convention: Taxonomist
name+affliation-abbreviation+submission date (yyyymmdd).xlsx (e.g.,
JSmith_ABCfirm_20180615.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'
names, date of publication, title of document, name of journal or publisher, volume and page
numbers, or ISBN number, as appropriate. If using on-line resource, include a screen capture of
the URL/data source. Keep the references on file, and provide to EPA if requested.
Table 7.2 Fish voucher: required data elements - data submission worksheet
FIELD FORMAT DESCRIPTION
LAB ID
text
Name or abbreviation for QC laboratory
DATE RECEIVED
text
Date sample was received by lab
SITE ID
text
NRSA site ID code as recorded on sample label or tracking form
VISIT NUMBER
numeric
Sequential visits to site (1 or 2)
SAMPLE ID
numeric
6-digit Sample ID number as recorded on sample jar or tracking form
TAG NUMBER
text
Voucher tag number (01, 02, etc.) included with tagged lot (bag in
sample jar) or digital image
DATE COLLECTED
MMDDYY
Date sample was collected
TAXONOMIST NAME
text
Name of QC taxonomist
DATE IDENTIFIED
MMDDYY
Date first specimen identified in sample
NEW TAXON
Text (Y/blank)
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.
COMMON OR
SCIENTIFIC NAME
text
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.
NUMBER OF
INDIVIDUALS
numeric
The number of individuals of taxa noted
CONDITION CODE
text
Condition codes associated with the condition of the sample upon
arrival at the laboratory.

Flag
Definition

OK
Sample is in good condition

C
Sample container is cracked

L
Sample or container is leaking

ML
Sample label is missing

NP
Sample is not or insufficiently preserved

S
Sample shipping problem (explain in QA_COMMENTS field)

UN
Unknown. Specimen has been sent to expert taxonomist for
definitive identification.

Q
Other quality concerns, not identified above
DATA FLAG (if
appropriate)
text
Data qualifier codes associated with specific identifications of voucher
samples. These codes provide more information that those used when
u
LU
CL
LT)
CC
U
3
o
>
84
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 85 of 185
: 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 Definition
¦ CF Tentative identification due to inconsistent characters; no
comprehensive reference for genus; etc. (explain in COMMENTS
field)
DD Damaged Specimen
IM Young-of year or too young/small to identify
L Identification based upon location
NP Not enough preservative used
UN Specimen/ or tagged lot could not be positively identified, even
after consultations with other taxonomic specialists
Q Other quality concerns, not identified above
COMMENTS text Explanation for data flag(s) (if needed) or other comments regarding the
taxon lot.
CITATION text Citation for reference used to identify organism, if taxon not present in
NRSA list offish names (APPENDIX D: OTHER PERTINENT
ATTACHMENTS)
7.6 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.
7.7 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.
7.8 Summary of QC Requirements for Fish Voucher Specimens
Percent taxonomic disagreement (PTD): measure of taxonomic precision comparing the number of
agreements (positive comparisons, compp0s) 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-
comp
N
pos
x 100
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 86 of 185
Table 7.3 Fish voucher: measurement data quality objectives
Variable or Measurement
Precision
Accuracy
Completeness
Identification
85% a
85% a
99%
a As measured by (100%-PTD)
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 87 of 185
Table 7.4 Fish Voucher: quality control -taxonomic identification
Check or Sample
Description
Frequency
Acceptance Criteria
Corrective Action
Use
widely/commonly
accepted taxonomic
references
All identifications
All keys and references used
must be on bibliography
prepared by the field and QC
taxonomists
For all field crew identifications,
EPA will convert field crew's use
of common names to taxonomic
references
Independent
identification by
outside, expert,
laboratory fish
taxonomist ("QC
taxonomist")
When field taxonomist
cannot identify
specimen
Identification by QC
taxonomist (who must be a
different individual than the
field taxonomist)
Replace field crew's "unknown"
identification with
determination by QC
taxonomist
External QC
Approximately 10% of
all sites sampled by
each field taxonomist
PTD < 15%
If PTD > 15%, review data for
possible explanations;
otherwise, insert data qualifier
for field crew identifications
Calculate average
PTD for field
taxonomist
Each sample
submitted to the QC
taxonomist
PTD < 15%
If PTD > 15%, consult with NARS
QA Officer for appropriate
action.
Conduct assistance
visit
EPA may choose to
visit any laboratory
Visit conducted using checklist
Performance and any
recommended improvements
described in debrief with
laboratory staff
Table 7.5 Fish voucher: data validation
Check or Sample
Description
Frequency
Acceptance Criteria
Corrective Action
Taxonomic
"reasonable-ness"
checks
All data sheets
Genera known to occur in
given rivers/streams or
geographic area
Data qualifiers on data that fail
reasonableness check. No
further corrective action steps.
7.9 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. Crossman, 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.
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Information
Sample Information
Labname

Sample ID

QC Taxonomist

Site ID

Project ID

Visit Number

Internal Sample ID

Date Collected

Taxonomist Name Date 1st Specimen Identified in Sample:

New
Taxon?
Common or Scientific Name
Condition
Code
Data
Flag
Comment Including Citation if
Appropriate

Figure 7.1 Fish voucher: example QC fish taxonomy bench sheet (optional)
Additional Comments
Laboratory Operations Manual
Page 88 of 185
-------
2018/19 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.1, June 2018 Page 89 of 185
8.0 FISH TISSUE FILLET (WHOLE FISH COLLECTION)
Laboratory Methods incorporated in OST Manuals.
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 90 of 185
9.0 FISH TISSUE PLUG
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 G: EXAMPLE SOPS
FOR MERCURY IN FISH TISSUE PLUG ANALYSIS.
Table 9.1 Fish tissue plug: measurement data quality objectives
Variable or Measurement
MDL
Quantitation Limit
Mercury
0.47 ng/g
5.0 ng/g
Table 9.2 Fish tissue plug: quality control
Activity
Evaluation/Acceptance Criteria
Corrective Action
Demonstrate competency
for analyzing fish samples to
meet the performance
measures
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 -20° C.
Check the temperature of the
freezer per laboratory's standard
operating procedures.
Record temperature of sample upon arrival at
the laboratory. If at any other time, samples
are warmer than required, note temperature
and duration in comment field.
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.
If data do not meet all QC requirements, rerun
sample or qualify data. If the lab believes the
data are to be qualified without rerunning
sample, the lab must consult with the EPA
Survey QA Lead before proceeding.
Maintain the required MDL
Evaluate for each sample
If MDL could not be achieved, then provide
dilution factor or QC code and explanation in
the comment field.
Use consistent units for QC
samples and field samples
Verify that all units are provided
in wet weight units and
consistently
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
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 91 of 185
Maintain completeness Completeness objective is 95%
for all parameters.
different units in the comments field of the
database.
Contact the EPA Survey QA Lead immediately
if issues affect laboratory's ability to meet
completeness objective.
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 92 of 185
10.0 DIATOMS
This procedure is applicable to the analysis of the diatom samples collected for the National Rivers and
Streams Assessment (NRSA) 2018-2019. NRSA field crews collect composite samples of the algae
present on benthic substrate; 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
diatoms benthic substrate. This procedure describes the steps for identifying and enumerating diatoms
in the samples.
In the following discussion, Sections 10.1, 10.2, and 10.3 summarize the procedure; health and safety
concerns; and required resources. Section 10.4 provides the steps for acknowledging sample receipt.
Section 10.5 provides the steps for subsampling and preparing the vials for the diatom. Section 10.6
provides the steps for the diatom cleaning and slide mounting. Section 10.7 describes the steps for
diatom analysis. Sections 10.8 and 10.9 provide the steps for the data entry, and sample and record
retention.
10.1 Summary of Procedure
The procedure describes the extraction and separate analysis of the diatom samples. Diatom samples
are digested using concentrated nitric acid to remove organic matter. After digestion samples are
centrifuged and supernatant decanted, and then refilled with distilled water. The rinsing process is
performed a minimum of six times. Cleaned material is place on a glass microscope slide and a cover slip
is mounted on to the cleaned material using Naphrax™. Once permanent slide mounts have been
created, analysts can then begin to identify and enumerate diatom taxa for a given sample. To ensure
transparent taxonomic consistency between analysts, both regionally and nationally, samples will be
randomly distributed among analysts; and analysts will use pre-count voucher flora to assign operational
taxonomic units (OTUs) to diatoms identified in individual samples. As new taxa are encountered,
analysts can add new OTU codes to the pre-count voucher flora. In additional to the use of the pre-
count voucher flora, 10% of samples are re-analyzed by the same analysts, and 10% of samples are re-
analyzed by a second analyst. Both the 10% same analyst re-analyzed and 10% second analyst re-
analyzed samples are randomly assigned to each analyst.
10.2 Health and Safety Warnings
In addition to the laboratory's usual requirements, personnel must implement the following health and
safety procedures for this procedure:
• Wear or apply proper personal protection clothing and equipment (e.g. lab coat, protective
eyewear, gloves).
• When working with potential hazardous chemicals (e.g. a mounting medium with a high
refractive index such as ™) 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.
10.3 Required Equipment
10.3.1 Laboratory
i/i
To participate in NRSA, each laboratory first must demonstrate to EPA that it has appropriate ^
qualifications as follows: ^
Q
92
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 93 of 185
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).
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.
10.3.2 Equipment/Materials
The procedure requires the following equipment and materials for sample preparation (subsampling)
and taxonomic identifications:
10.3.2.1 Subsampling Equipment/Materials
• 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
• 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
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 94 of 185
10.3.2.2 Diatoms
10.3.2.2.1 Preparation of Diatom Slides
• 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 (1.7) index (e.g., Naphrax™)
• Diamond scribe
• Disposable plastic pipettes
• Adjustable pipettor (0 - 250^1); adjustable pipettor (200 -1000^1)
• 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., Kimwipe®)
• Slide labels
• Wax (the kind commonly used for candle making and canning foods)
• 10% HCI
10.3.2.2.2 Analysis of Diatoms
• Compound microscope with transmitted light including:
o Objective lenses of at minimum (lOx, 40x, lOOx)
o Oil immersion objective (lOOx) with a minimum numerical aperture of 1.3
o Substage condenser lens with a minimum numerical aperture of 1.3
o Ocular lenses of 10-15x
o DIC (differential interference contrast) illumination
o Diamond objective marker mounted on the objective turret
o 100 watt halogen illumination, or equivalent
o Digital camera
10.4 Sample Receipt
Under U.S. regulations, shippers must classify samples preserved in formalin as "Dangerous Goods
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
" and
O
I—
94
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 95 of 185
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 10.1).
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).
4. Verify that the login information includes the required data elements in Table 10.1. After
completing all required elements, provide the information to the data entry personnel.
Table 10.1 Required data elements for diatom sample login
FIELD FORMAT DESCRIPTION
LAB ID
text
Name or abbreviation for QC laboratory
DATE RECEIVED
MMDDYY
Date sample was received by lab
SITE ID
text
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 (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.

CODE
Definition

OK
Sample is in good condition

C
Sample container is cracked

HM
Sample contains heavy amounts of sand, silt, or other
heavy material that may interfere with the algal analysis

L
Sample or container is leaking

TL
Too little sample left for the procedure

ML
Sample label is missing

PA
Preservative added, add amount in comments field

Q
Other quality concerns, not identified above
O
I—
<
Q
95
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 96 of 185
FORMALIN_ADDED
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.
COMMENTS_ARRIVAL
text
Any comments about the condition of the sample upon arrival.
10.5 Sample Preparation
Before the sample is digested, the volume received is measure in a 100 mL graduated cylinder. The
volume is recored to the nearest 1 mL. Once the total volume is recorded on the Table 10.2 after
completing all required elements, provide the information to the data entry personnel.
Table 10.2 Required data elements for diatom subsampling
FIELD
FORMAT
DESCRIPTION
LABJD
text
Name or abbreviation for QC laboratory
SITE ID
text
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 (on sample label)
VIALJD
numeric or
text
Laboratory assigned ID to the vial holding the subsample
SAMPLE_VOLUME
numeric
Total volume of sample before any processing (from the
sample label)
SUBSAMPLE_VOLUME
numeric
Volume of subsample in the vial
DESTINATION
text
Name of the receiving laboratory (i.e., if the vial will be
shipped to another location)
COMMENTS_SUBSAMP
LE
text
any comments about the subsampling
10.6 Diatom C eaning 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.
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 97 of 185
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
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 10.3.2 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 Naphrax™ is a commercially-available toluene-based mounting medium with high refractive index.
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 98 of 185
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).
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 10.5. 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 lOOOx 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.
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 99 of 185
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.
c) When the sample is dry, place the coverslip onto a hot plate for about 30
min at approximately 570°C.
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., Kimwipe®c).
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 22 x 22 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 piL 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.
c Kimwipe® is a commercially-available product.
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 100 of 185
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;
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 3002F.
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.
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. q
100
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 101 of 185
i. Set aside the mount to finish cooling.
8. 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.
9. 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.
10. Wipe the mount clean with a tissue (e.g., Kimwipe®).
11. Add a paper label to slides before analyzing the slides.
12. Mark the sample ID number on the side of the slide without a label with a diamond pencil.
13. 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.
14. Preserve and store cleaned material.
15. Record the data elements described in Table 10.3. 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 10.3 Required data elements for diatom cleaning & mounting
FIELD FORMAT DESCRIPTION
LAB ID
text
Name or abbreviation for QC laboratory
SITE ID
text
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 (on sample label)
VIAL ID
numeric or
Laboratory assigned ID of the diatom vial used for the

text
cleaning
SUBSAMPLE_DCF
numeric
Dilution or concentration factor. Record as 1 if sample
was not diluted or concentrated.
SLIDEJD
numeric or
text
Laboratory assigned ID for the slide
SLIDE DATE
date
Date that slide preparation was completed
TECHNICIAN
text
Name or initials of technician who prepared the slide
MOUNT MEDIUM
text
Mounting medium used (e.g., Naphrax™)
INTERNAL
text
Name or initials of QC Taxonomist who reviewed the
QCJAXONOMIST

slide
SLIDE_CHECK
Y/N
Is slide acceptable for diatom analysis? If no, still record
the slide number, but note in the comments that the
slide has been destroyed.
O
I—
<
Q
101
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 102 of 185
EJECT_AMOUNT
numeric
I Final amount of cleaned diatom material ejected on
| coverslip
EJECT_DCF
numeric
Dilution or concentration factor. Record as 1 if ejected
amount was not diluted or concentrated separately
from any dilution or concentration of the subsample.
DESTINATION
text
Name of the receiving laboratory (i.e., if the slide will be
shipped to another location)
COMMENTS_DIATOM_PRE
P
text
any comments about the diatom cleaning and mounting
10.7 Analysis of Diatoms
The taxonomist should count 600 valves for any properly concentrated and prepared sample as
described in Section 10.6. If a taxonomist is unable to count 600 values after analyzing a complete slide,
then the taxonomist should make note of this in the sample analysis data file comments section.
10.7.1 Creation of Pre-Count Regional Voucher Flora and Develop Diatom
Analyst Schedule
For analyses of diatoms NRSA, EPA requires that all laboratories to develop pre-count regional voucher
flora. The regional voucher flora documents must be completed before counting of diatom samples
begins. The following section is an overview of how the voucher floras are created:
1. The project lead taxonomist obtains light micrographs (LM) images from a minimum of 80% of
the project prepared slides.
a. For common taxa images should be limited to 1-2, for example, Navicula
rhynchocephala, Cocconeis placentula.
b. The goal of the regional voucher flora document is to capture images taxa that analysts
are most likely to encounter during the counting process. A target effort is for the
taxonomist to spend 15 minutes per slide searching for species that need to be
documented.
c. To save time, use the microscope camera in its fixed position (that is, it does not need to
be rotated to orient cells vertically or horizontally), as image post-processing is more
efficient.
d. Image filename should include slide number, date, and sequential number (e.g.
"15565a_12092016_01").
2. Once all images are captured, process images as a group using GIMP, Photoshop or other image
software. Rotate, crop and add a 10 pim scale bar to each raw image, then export as a jpeg file
(Fig. 9.1)
a. It is important that these images are of optimal resolution and contrast to capture the
morphological features of each taxon. These images will be used in project voucher flora
publications, as well as in Diatoms of the US taxon pages.
3. Create image "plates" for each genus, or other grouping (Fig. 9.2), by gathering jpeg images into
Photoshop (.psd) or gimp (.xcf) plates. Scale all images equally to a standard lOOx, which allows
users to measure cells directly on the digital plate.
a. Use a template with the dimensions 17x25cm, 600ppi, greyscale, white background. i/i
b. Note that it is easiest to import all the jpeg images at once, each as a layer labelled with o
their sample identification information. Layers can be immediately locked together and <
Q
102
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 103 of 185
scaled to lOOx. Large cells may need to be scaled separately, for example, we capture
some valves using the 60x objective.
4. Sort images into their "best" morphological groups, or genera.
5. Provide a list of additional sequential provisional names on the plate (Fig. 9.2)
6. Once the regional voucher flora documents have been developed, then the project lead
taxonomist will develop an analysts schedule.
a. Samples will be randomized across analysts, and laboratories if more than one, to
ensure that samples are distributed randomly across geographic areas (i.e. avoiding one
analyst getting all or most samples from one ecoregion or state).
b. The project lead taxonomist will also determine which samples are re-counted by
randomly selecting 10% of analyst's processed samples to be re-counted by the sample
analyst and 10% of an analyst's processed samples to be re-counted by a second analyst
involved in the project.
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 104 of 185
Figure 10,1 Example of light micrograph image after rotation, histogram, and scale bar processing.
O
H
<
Q
104
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 105 of 185
EUN03
i
EUN04
1 r ] /
' /

EUN07
I]
EUN12 EUN13
A»N11
^112
E>fll3
^N14
Iff
EUN16
EUN17
EUN18
EUN19
EUN20
EUN21
EUN22
EUN23
EUN24
EUN25
EUN26
EUN27
EUN28
EUN29
EUN30
I
Figure 10.2 Example plate of a regional voucher flora collected before diatom sampling counting has begun.
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 106 of 185
10.7.2 Diatom Sample Count
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
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 10.3.2 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 10.6 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. Alternatively, maintain a spreadsheet that identifies the starting location and type of
coordinates used in specifying the starting point. Either etching or documenting the starting
location 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
visible (see diagram below).
b) Etch a circle around the last field counted in the first row and at the start and
end of all other rows.
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 107 of 185
iii. Verify that the etching is clearly visible so that another taxonomist can easily locate
the circles and lines.
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 regional voucher
flora documents. If the valve is the first occurrence of the species/taxon, take a photograph
following the specifications in Section 10.7.1.
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. Verify that the SUBSAMPLE_DCF value reflects all dilution and concentration of the subsample.
Make any necessary corrections to the value.
6. Record the required data elements in described in Section 10.7.3 (Table 10.4).
7. Once all samples have been counted and any new OTU taxa have been added to the regional
voucher flora by analysts, the lead taxonomist will develop a project species list that will
coordinate the project OTU list with valid scientific names from Biodata. The taxa list created
from this final process will become the final project taxa list for NRSA 2018-2019. A current list
of diatom taxa can be found at the biodata website: http://aquatic.biodata.usgs.gov
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 108 of 185
10.7.3 Internal Quality Control
For each sample, the laboratory must perform internal quality control evaluations. The lead taxonomist
must randomly select 10% of each analyst's processed samples to be re-counted by the same analyst,
and randomly select 10% of each analyst's processed samples to be re-count by a secondary (internal)
analyst. Calculate the PDE, and PTD listed below. If any samples do not meet the QA requirements listed
in the Attachment, perform a third re-count for the sample.
Percent disagreement in enumeration (PDE): measure of taxonomic precision for diatoms comparing
the number of organisms, rii, counted in a sample by the primary taxonomist with the number of
organisms, n2, counted by the secondary taxonomist.
PDE =
\ni ~n2\
100
Hi

Percent taxonomic disagreement (PTD): measure of taxonomic precision for diatoms 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 =
1-
compp
N
x 100
Table 10.4 Diatom sample analysis required data elements
FIELD FORMAT DESCRIPTION
LAB ID
text
Name or abbreviation for QC laboratory
SITE ID
text
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 (on sample label)
SAMPLE_VOLUME
numeric
Original sample volume measured during
subsampling step in Section 10.5.
VIALJD
numeric or
text
Laboratory assigned ID of the diatom vial
SUBSAMPLE VOLUME
numeric
Volume of the subsample in the vial
SLIDEJD
numeric or
text
Laboratory assigned ID for the slide
TAXONOMIST
text
Name or initials of taxonomist or Internal QC
Taxonomist analyzing the sample.
QC_CHECK
Y/N
Y if results were provided by the Internal QC
Taxonomist for a sample selected for the 10% QC
check.
DATE ANALYSIS
date
Date when the analysis was completed
OTUCODE
numeric or
An identifier assigned by the laboratory to provide a

text
unique number for each taxon identified for NRSA.
O
I—
<
Q
108
-------
2018/19 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.1, June 2018 Page 109 of 185
LAB_TAXON_NAME
text
Taxon name. If BiodataTaxonName is available, this
field is optional.
SPECIES
text
Genus and species
TRANSECTS COUNTED
numeric
Number of transects counted
TRANS LENGTH
numeric
Length (mm) of transects counted
TRANS WIDTH
numeric
Width (mm) of transects counted
COUNT
numeric
Number of diatom valves
COUNT_CENSORED
text
Code for a sample for which the taxonomist
counted fewer than 600 valves
CODE Definition

SS Sparse sample

OT ¦ Other. Identify other reason in

; COMMENT_ANALYSIS
COMMENTS ANALYSIS
text
any comments about the analysis
10.8 Data Entry
Table 10.2, and Table 10.3 identify the required data elements that the laboratories must provide to
EPA. Table 10.4 provides a list of required data elements for the diatom count data. 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 ci/for each taxonomist is
submitted once to EPA's External QC Coordinator.
10.9 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:
5. The sample materials, including vials and slides, for a minimum of 1 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.
6. 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 deposit sample material in a public herbarium, such as
the Academy of Natural Sciences of Drexel University.
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 110 of 185
11.0 PERIPHYTON BIOMASS
Samples for periphyton biomass are processed alongside the periphyton Chloryphyll a and water column
Chloryphyll a samples as Ash Free Dry Mass (AFDM). This procedure is applicable to the analysis of
periphyton biomass from the filtered, composite periphyton sample collected in the field. The method is
performance based (Table 11.1). Laboratories may use any method that meets the requirements below
to analyze the samples. An example SOP is provided in APPENDIX F: EXAMPLE SOP FOR ASH FREE DRY
MASS ANALYSIS OF PERIPHYTON BIOMASS.
Table 11.1 Quality assurance measures for AFDM
Demonstrate competency
for analyzing AFDM to meet
the performance measures
Check condition of sample
when it arrives.
Store sample appropriately.
While stored at the
laboratory, the sample must
be kept at a maximum
temperature of -20° C.
Analyze sample within
holding time.
Maintain quality control
specifications from selected
method/SOP (that meets
the measurement data
quality objectives).
Maintain completeness
Demonstration of past
experience with AFDM samples
in applying the laboratory SOP in
achieving the method detection
limit
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.
Check the temperature of the
freezer per laboratory's standard
operating procedures.
The test must be completed
within the holding time. If the
original test fails, then the retest
also must be conducted within
the holding time.
Data meet all QC specifications
in the selected method/SOP.
Completeness objective is 95%
for all parameters.
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.
Assign appropriate condition code identified in
Table 11.2.
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.
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.
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.
Contact the EPA Survey QA Lead immediately
if issues affect laboratory's ability to meet
completeness objective.
i/i
i/i
<
O
CO
Z
0
£
1
CC
110
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 111 of 185
Table 11.2 Data field requirements from labs for AFDM
FIELD
FORMAT
DESCRIPTION
LABORATORY ID
text
Name or abbreviation for QC laboratory
DATE RECEIVED
MMDDYY
Date sample was received by laboratory
SITE ID
text
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 (on sample label)
DATE COLLECTED
MMDDYY
Date sample was collected
CONDITION CODE
text
Condition codes describing the condition of the
sample upon arrival at the laboratory.

Flag
Definition

OK
Sample is in good condition

C
Sample container is cracked

L
Sample or container is leaking

ML
Sample label is missing

NF
Sample not frozen

Q
Other quality concerns, not identified
above
CONDITION
COMMENT
text
Comments about the condition of the sample.
-------
2018/19 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.1, June 2018 Page 112 of 185
12.0 PERIPHYTON META-GENOMICS (RESEARCH EFFORT)
Laboratory methods incorporated in an ORD Quality Assurance Project Plan.
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 113 of 185
13.0 WATER CHEMISTRY AND CHLOROPHYLL A
13.1 Analytical Parameters
A total of 19 parameters are determined from each bulk water chemistry sample collected (Table 13.1).
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 13.1 Water chemistry: NRSA 2018/19 parameters
Analyte Units Comments
Conductivity
|j.S/cm at 25°C

pH (laboratory)
Standard (Std) Units

Turbidity
Nephelometric Turbidity Units (NTU)

Acid Neutralizing Capacity
(ANC)
|aeq/L (20 |aeq/L-l mg as CACO3)

Dissolved Organic Carbon
(DOC)
mg C/L

Ammonia-N (NH3-N)
mg N/L
The method measures ammonia and
ammonium; the relative proportion
between these two analytes depends on
pH. Typically, NRSA (and other NARS)
samples consist mostly of ammonium.
Nitrate-Nitrite (N03-N02)
mg N/L
Note different preservation methods and
holding times depending on whether the
lab is using ion chromatography (IC) or
flow injection analysis (FIA).
Total Nitrogen (TN)
mg/L

Total Phosphorus (TP)
M-g/L

Sulfate (S04)
mg/L

Chloride (CI)
mg/L

Nitrate (N03)
mg/L
May be obtained as part of nitrate-nitrite
determination (use FIA to obtain nitrate-
nitrite separately, then calculate
difference for nitrate), or as a direct
measurement (e.g., IC)
Calcium (Ca)
Mg/L

Magnesium (Mg)
Mg/L

Sodium (Na)
mg/L

Potassium (K)
mg/L

Silica (Si02)
mg/L

Total Suspended Solids (TSS)
mg/L

True Color
PCU
Performance objective based on use of
visual estimation method
Chlorophyll a
|ag/L (in extract)

-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 114 of 185
13.2 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 inspect 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 4°C
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
reject results from samples that exceeded target holding times outright; the team will review the data
and holding times and decide whether EPA will use the data in the NRSA assessment.
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 115 of 185
13.2.1 Water Chemistry Samples
Sample Receipt
4 L Bulk Sample
Inspect samples and complete
tracking form
I

Process Sample
within 24 hours
Filtration
HDPE
bottle
Acid
washed
Preserve
with HNC>
Analyses
Calcium (180
days)
Magnesium
(180 days)
Sodium (180
days)
Potassium
(180 days)
i—.
HDPE bottle
Not acid
washed
Store at 4 °C
in darkness
HDPE bottle
Acid washed
Preserve
with H2SO4

Analyses
Chloride (28
days)
Nitrate (7 days)
Sulfate (28
days)
Silica (28 days)
Nitrate-Nitrite
(IC) (7 days)
True Color (3
davs)
Analyses
Ammonia-N (28 days)
Dissolved Organic
Carbon (28 days)
Nit rate-Nit rite (with FIA)
(28 days)
Not Filtered
1
~
\

HDPE
' HDPE
bottle
1 bottle
Acid
1 Not acid
washed
washed
Preserve
Store at 4
with
°C in
.... /

Analyses
Total Phosphorus
(28 days)
Total Nitrogen (28
days)
Analyses
pH (3 days)
ANC (7 days)
Conductivity
(28 days)
TSS (7 days)
Turbidity (3
days)
Figure 13.1 Water chemistry: sample processing procedures
Figure 13.1 illustrates sample preparation processing for the water chemistry indicators, including
filtering and acidifying, for the various analytes.
1. Use 0.4nm pore size polycarbonate filters for all filtration.
2. Rinse vacuum filter funnel units thoroughly with reverse-osmosis (RO) or de-ionized (Dl) water
(ASTM Type II reagent water) five times before each use and in between samples. After placing a
filter in the funnel unit, run approximately 100 mL of RO or Dl water through the filter, with
vacuum pressure, to rinse the filter. Discard the rinse water.
3. Place the appropriate sample bottle under the funnel unit and filter sample directly into the
bottle. If a new filter is needed, remove the sample bottle, and rinse the new filter with 100 mL
of RO or Dl water before continuing.
4. After all filtered and unfiltered aliquots are collected, add ultra-pure acid (HN03 or H2S04,
depending on the analyte, see Table 13.2) to the sample in the aliquot container. Cap tightly and
invert the bottle several times to mix.
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 116 of 185
5. Store all aliquots except the cation aliquot (filtered, acidified with HN03) in a refrigerator at 4°C.
Table 13.2 Water chemistry: acid preservatives added for various analytes
Preservatives
H2S04
HNO3
DOC
Ca
NH3-N
Mg
Total N
Na
Total P
K
NO2-NO3

13.2.2 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 ±
2°C for no more than thirty days before analysis.
13.3 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 13.4). 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 lower H+ (or OH") concentrations (pH between 5.75 and 8.25) and higher H+
(or OH") concentrations (<5.75 or >8.25).
For duplicate samples, precision is estimated as the pooled standard deviation (calculated as the root-
mean square) of all samples at the lower concentration range, and as the pooled percent relative
standard deviation of all samples at the higher concentration range. For standard samples (of known
concentration), precision is estimated as the standard deviation of repeated measurements across
batches at the lower concentration range, and as percent relative standard deviation of repeated
measurements across batches at the higher concentration range. Bias (i.e., systematic error) is
estimated as the difference between the mean measured 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 13.3.
Participating laboratories may use alternative analytical methods for each target analyte if they can
satisfactorily demonstrate the alternative method can achieve the performance requirements as listed
in Table 13.4. 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 2018/19 QA files by the EPA Laboratory Review Coordinator.
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 117 of 185
Table 13.3 Water chemistry: NRSA 2018/19 analytical methods (Central Laboratory, EPA ORD-Corvallis)
Analyte Summary of Methodd References6 WRSSOPf
pH (lab)
Automated, using ManSci PC-Titrate w/ Titra-Sip
autotitrator and Ross combination pH electrode.
Initial pH determination for ANC titration
EPA 150.6 (modified)
WRS 16A.0 (April
2011)
Specific conductance
(conductivity) @ 25°C
Electrolytic, Man-Tech TitraSip automated analysis
OR manual analysis, electrolytic
EPA 120.6
WRS 16A.0 (April
2011)
WRS 11A.4 (April
2011)
Acid neutralizing
capacity (ANC)
Automated acidimetric titration to pH<3.5, with
modified Gran plot analysis
U.S. EPA (1987)
WRS 16A.0 (April
2011)
Turbidity
Nephelometric; Man-Tech TitraSip automated
analysis,
OR
Manual analysis using Hach turbidimeter (high
turbidity samples)
APHA 214 A, EPA 180.1 U.S. EPA
(1987)
WRS 16A.0 (April
2011)

WRS 13A.3 (April
2011)
Total suspended solids
(TSS)
Gravimetric, dried at 104 °C
EPA 160.2; APHA 209-C
WRS 14B.4
(February 2011)
True color (Hach Kit)
Visual comparison to calibrated glass color disk.
APHA 204 A (modified), EPA
110.2 (modified), U.S. EPA (1987)
WRS 15A.3 (April
2011)
Dissolved Organic
Carbon (DOC)g
UV promoted persulfate oxidation to CO2 with
infrared detection
APHA 5310-C
U.S. EPA (1987)
WRS 21A.4 (May
2011)
Nitrate+Nitrite, as N
(fresh waters)
Ion Chromatography
OR
FIA automated colorimetric (cadmium reduction)
EPA 300.6; SW-846 9056A; APHA
4110B
EPA 353.2
APHA 4500-N03-N-E
Lachat 10-107-04-1-C
WRS 36A.0 (April
2011
WRS 40A.5 (May
2011)
Nitrate
Measured as part of Nitrate-Nitrite when using IC or
calculated after measuring Nitrate+Nitrite using FIA.
See above
See above
Ammonia, as N (fresh
waters)
FIA automated colorimetric (salicylate,
dichloroisocyanurate)
Lachat 10-107-06-3-D
WRS 30A.4 (April
2011)
Silica, dissolved (SiOz)
Fresh waters
FIA automated colorimetric (molybdate, stannous
chloride)
EPA366.0, APHA 425 C
Lachat 10-114-27-1-B
WRS 32A.5
(February 2010)
Total nitrogen (TN)
Persulfate Digestion; FIA Automated Colorimetric
Analysis (Cadmium Reduction, sulfanilamide)
EPA353.2 (modified)
APHA 4500-N-C (modified)
ASTM WK31786
U.S. EPA (1987)
Lachat 10-107-04-1-C (modified)
WRS 34A.5 (April
2011)
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)
g For DOC, "dissolved" is defined as that portion passing through a 0.45 nm nominal pore size filter. For other
analytes, "dissolved" is defined as that portion passing through a 0.4 nm pore size filter (Nucleopore or
equivalent).
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 118 of 185
Analyte
Summary of Methodd
References6
WRS SOPf
Total phosphorus (TP)
Persulfate Digestion; Automated Colorimetric
Analysis (molybdate, ascorbic acid)
APHA 4500-P-E
USGS 1-4650-03
U.S. EPA (1987)
Lachat 115-01-1-B (modified)
WRS 34A.5 (April
2011)
Major anions,
dissolved
chloride, nitrate,
nitrite, sulfate
Ion Chromatography
EPA 300.6; SW-846 9056A; APHA
4110B
WRS 40A.5 (May
2011)
Major cations,
dissolved
calcium, sodium,
potassium,
magnesium
Inductively-coupled Plasma Atomic Emission
Spectroscopy (ICP-AES)
OR
Flame AAS
EPA 200.7; EPA 6010B
U.S. EPA (1987), EPA 215.1
EPA 273.1, EPA 258.1
EPA 242.1
WRS SOP 3.04 v3
(October 2011)
WRS 50A.4 (March
2007)
Chlorophyll-a
(Chl-a)
Extraction 90% acetone analysis by fluorometry
EPA 445.0, EPA 446.0
WRS 71A.3 (April
2011)
13.4 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 2018/19 QAPP will be followed to ensure these
LRLs are met for the NRSA 2018/19.
13.4.1 Laboratory Performance Requirements
Table 13.4 summarizes the pertinent laboratory performance requirements for the water chemistry and
chlorophyll-a indicators.
13.4.2 Laboratory Quality Control Samples
Table 13.5 summarizes the pertinent laboratory quality control samples for the water chemistry and
chlorophyll-a indicators.
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 119 of 185
Table 13.4 Water chemistry & chlorophyll-a: laboratory method performance requirements
Potential
Range of
Analyte Units Samplesh
Lower
Reporting Transition Precision Bias
Limit1 ValueJ Objectivek Objective1
Conductivity
|j.S/cm at 25°C
1 to 15,000
2.0
20
± 2 or±10%
± 2 or 5%
pH (laboratory)
Std Units
3.5 to 10
N/A
5.75, 8.25
>5.75 or
<8.25= ±0.15
<5.75 or
>8.25=±0.07
>5.75 and
<8.25 =
±0.05
<5.75 or
>8.25 =
±0.15
Turbidity
Nephelometric
Turbidity Units
(NTU)
0 to 44,000
2.0
20
± 2 or±10%
± 2 or ±10%
Acid
Neutralizing
Capacity (ANC)
Heq/L
(1 mg/Las
CaCO3=20 neq/L
-300 to
+75,000
(-16 to
3,750 mg as
CaC03)
N/A
±50
± 5 or ±10%
± 5 or ±10%
Dissolved
Organic Carbon
(DOC)
mg/L
0.1 to 109
0.20
< 1
> 1
±0.10 or
±10%
±0.10 or
±10%
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.
<
1 Value at which performance objectives for precision and bias switch from absolute (< transition value) to relative —j
> 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. ^
cc
K For duplicate samples, precision is estimated as the pooled standard deviation (calculated as the root-mean O
square) of all samples at the lower concentration range, and as the pooled percent relative standard deviation of
all samples at the higher range. For standard samples, precision is estimated as the standard deviation of repeated u
measurements across batches at the lower concentration range, and as percent relative standard deviation of §
repeated measurements across batches at the higher concentration range. ^
I—
CO
For pH precision, the looser criteria apply to more highly alkaline samples. For NRSA, that is less of a concern than ^
the ability to measure acidic samples accurately and precisely. w
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 h
sample batches, and as the percent difference at the higher concentration range. ^
119
u
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 120 of 185

Potential
Lower

Range of
Reporting
Transition
Precision
Bias
Analyte
Units
Samplesh
Limit1
ValueJ
Objectivek
Objective1
Ammonia as
N(NHj-N)
mg/L
Oto 17
0.02 (1.4
Heq/L)
0.10
±0.01 or
±10%
±0.01 or
±10%
Nitrate-Nitrite
(N03-N02)
mg/L
0 to 360 (as
nitrate)
0.02
0.10
±0.01 or
±10%
±0.01 or
±10%
Total Nitrogen
(TN)
mg/L
0.1 to 90
0.02
0.10
±0.01 or
±10%
±0.01 or
±10%
Total
Phosphorus
(TP)
Hg/L
0 to 22,000
4
20
± 2 or±10%
± 2 or ±10%
Sulfate (S04)
mg/L
0 to 5,000
0.50 (10
Heq/L)
2.5
±0.25 or
±10%
±0.25 or
±10%
Chloride (CI)
mg/L
0 to 5,000
0.20 (6
Heq/L)
1
±0.10 or
±10%
±0.10 or
±10%
Nitrate (N03)
mg/L
0 to 360
0.02 (4
Heq/L)
0.1
±0.01 or
±10%
± 0.01 ±10%
Calcium (Ca)
mg/L
0.04 to
5,000
0.10 (5
Heq/L)
0.5
± 0.05 or
±10%
±0.05 or
±10%
Magnesium
(Mg)
mg/L
0.1 to 350
0.10 (8
Heq/L)
0.5
± 0.05 or
±10%
±0.05 or
±10%
Sodium (Na)
mg/L
0.08 to
3,500
0.10 (4
Heq/L)
0.5
± 0.05 or
±10%
±0.05 or
±10%
Potassium (K)
mgL
0.01 to 120
0.10 (2
Heq/L)
0.5
± 0.05 or
±10%
±0.05 or
±10%
Silica (Si02)
mg/L
0.01 to 100
0.10
0.5
± 0.05 or
±10%
±0.05 or
±10%
Total
Suspended
Solids (TSS)
mg/L
0 to 27,000
2
10
±1 or ±10%
± 1 or ±10%
True Color
PCU
0 to 350
5
50
±5 or ±10%
±5 or ±10%
Chlorophyll a
|ag/L (in extract)
0.7 to
11,000
0.5
15
± 1.5 or ±10%
± 1.5 or
±10%
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 121 of 185
Table 13.5 Water chemistry: quality control - laboratory samples
QC Sample

Type and

Acceptance

Description Analytes
Description
Frequency
Criteria
Corrective Action
Laboratory/
All except TSS

Once per day
Control limits
Prepare and analyze new
Reagent
(ForTSS, the lab

prior to
< LRL
blank. Determine and
Blank
will filter a

sample

correct problem (e.g.,

known volume

analysis

reagent contamination,

of reagent water

instrument calibration, or

and process the

contamination introduced

filters per

during filtration) before

method)

proceeding with any

sample analyses.

Reestablish statistical

control by analyzing three

blank samples.
Filtration
All dissolved
ASTM Type II
Prepare once
Measured
Measure archived
Blank
analytes
reagent
per week
concentrations
samples if review of other

water
and archive
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 122 of 185
QC Sample

Type and

Acceptance

Description Analytes
Description
Frequency
Criteria
Corrective Action
Laboratory
Duplicate
Sample
All analyses

One per
batch
Control limits
< precision
objective
If results are below LRL:
Prepare and analyze split
from different sample
(volume permitting).
Review precision of QCCS
measurements for batch.
Check preparation of split
sample. Qualify all
samples in batch for
possible reanalysis.
Standard
Reference
Material
(SRM)
When available
for a particular
analyte

One analysis
in a
minimum of
five separate
batches
Manufacturers
certified range
Analyze standard in next
batch to confirm
suspected imprecision or
bias. Evaluate calibration
and QCCS solutions and
standards for
contamination and
preparation error. Correct
before any further
analyses of routine
samples are conducted.
Reestablish control by
three successive
reference standard
measurements that are
acceptable. Qualify all
sample batches analyzed
since the last acceptable
reference standard
measurement for possible
reanalysis.
Matrix
Spike
Samples
Only prepared
when samples
with potential
for matrix
interferences are
encountered

One per
batch
Control limits
for recovery
cannot exceed
100±20%
Select two additional
samples and prepare
fortified subsamples.
Reanalyze all suspected
samples in batch by the
method of standard
additions. Prepare three
subsamples (unfortified,
fortified with solution
approximately equal to
the endogenous
concentration, and
fortified with solution
approximately twice the
endogenous
concentration).
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 123 of 185
13.4.3 Data Reporting, Review, and Management
Checks made of the data in the process of review and verification are summarized in Table 13.6. Data
reporting units and significant figures are given in Table 13.7. The NRSA 2018/19 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 13.6 Water chemistry: quality control - data validation
Activity or Procedure
Requirements and Corrective Action
Range checks, summary statistics, and/or
exploratory data analysis (e.g., box and
whisker plots)
Correct reporting errors or qualify as suspect or invalid
Review holding times
Qualify value for additional review
Ion balance:
Calculate percent ion balance difference
(%IBD) using data from cations, anions,
pH, and ANC (see equation 11.1).
Requirements:
• If total ionic strength <100 |aeq/L
- %IBD < ±25%.
• If total ionic strength > 100 |aeq/L
- %IBD <±10%.
Corrective actions:
• Determine which analytes, if any, are the largest contributors to
the ion imbalance. Review suspect analytes for analytical error
and reanalyze.
— Flag = unacceptable %IBD
• If analytical error is not indicated, qualify sample to attribute
imbalance to unmeasured ions. Reanalysis is not required.
— Flag = %IBD outside acceptance criteria due to unmeasured
ions
Conductivity check:
Compare measured conductivity of each
sample to a calculated conductivity
based on the equivalent conductance of
major ions in solution (Hillman et al.,
1987).
Requirements:
• If measured conductivity < 25 |j.S/cm,
— ([measured - calculated] -r- measured) < ±25%.
• If measured conductivity > 25 |j.S/cm,
— ([measured - calculated] -r- measured) < ±15%.
Corrective Actions:
• Determine which analytes, if any, are the largest contributors to
the difference between calculated and measured conductivity.
• Review suspect analytes for analytical error and reanalyze.
• If analytical error is not indicated, qualify sample to attribute
conductivity difference to unmeasured ions. Reanalysis is not
required.
Review data from QA samples (laboratory
PE samples, and inter-laboratory
comparison samples)
Indicator QC Coordinator determines impact and possible
limitations on overall usability of data based on the specific issue.
Table 13.7 Water chemistry: data reporting criteria
Measu rement
Units
No. Significant
Figures
Maximum No.
Decimal Places
DO
mg/L
2
1
Temperature
°C
2
1
PH
pH units
3
2
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 124 of 185

No. Significant
Maximum No.
Measu rement
Units
Figures
Decimal Places
Carbon, total & dissolved organic
mg/L
3
1
ANC
|aeq/L
3
1
Conductivity
|j.S/cm at 25 °C
3
1
Calcium, magnesium, sodium, potassium,
chloride, nitrate, and sulfate
|aeq/L
3
1
Silica
mg/L
3
2
Total phosphorus
M-g/L
3
0
Total nitrogen
mg/L
3
2
Nitrate-Nitrite
mg/L
3
2
Ammonia-N
mg/L
3
2
Turbidity
NTU
3
0
True color
PCU
2
0
TSS
mg/L
3
1
Chlorophyli-a
ug/l
3
2
The ion balance for each sample is computed using the results for major cations, anions, and the
measured acid neutralizing capacity. The percent ion difference (%IBD) for a sample is calculated as:
Equation 13.1 Percent ion difference (%IBD)
(y cations - ^ anions ) - ANC
%IBD =
ANC + anions + ^ cations + 2\h j
where ANC is the acid neutralization capacity; cations are the concentrations of calcium, magnesium,
sodium, potassium, and ammonium (converted from mg/L to |a,eq/L); anions are the concentrations of
chloride, nitrate, and sulfate (converted from mg/L to |a,eq/L), and H+ is the hydrogen ion concentration
calculated from the antilog of the sample pH. Factors to convert major ions from mg/L to |a,eq/L are
presented in Table 13.8. For the conductivity check, equivalent conductivities for major ions are
presented in Table 13.9.
Table 13.8 Water chemistry: constants for converting major ion concentration from mg/L to |ieq/L
Analyte
Conversion from mg/L to (ieq/Lm
Calcium
49.9
Magnesium
82.3
Potassium
25.6
Sodium
43.5
Ammonia-N
71.39
<
i
i
>
CL
o
QC
0
I
1
u
T3
c
tc
>
QC
m Measured values are multiplied by the conversion factor. For ammonia and nitrate, two factors are provided,
one if results are reported as mg N/L, the other if the ion is reported directly.
u
QC
124
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Ammonium
55.4
Chloride
28.2
Nitrate-N
71.39
Nitrate
16.1
Sulfate
20.8
Table 13.9 Water chemistry: factors to calculate equivalent conductivities of major ions."
Ion
Equivalent Conductance per mg/L
(|aS/cm at 25 °C)
Ion
Equivalent Conductance per
mg/L (|j.S/cm at 25 °C)
Calcium
2.60
Nitrate
1.15
Magnesium
3.82
Sulfate
1.54
Potassium
1.84
Hydrogen
3.5 x 105 0
Sodium
2.13
Hydroxide
1.92 x 105
Ammonium
4.13
Bicarbonate
0.715
Chloride
2.14
Carbonate
2.82
13.5 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.
Hillman, D.C., J.F. Potter, and S.J. Simon. 1986. National Surface Water Survey, Eastern Lake Survey-
Phase I, Analytical Methods Manual. EPA/600/4-86/009. U.S. Environmental Protection Agency, Las,
Vegas, NV.
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.
Oblinger Childress, C. J., W. T. Foreman, B. F. Connor, and T. J. Maloney. 1999. New reporting
procedures based on long-term method detection levels and some considerations for interpretations of
water-quality data provided by the U.S. Geological Survey National Water Quality Laboratory. Open-File
Report 99-193, US Geological Survey, Reston, Virginia.
Laboratory Operations Manual
Page 125 of 185
" From Hillman et al. (1987).
°Specific conductance per mole/L, rather than per mg/L.
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 126 of 185
APPENDIX A: CONTACT INFORMATION
Title
Name
Contact Information
EPA HQ NRSA
Project Lead
Richard Mitchell,
OW
mitchell.richardPepa.gov
202-566-0644
EPA HQ NRSA
Project QA
Coordinator
Sarah Lehmann,
OW
lehmann.sarahPepa.gov
202-566-1379
EPA HQ NRSA
Laboratory Review
Coordinator
Kendra Forde, OW
forde.kendra@epa.gov
202-564-0417
Information
Management
Center Coordinator
Marlys Cappaert,
SRA International
Inc.
cappaert.marlysPepa.gov
541-754-4467
541-754-4799 (fax)
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 127 of 185
APPENDIX B: LABORATORY REMOTE EVALUATION FORMS
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 128 of 185
NRSA 2018-2019 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 NRSA 2018-2019, 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 NRSA 2018-2019.
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 NRSA 2018-2019:
~ Water Chemistry, chlorophyll a, and Ash Free Dry Mass (AFDM) (all of the analytes identified in
the LOM and QAPP)
~ Microcystin
~ Fish Tissue Plugs
If your lab has been previously approved within the last 5 years for the water chemistry indicator:
~ 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 NRSA 2018-2019.
~ 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
water chemistry indicator, 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:
~ 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).
~ Documentation showing participation in a previous NARS for Water Chemistry for the same
parameters/methods.
Additionally, we request that all labs provide the following information in support of your
capabilities, (these materials are required if neither of the two items above are provided):
~ A copy of your Laboratory's accreditations and certifications if applicable (i.e. NELAC, ISO, state
certifications, NABS, etc.).
~ An updated copy of your Laboratory's QAPP.
~ Standard Operating Procedures (SOPs) for your lab for each analysis to be performed (if not
covered in NRSA 2018-2019 LOM).
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 129 of 185
~ Documentation attesting to experience running all analytes for the NRSA 2018-2019, 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
mitchell.richard@epa.gov and lehmann.sarah@epa.gov. Questions concerning this request can be
submitted forde.kendra@epa.gov (202-566-0417) or mitchell.richard@epa.gov (202-564-0644).
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 130 of 185
Lab Signature Form - Chemistry Labs
certify that the
lab
located in
j will abide by the following standards in
performing the following data analysis and reporting for the National Rivers and
Streams Assessment (NRSA) 2018-2019.
1.) Utilize procedures identified in the NRSA 2018-2019 Lab Operations Manual (or
equivalent). If using equivalent procedures, please provide procedures manual
and obtain approval from EPA.
2.) Read and abide by the NRSA 2018-2019 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, 2019 (for samples collected in 2018) and May 1, 2020 (for
samples collected in 2019) 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.
This applies to the
chemistry indicator.
Signature
Date
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 131 of 185
NRSA 2018-2019 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 NRSA 2018-2019, 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 NRSA 2018-2019.
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 NRSA 2018-2019:
~ Fish Voucher
~ Benthic Macroinvertabrates
~ Periphyton
If your lab has been previously approved within the last 5 years for the specific parameters:
~ 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 NRSA 2018-2019.
~ 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:
~ Resume of independent fish taxonomist who will verify fish vouchers (if applicable)
~ 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 Q
contact the individual listed below). ^
~ Documentation showing participation in previous NARS for this particular indicator. <
LU
Additionally, we request that all labs provide the following information in support of your capabilities, h
(these materials are required if neither of the two items above are provided): ^
LU
~ A copy of your Laboratory's accreditations and certifications if applicable (i.e. NELAC, ISO, state
QC
certifications, NABS, etc.). O
i_
~ Documentation of NABS (or other) certification for the taxonomists performing analyses (if ^
applicable). co
~ An updated copy of your Laboratory's QAPP. 7!
CO
~ Standard Operating Procedures (SOPs) for your lab for each analysis to be performed (if not ><
covered in NRSA 2018-2019 LOM). i
LU
O.
O.
<
131
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 132 of 185
This documentation may be submitted electronically via e-mail to forde.kendra@epa.gov with a cc: to
mitchell.richard@epa.gov and lehmann.sarah@epa.gov. Questions concerning this request can be
submitted forde.kendra@epa.gov (202-566-0417) or mitchell.richard@epa.gov (202-566-0644).
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 133 of 185
Lab Signature Form - Biology Labs
certify that the
lab
located in
_, will abide by the following standards in
performing biology data analysis and reporting for the National Rivers and Streams
Assessment (NRSA) 2018-2019.
9.) Utilize procedures identified in the NRSA 2018-2019 Lab Operations Manual (or
equivalent). If using equivalent procedures, please provide procedures manual
and obtain approval from EPA.
10.) Read and abide by the NRSA 2018-2019 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 NRSA 2018-2019 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, 2019 (for samples collected in 2018) and May 1, 2020 (for
samples collected in 2019) 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
2018-2019.
This applies to the
biological indicator.
Signature
Date
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 134 of 185
APPENDIX C: SAMPLE LABORATORY FORMS
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 135 of 185
Benthic Macroinvertebrate: Sorting Bench Sheet
Laboratory Information
Sample Information
Project ID

Sample ID

Station Name

Site ID

Station Location

Date Collected

Station Number

Field Crew ID

Sorter Name Proficiency: %
Date Sorting Initiated Proportion of Sample Sorted:

Grid #
(e.g., A5)
Quarter #
(if any)
Sorter's Results
# Organisms
Sorting QC Officer
(if QC check)
Sorting
Order
Each grid
has unique
identifier
Each quarter
has unique
identifier

Cumulative
(include sorter#)
1

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
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 136 of 185
Benthic Macroinvertebrates: Taxonomy Bench Sheet (optional)
Laboratory Information
Sample Information
Project ID

Sample ID

Station Name

Site ID

Station Location

Date Collected

Station Number

Field Crew ID

Taxonomist Name
Date 1st Organism Identified in Sample: QC Check? Y / N
TSN
(Use # in Taxon
Uniqueldentifier (see target taxon
) in Table 3)
Counts of Organisms in the Data
Taxon: Cumulative Qualifier

Number of
(Codes
Distinct

Organisms
in Table
(Y/N)

in Sample
4)
Is cumulative number of organisms > 500? If not, unless the sample was completed sorted, obtain
more sorted sample.
Comments:
136
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 137 of 185
Enterrococci (EPA Method 1606) Laboratory Bench Sheet
Site ID Sample ID Date
Purified DNA Extracts
Batch
Sample #
Sample
ID#
QA/QC
QualCode
Sample Vol
(mL) Filtered
Vol. SAE
Buffer Added
(HL)
Color of
Filter
25X Dilution
Needed?
Comments

137
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 138 of 185
Fish Voucher: Taxonomy Bench Sheet (optional)
Laboratory Information
Sample Information
Labname

Sample ID

QC Taxonomist

Site ID

Project ID

Visit Number

Internal Sample ID

Date Collected

Taxonomist Name:
Date 1st Specimen Identified in Sample:
Tag New
No. Taxon? Common or Scientific Name
Condi
tion Data Comment Including Citation
Code Flag if Appropriate
Additional Comments
138
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 139 of 185
APPENDIX D: OTHER PERTINENT ATTACHMENTS
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 140 of 185
Table D.0.1 Fish identification: standard common and scientific names
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
1
Perciformes
Cichlidae
Hemichromis letourneuxi
african jewelfish
2
Percopsiformes
Amblyopsidae
Speoplotyrhinus poulsoni
alabama cavefish
3
Perciformes
Percidae
Etheostomo romseyi
alabama darter
4
Cypriniformes
Catostomidae
Hypentelium etowonum
alabama hog sucker
5
Clupeiformes
Clupeidae
Aloso olabomoe
alabama shad
6
Cypriniformes
Cyprinidae
Cyprinello collistio
alabama shiner
7
Acipenseriformes
Acipenseridae
Scophirhynchus suttkusi
alabama sturgeon
8
Esociformes
Umbridae
Dollio pectorolis
alaska blackfish
9
Clupeiformes
Clupeidae
Aloso pseudohorengus
alewife
10
Lepisosteiformes
Lepisosteidae
Atroctosteus spotulo
alligator gar
11
Cypriniformes
Cyprinidae
Cyprinello xoenuro
altamaha shiner
12
Cypriniformes
Cyprinidae
Gilo olvordensis
alvord chub
13
Cyprinodontiformes
Cyprinodontidae
Cyprinodon nevodensis
amargosa pupfish
14
Cyprinodontiformes
Poeciliidae
Poecilio formoso
amazon molly
15
Siluriformes
Loricariidae
Pterygoplichthys pordolis
amazon sailfin catfish
16
Perciformes
Percidae
Percino ontesello
amber darter
17
Petromyzontiformes
Petromyzontidae
Lompetro appendix
american brook lamprey
18
Anguilliformes
Anguillidae
Anguillo rostroto
american eel
19
Clupeiformes
Clupeidae
Aloso sopidissimo
american shad
20
Cyprinodontiformes
Poeciliidae
Gombusio omistodensis
amistad gambusia
21
Cypriniformes
Cyprinidae
Pteronotropis grondipinnis
apalachee shiner
22
Perciformes
Percidae
Percino gymnocepholo
appalachia darter
23
Salmoniformes
Salmonidae
Solvelinus alp in us
arctic char
24
Salmoniformes
Salmonidae
Coregonus autumnal is
arctic cisco
25
Salmoniformes
Salmonidae
Thy ma II us orcticus
arctic grayling
26
Petromyzontiformes
Petromyzontidae
Lampetro camtschotica
arctic lamprey
27
Perciformes
Percidae
Etheostomo cragini
arkansas darter
28
Cypriniformes
Cyprinidae
Notropis girardi
arkansas river shiner
29
Perciformes
Percidae
Etheostomo euzonum
arkansas saddled darter
30
Perciformes
Percidae
Etheostomo sogitta
arrow darter
31
Perciformes
Gobiidae
Clevelondia ios
arrow goby
32
Cypriniformes
Cyprinidae
Gila orcuttii
arroyo chub
33
Cyprinodontiformes
Goodeidae
Empetrichthys merriomi
ash meadows poolfish
34
Perciformes
Percidae
Etheostomo cine re um
ashy darter
35
Synbranchiformes
Synbranchidae
Monopterus olbus
asian swamp eel
36
Perciformes
Sciaenidae
Micropogonias undulotus
atlantic croaker
37
Beloniformes
Belonidae
Strongylura marina
atlantic needlefish
-------
2018/19 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.1, June 2018 Page 141 of 185
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
38
Salmoniformes
Salmonidae
Solmo solor
atlantic salmon
39
Myliobatiformes
Dasyatidae
Dasyatis sobino
atlantic stingray
40
Acipenseriformes
Acipenseridae
Acipenser oxyrinchus
atlantic sturgeon
41
Clupeiformes
Clupeidae
Opisthonemo oglinum
atlantic thread herring
42
Gadiformes
Gadidae
Microgodus tomcod
atlantic tomcod
43
Perciformes
Percidae
Etheostomo zonifer
backwater darter
44
Perciformes
Sciaenidae
Bairdiella icistio
bairdiella
45
Perciformes
Cichlidae
Heros severus
banded cichlid
46
Perciformes
Percidae
Etheostomo zonole
banded darter
47
Cypririodontiformes
Fundulidae
Fundulus diophonus
banded killifish
48
Perciformes
Elassomatidae
Elossomo zona turn
banded pygmy sunfish
49
Scorpaeniformes
Cottidae
Cottus carolinae
banded sculpin
50
Perciformes
Centrarchidae
Enneocanthus obesus
banded sunfish
51
Cypririodontiformes
Fundulidae
Fundulus cingulotus
banded topminnow
52
Perciformes
Percidae
Etheostomo zonistium
bandfin darter
53
Cypriniformes
Cyprinidae
Luxilus zonistius
bandfin shiner
54
Cypriniformes
Cyprinidae
Cyprinella leedsi
bannerfin shiner
55
Perciformes
Centrarchidae
Lepomis symmetricus
bantam sunfish
56
Perciformes
Percidae
Etheostomo obeyense
barcheek darter
57
Perciformes
Percidae
Etheostomo forbesi
barrens darter
58
Cypririodontiformes
Fundulidae
Fundulus julisia
barrens topminnow
59
Clupeiformes
Engraulidae
Anchoo mitchilli
bay anchovy
60
Pleuronectiformes
Paralichthyidae
Cithorichthys spilopterus
bay whiff
61
Perciformes
Percidae
Etheostomo rubrum
bayou darter
62
Cyprinodontiformes
Fundulidae
Fundulus pulvereus
bayou killifish
63
Cyprinodontiformes
Fundulidae
Fundulus nottii
bayou topminnow
64
Scorpaeniformes
Cottidae
Cottus extensus
bear lake sculpin
65
Salmoniformes
Salmonidae
Prosopium abyssicola
bear lake whitefish
66
Cypriniformes
Cyprinidae
Cyprinella formoso
beautiful shiner
67
Cypriniformes
Cyprinidae
Notropis rupestris
bedrock shiner
68
Salmoniformes
Salmonidae
Coregonus laurettae
bering cisco
69
Cyprinodontiformes
Poeciliidae
Gambusio goigei
big bend gambusia
70
Cypriniformes
Cyprinidae
Hy bops is omblops
bigeye chub
71
Cypriniformes
Catostomidae
Moxostomo oriommum
bigeye jumprock
72
Cypriniformes
Cyprinidae
Notropis boops
bigeye shiner
73
Cypriniformes
Cyprinidae
Hypophtholmichthys nobilis
bighead carp
74
Cypriniformes
Catostomidae
Ictiobus cyprinellus
bigmouth buffalo
75
Cypriniformes
Cyprinidae
Nocomis plotyrhynchus
bigmouth chub
-------
2018/19 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.1, June 2018 Page 142 of 185
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
76
Cypriniformes
Cyprinidae
Notropis dorsolis
bigmouth shiner
77
Perciformes
Eleotridae
Gobiomorus dormitor
bigmouth sleeper
78
Perciformes
Percidae
Percino macrolepida
bigscale logperch
79
Cypriniformes
Cyprinidae
Rhodeus sericeus
bitterling
80
Perciformes
Cichlidae
Cichlosomo bimoculotum
black acara
81
Cypriniformes
Catostomidae
Ictiobus niger
black buffalo
82
Siluriformes
Ictaluridae
Ameiurus melos
black bullhead
83
Perciformes
Centrarchidae
Pomoxis nigromoculotus
black crappie
84
Siluriformes
Ictaluridae
Noturus funebris
black madtom
85
Cypriniformes
Catostomidae
Moxostomo duquesnei
black redhorse
86
Scorpaeniformes
Cottidae
Cottus boileyi
black sculpin
87
Perciformes
Percidae
Percino nigrofoscioto
blackbanded darter
88
Perciformes
Centrarchidae
Enneocanthus choetodon
blackbanded sunfish
89
Cypriniformes
Cyprinidae
Notropis heterodon
blackchin shiner
90
Perciformes
Cichlidae
So roth e rodon mefonotheron
blackchin tilapia
91
Salmoniformes
Salmonidae
Coregonus nigripinnis
blackfin cisco
92
Perciformes
Percidae
Etheostomo nigripinne
blackfin darter
93
Cypriniformes
Catostomidae
Thoburnio otripinnis
blackfin sucker
94
Cypriniformes
Cyprinidae
Notropis melonostomus
blackmouth shiner
95
Cypriniformes
Cyprinidae
Notropis heterolepis
blacknose shiner
96
Cypriniformes
Cyprinidae
Ph ox in us cumberlondensis
blackside dace
97
Perciformes
Percidae
Percino moculoto
blackside darter
98
Perciformes
Percidae
Etheostomo duryi
blackside snubnose darter
99
Cypriniformes
Cyprinidae
Notropis otrocoudolis
blackspot shiner
100
Cyprinodontiformes
Fundulidae
Fundulus olivaceus
blackspotted topminnow
101
Cyprinodontiformes
Fundulidae
Fundulus nototus
blackstripe topminnow
102
Cypriniformes
Catostomidae
Moxostomo poecilurum
blacktail redhorse
103
Cypriniformes
Cyprinidae
Cyprinella venusto
blacktail shiner
104
Cypriniformes
Catostomidae
Moxostomo cervinum
blacktip jumprock
105
Cypriniformes
Cyprinidae
Lythrurus atrapiculus
blacktip shiner
106
Cypriniformes
Cyprinidae
Luxilus zonotus
bleeding shiner
107
Perciformes
Percidae
Etheostomo blennius
blenny darter
108
Salmoniformes
Salmonidae
Coregonus hoyi
bloater
109
Perciformes
Percidae
Etheostomo sanguifluum
bloodfin darter
110
Perciformes
Gobiidae
Ctenogobius fasciatus
blotchcheek goby
111
Cypriniformes
Cyprinidae
Erimystox insignis
blotched chub
112
Cyprinodontiformes
Poeciliidae
Gambusia senilis
blotched gambusia
113
Perciformes
Percidae
Percino burtoni
blotchside logperch
-------
2018/19 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.1, June 2018 Page 143 of 185
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
114
Siluriformes
Ictaluridae
Ictolurus furcotus
blue catfish
115
Cypriniformes
Cyprinidae
Gilo coeruleo
blue chub
116
Scorpaeniformes
Cottidae
Cottus coeruleomentum
blue ridge sculpin
117
Cypriniformes
Cyprinidae
Cyprinello caerulea
blue shiner
118
Cypriniformes
Catostomidae
Cycleptus elongotus
blue sucker
119
Perciformes
Cichlidae
Oreochromis aureus
blue tilapia
120
Clupeiformes
Clupeidae
Aloso aestivalis
blueback herring
121
Perciformes
Elassomatidae
Elassoma okatie
bluebarred pygmy sunfish
122
Perciformes
Percidae
Etheostoma camurum
bluebreast darter
123
Cyprinodontiformes
Fundulidae
Lucania goodei
bluefin killifish
124
Cypriniformes
Cyprinidae
Campostoma pauciradii
bluefin stoneroller
125
Perciformes
Centrarchidae
Lepomis macrochirus
bluegill
126
Cypriniformes
Cyprinidae
Nocomis leptocephalus
bluehead chub
127
Cypriniformes
Cyprinidae
Pteronotropis hubbsi
bluehead shiner
128
Cypriniformes
Catostomidae
Catostomus discobolus
bluehead sucker
129
Cypriniformes
Cyprinidae
Pteronotropis welaka
bluenose shiner
130
Perciformes
Percidae
Etheostoma jessiae
blueside darter
131
Perciformes
Centrarchidae
Enneacanthus gloriosus
bluespotted sunfish
132
Perciformes
Percidae
Percina cymatotaenia
bluestripe darter
133
Cypriniformes
Cyprinidae
Cyprinella callitaenia
bluestripe shiner
134
Cypriniformes
Cyprinidae
Cyprinella camura
bluntface shiner
135
Perciformes
Percidae
Etheostoma chlorosoma
bluntnose darter
136
Cypriniformes
Cyprinidae
Pimephales notatus
bluntnose minnow
137
Cypriniformes
Cyprinidae
Notropis simus
bluntnose shiner
138
Salmoniformes
Salmonidae
Prosopium gemmifer
bonneville cisco
139
Salmoniformes
Salmonidae
Prosopium spilonotus
bonneville whitefish
140
Cypriniformes
Cyprinidae
Gila elegans
bonytail
141
Cypriniformes
Cyprinidae
Gila boraxobius
borax lake chub
142
Perciformes
Percidae
Etheostoma wapiti
boulder darter
143
Amiiformes
Amiidae
Amia calva
bowfin
144
Cypriniformes
Cyprinidae
Hybognathus ha n kin son i
brassy minnow
145
Cypriniformes
Catostomidae
Catostomus columbianus
bridgelip sucker
146
Cypriniformes
Cyprinidae
Notropis bifrenatus
bridle shiner
147
Perciformes
Percidae
Etheostoma lynceum
brighteye darter
148
Siluriformes
Ictaluridae
Noturus miurus
brindled madtom
149
Salmoniformes
Salmonidae
Coregonus nasus
broad whitefish
150
Cypriniformes
Cyprinidae
Pteronotropis euryzonus
broadstripe shiner
151
Cyprinodontiformes
Fundulidae
Fundulus euryzonus
broadstripe topminnow
-------
2018/19 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.1, June 2018 Page 144 of 185
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
152
Perciformes
Percidae
Percino polmoris
bronze darter
153
Perciformes
Percidae
Etheostomo burri
brook darter
154
Atheriniformes
Atherinopsidae
Labidesthes sicculus
brook silverside
155
Gasterosteiformes
Gasterosteidae
Culaeo inconstons
brook stickleback
156
Salmoniformes
Salmonidae
Sol velin us fon final is
brook trout
157
Siluriformes
Ictaluridae
Ameiurus nebulosus
brown bullhead
158
Perciformes
Percidae
Etheostomo edwini
brown darter
159
Siluriformes
Callichthyidae
Hoplosternum littorole
brown hoplo
160
Siluriformes
Ictaluridae
Noturus phoeus
brown madtom
161
Salmoniformes
Salmonidae
Solmo trutto
brown trout
162
Perciformes
Percidae
Etheostomo bison
buffalo darter
163
Cypriniformes
Cyprinidae
Nocomis roneyi
bull chub
164
Carcharhiniformes
Carcharhinidae
Corchorhinus leu cos
bull shark
165
Salmoniformes
Salmonidae
Sol velin us confluentus
bull trout
166
Cypriniformes
Cyprinidae
Pimepholes vigilox
bullhead minnow
167
Perciformes
Channidae
Chan no morulius
bullseye snakehead
168
Gadiformes
Gadidae
Loto loto
burbot
169
Cypriniformes
Cyprinidae
Mocrhybopsis morconis
burrhead chub
170
Cypriniformes
Cyprinidae
Notropis osperifrons
burrhead shiner
171
Perciformes
Cichlidae
Cichlo ocelloris
butterfly peacock bass
172
Siluriformes
Ictaluridae
Noturus toylori
caddo madtom
173
Cypriniformes
Cyprinidae
Notropis cohoboe
cahaba shiner
174
Cypriniformes
Cyprinidae
Hesperoleucus symmetricus
California roach
175
Perciformes
Percidae
Etheostomo osburni
candy darter
176
Cypriniformes
Cyprinidae
Notropis mekistocholos
cape fear shiner
177
Cypriniformes
Cyprinidae
Luxilus cardinal is
cardinal shiner
178
Cypriniformes
Cyprinidae
Notropis percobromus
carmine shiner
179
Perciformes
Percidae
Etheostomo col lis
Carolina darter
180
Siluriformes
Ictaluridae
Noturus furiosus
Carolina madtom
181
Perciformes
Elassomatidae
Elassoma boehlkei
Carolina pygmy sunfish
182
Esociformes
Umbridae
Umbra limi
central mudminnow
183
Cypriniformes
Cyprinidae
Campostoma anomalum
central stoneroller
184
Esociformes
Esocidae
Esox niger
chain pickerel
185
Perciformes
Percidae
Percina nevisense
chainback darter
186
Siluriformes
Ictaluridae
Ictalurus punctatus
channel catfish
187
Perciformes
Percidae
Percina copelandi
channel darter
188
Cypriniformes
Cyprinidae
Notropis wickliffi
channel shiner
189
Siluriformes
Ictaluridae
Noturus flavater
checkered madtom
-------
2018/19 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.1, June 2018 Page 145 of 185
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
190
Perciformes
Percidae
Etheostomo scotti
cherokee darter
191
Perciformes
Percidae
Etheostomo etnieri
cherry darter
192
Cypriniformes
Cyprinidae
Lythrurus roseipinnis
cherryfin shiner
193
Petromyzontiformes
Petromyzontidae
Ichthyomyzon costoneus
chestnut lamprey
194
Perciformes
Percidae
Etheostomo cervus
chickasaw darter
195
Cypriniformes
Cyprinidae
Gilo nigrescens
chihuahua chub
196
Cypriniformes
Cyprinidae
Notropis chihuohuo
chihuahua shiner
197
Salmoniformes
Salmonidae
Oncorhynchus tshowytscho
chinook salmon
198
Cypriniformes
Cyprinidae
Acrocheilus olutoceus
chiselmouth
199
Perciformes
Percidae
Etheostomo davisoni
choctawhatchee darter
200
Perciformes
Percidae
Etheostomo hopkinsi
Christmas darter
201
Cypriniformes
Cyprinidae
Notropis potteri
chub shiner
202
Salmoniformes
Salmonidae
Oncorhynchus keto
chum salmon
203
Salmoniformes
Salmonidae
Coregonus artedi
cisco
204
Cypriniformes
Cyprinidae
Hybopsis wincheili
clear chub
205
Cyprinodontiformes
Poeciliidae
Gambusia heterochir
clear creek gambusia
206
Cypriniformes
Cyprinidae
Pogonichthys ciscoides
clear lake splittail
207
Perciformes
Gobiidae
Microgobius gulosus
clown goby
208
Osteoglossiformes
Notopteridae
Chita la ornata
clown knifefish
209
Perciformes
Percidae
Percina brevicauda
coal darter
210
Perciformes
Percidae
Etheostomo colorosum
coastal darter
211
Cypriniformes
Cyprinidae
Notropis petersoni
coastal shiner
212
Scorpaeniformes
Cottidae
Cottus oleuticus
coastrange sculpin
213
Salmoniformes
Salmonidae
Oncorhynchus kisutch
coho salmon
214
Perciformes
Percidae
Etheostomo d it re mo
coldwater darter
215
Cypriniformes
Cyprinidae
Ptychocheilus lucius
Colorado pikeminnow
216
Scorpaeniformes
Cottidae
Cottus hubbsi
Columbia sculpin
217
Cyprinodontiformes
Cyprinodontidae
Cyprinodon elegons
comanche springs pupfish
218
Cypriniformes
Cyprinidae
Notropis omoenus
comely shiner
219
Cypriniformes
Cyprinidae
Cyprinus corpio
common carp
220
Cypriniformes
Cyprinidae
Luxilus cornutus
common shiner
221
Perciformes
Centropomidae
Centropomus undecimolis
common snook
222
Perciformes
Percidae
Percina jenkinsi
conasaugalogperch
223
Cyprinodontiformes
Cyprinodontidae
Cyprinodon eximius
conchos pupfish
224
Perciformes
Cichlidae
Cichlasoma nigrofasciatum
convict cichlid
225
Perciformes
Percidae
Etheostomo coosoe
coosa darter
226
Cypriniformes
Cyprinidae
Notropis xoenocepholus
coosa shiner
227
Perciformes
Percidae
Etheostomo oquoli
coppercheek darter
-------
2018/19 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.1, June 2018 Page 146 of 185
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
228
Perciformes
Percidae
Etheostomo bosilore
corrugated darter
229
Cypriniformes
Cyprinidae
Semotilus otromoculotus
creek chub
230
Cypriniformes
Catostomidae
Erimyzon oblong us
creek chubsucker
231
Perciformes
Percidae
Etheostomo collettei
Creole darter
232
Cypriniformes
Cyprinidae
Luxilus cerosin us
crescent shiner
233
Perciformes
Gobiidae
Lophogobius cyprinoides
crested goby
234
Perciformes
Belontiidae
Trichopsis vittoto
croaking gourami
235
Perciformes
Percidae
Etheostomo corona
crown darter
236
Perciformes
Percidae
Crystollorio osprello
crystal darter
237
Cypriniformes
Catostomidae
Chasmistes cujus
cui-ui
238
Perciformes
Percidae
Etheostomo susonoe
Cumberland darter
239
Perciformes
Percidae
Etheostomo uniporum
current darter
240
Cypriniformes
Cyprinidae
Exoglossum maxillinguo
cutlip minnow
241
Salmoniformes
Salmonidae
Oncorhynchus clorkii
cutthroat trout
242
Perciformes
Percidae
Etheostomo proeliore
cypress darter
243
Cypriniformes
Cyprinidae
Hybognothus hoyi
cypress minnow
244
Perciformes
Gobiidae
Ctenogobius boleosomo
darter goby
245
Salmoniformes
Salmonidae
Coregonus johonnoe
deepwater cisco
246
Scorpaeniformes
Cottidae
Myoxocephoius thompsonii
deepwater sculpin
247
Salmoniformes
Osmeridae
Hypomesus tronspocificus
delta smelt
248
Cypriniformes
Cyprinidae
Eremichthys ocros
desert dace
249
Cyprinodontiformes
Cyprinodontidae
Cyprinodon moculorius
desert pupfish
250
Cypriniformes
Catostomidae
Cotostomus clorkii
desert sucker
251
Cyprinodontiformes
Cyprinodontidae
Cyprinodon diobolis
devils hole pupfish
252
Cypriniformes
Cyprinidae
Dionda diaboli
devils river minnow
253
Cypriniformes
Cyprinidae
Semotilus thoreouionus
dixie chub
254
Perciformes
Centrarchidae
Lepomis morginotus
dollar sunfish
255
Salmoniformes
Salmonidae
Solvelinus molmo
dolly varden
256
Perciformes
Percidae
Percino sciero
dusky darter
257
Cypriniformes
Cyprinidae
Notropis cummingsoe
dusky shiner
258
Cypriniformes
Cyprinidae
Luxilus pilsbryi
duskystripe shiner
259
Perciformes
Percidae
Etheostomo percnurum
duskytail darter
260
Cypriniformes
Cyprinidae
Rhinichthys otrotulus
eastern blacknose dace
261
Cyprinodontiformes
Poeciliidae
Gambusio holbrooki
eastern mosquitofish
262
Esociformes
Umbridae
Umbro pygmoeo
eastern mudminnow
263
Perciformes
Percidae
Ammocrypto pellucido
eastern sand darter
264
Cypriniformes
Cyprinidae
Hybognothus regius
eastern silvery minnow
265
Perciformes
Percidae
Etheostomo pseudovulotum
egg-mimic darter
-------
2018/19 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.1, June 2018 Page 147 of 185
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
266
Siluriformes
Ictaluridae
Noturus el eg on s
elegant madtom
267
Perciformes
Percidae
Etheostomo baileyi
emerald darter
268
Cypriniformes
Cyprinidae
Notropis otherinoides
emerald shiner
269
Gasterosteiformes
Gasterosteidae
Gosterosteus oculeotus
espinocho
270
Perciformes
Percidae
Etheostomo etowohoe
etowah darter
271
Salmoniformes
Osmeridae
Tholeichthys pocificus
eulachon
272
Perciformes
Elassomatidae
Elossomo everglodei
everglades pygmy sunfish
273
Cypriniformes
Cyprinidae
Semotilus corporal is
fallfish
274
Perciformes
Percidae
Etheostomo flobellore
fantail darter
275
Perciformes
Eleotridae
Dormitotor mocu lotus
fat sleeper
276
Cypriniformes
Cyprinidae
Pimepholes promelos
fathead minnow
277
Cypriniformes
Cyprinidae
Phenacobius crossilobrum
fatlips minnow
278
Cypriniformes
Cyprinidae
Cyprinello pyrrhomelos
fieryblack shiner
279
Cypriniformes
Cyprinidae
Ph oxin us neogoeus
finescale dace
280
Perciformes
Percidae
Etheostomo pyrrhogoster
firebelly darter
281
Perciformes
Cichlidae
Cichlosomo meeki
firemouth cichlid
282
Cypriniformes
Cyprinidae
Pteronotropis signipinnis
flagfin shiner
283
Cyprinodontiformes
Cyprinodontidae
Jordanella floridoe
flagfish
284
Cypriniformes
Cyprinidae
He mitre mio flommea
flame chub
285
Cypriniformes
Catostomidae
Cotostomus lotipinnis
flannelmouth sucker
286
Siluriformes
Ictaluridae
Ameiurus plotycepholus
flat bullhead
287
Siluriformes
Ictaluridae
Pylodictis olivoris
flathead catfish
288
Cypriniformes
Cyprinidae
Platygobio gracilis
flathead chub
289
Perciformes
Centrarchidae
Centrarchus macropterus
flier
290
Lepisosteiformes
Lepisosteidae
Lepisosteus platyrhincus
florida gar
291
Perciformes
Percidae
Ammocrypta bifascia
florida sand darter
292
Cypriniformes
Cyprinidae
Notropis edwardraneyi
fluvial shiner
293
Perciformes
Percidae
Etheostomo fonticolo
fountain darter
294
Scorpaeniformes
Cottidae
Myoxocepholus q uodricorn is
fourhorn sculpin
295
Gasterosteiformes
Gasterosteidae
Apeltes quodrocus
fourspine stickleback
296
Perciformes
Percidae
Percino stictogoster
frecklebelly darter
297
Siluriformes
Ictaluridae
Noturus munitus
frecklebelly madtom
298
Perciformes
Percidae
Percino lenticulo
freckled darter
299
Siluriformes
Ictaluridae
Noturus nocturnus
freckled madtom
300
Perciformes
Sciaenidae
Aplodinotus grunniens
freshwater drum
301
Perciformes
Gobiidae
Ctenogobius shufeldti
freshwater goby
302
Perciformes
Percidae
Etheostomo crossopterum
fringed darter
303
Cypriniformes
Cyprinidae
Notropis buchanani
ghost shiner
-------
2018/19 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.1, June 2018 Page 148 of 185
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
304
Cyprinodontiformes
Aplocheilidae
Rivulus hortii
giant rivulus
305
Cypriniformes
Cyprinidae
Gilo intermedia
gila chub
306
Cyprinodontiformes
Poeciliidae
Poeciliopsis occidentolis
gila topminnow
307
Salmoniformes
Salmonidae
Oncorhynchus gitae
gila trout
308
Perciformes
Percidae
Percino evides
gilt darter
309
Clupeiformes
Clupeidae
Dorosoma cepedionum
gizzard shad
310
Perciformes
Percidae
Etheostomo vitreum
glassy darter
311
Perciformes
Percidae
Etheostomo denoncourti
golden darter
312
Cypriniformes
Catostomidae
Moxostomo erythrurum
golden redhorse
313
Cypriniformes
Cyprinidae
Notemigonus crysoleucos
golden shiner
314
Cyprinodontiformes
Fundulidae
Fundulus chrysotus
golden topminnow
315
Hiodontiformes
Hiodontidae
Hiodon olosoides
goldeye
316
Cypriniformes
Cyprinidae
Corossius ourotus
goldfish
317
Perciformes
Percidae
Percino auroiineata
goldline darter
318
Perciformes
Percidae
Etheostomo porvipinne
goldstripe darter
319
Cypriniformes
Cyprinidae
Ctenopharyngodon idella
grass carp
320
Cypriniformes
Cyprinidae
Erimystax x-punctotus
gravel chub
321
Cypriniformes
Catostomidae
Moxostomo congestum
gray redhorse
322
Perciformes
Lutjanidae
Lutjonus g rise us
gray snapper
323
Cypriniformes
Catostomidae
Moxostomo lochneri
greater jumprock
324
Cypriniformes
Catostomidae
Moxostomo volenciennesi
greater redhorse
325
Acipenseriformes
Acipenseridae
Acipenser medirostris
green sturgeon
326
Perciformes
Centrarchidae
Lepomis cyonellus
green sunfish
327
Cyprinodontiformes
Poeciliidae
Xiphophorus hellerii
green swordtail
328
Perciformes
Percidae
Etheostomo jordani
greenbreast darter
329
Perciformes
Percidae
Etheostomo chlorobranchium
greenfin darter
330
Cypriniformes
Cyprinidae
Cyprinello chloristio
greenfin shiner
331
Cypriniformes
Cyprinidae
Notropis chiorocephalus
greenhead shiner
332
Perciformes
Percidae
Etheostomo blennioides
greenside darter
333
Perciformes
Percidae
Etheostomo lepidum
greenthroat darter
334
Perciformes
Centrarchidae
Micropterus treculii
guadalupe bass
335
Cyprinodontiformes
Fundulidae
Fundulus porvipinnis
guadalupe cardinalfish
336
Cypriniformes
Cyprinidae
Dionda nigrotoenioto
guadalupe roundnose minnow
337
Perciformes
Percidae
Etheostomo oophylox
guardian darter
338
Perciformes
Eleotridae
Guavino guovino
guavina
339
Perciformes
Percidae
Etheostomo swoini
gulf darter
340
Cyprinodontiformes
Fundulidae
Fundulus grand is
gulf killifish
341
Perciformes
Percidae
Percino suttkusi
gulf logperch
-------
2018/19 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.1, June 2018 Page 149 of 185
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
342
Gasterosteiformes
Syngnathidae
Syngnothus scovelli
gulf pipefish
343
Cyprinodontiformes
Poeciliidae
Poecilia reticulata
guppy
344
Cypriniformes
Cyprinidae
Mylopharodon conocephalus
hardhead
345
Siluriformes
Ariidae
Ariopsis felis
hardhead catfish
346
Cypriniformes
Catostomidae
Moxostoma lace rum
harelip sucker
347
Perciformes
Percidae
Etheostoma histrio
harlequin darter
348
Siluriformes
Ictaluridae
Ictalurus lupus
headwater catfish
349
Cypriniformes
Cyprinidae
Gila nigra
headwater chub
350
Perciformes
Percidae
Etheostoma law re nee i
headwater darter
351
Clupeiformes
Clupeidae
Alosa mediocris
hickory shad
352
Cypriniformes
Cyprinidae
Hybopsis hypsinotus
highback chub
353
Cypriniformes
Catostomidae
Carpiodes velifer
highfin carpsucker
354
Cypriniformes
Cyprinidae
Notropis altipinnis
highfin shiner
355
Perciformes
Percidae
Etheostoma kantuckeense
highland rim darter
356
Cypriniformes
Cyprinidae
Notropis micropteryx
highland shiner
357
Cypriniformes
Cyprinidae
Notropis hypsilepis
highscale shiner
358
Cypriniformes
Cyprinidae
Lavinia exilicauda
hitch
359
Pleuronectiformes
Achiridae
Trinectes maculatus
hogchoker
360
Perciformes
Percidae
Etheostoma brevirostrum
holiday darter
361
Cypriniformes
Cyprinidae
Nocomis biguttatus
hornyhead chub
362
Cypriniformes
Cyprinidae
Gila cypha
humpback chub
363
Salmoniformes
Salmonidae
Coregonus pidschian
humpback whitefish
364
Cypriniformes
Cyprinidae
Leuciscus id us
ide
365
Salmoniformes
Salmonidae
Stenodus leucichthys
inconnu
366
Atheriniformes
Atherinopsidae
Menidia beryllina
inland silverside
367
Perciformes
Percidae
Etheostoma exile
iowa darter
368
Perciformes
Gerreidae
Diapterus auratus
irish pompano
369
Cypriniformes
Cyprinidae
Notropis chalybaeus
ironcolor shiner
370
Perciformes
Cichlidae
Cichlasoma octofasciatum
jack dempsey
371
Perciformes
Cichlidae
Cichlasoma managuense
jaguar guapote
372
Perciformes
Percidae
Etheostoma nigrum
johnny darter
373
Cypriniformes
Catostomidae
Chasmistes liorus
june sucker
374
Perciformes
Percidae
Etheostoma kanawhae
kanawha darter
375
Cypriniformes
Cyprinidae
Phenacobius teretulus
kanawha minnow
376
Perciformes
Percidae
Etheostoma rafinesquei
kentucky darter
377
Petromyzontiformes
Petromyzontidae
Lampetra hubbsi
kern brook lamprey
378
Cypriniformes
Cyprinidae
Notropis ortenburgeri
kiamichi shiner
379
Salmoniformes
Salmonidae
Coregonus kiyi
kiyi
-------
2018/19 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.1, June 2018 Page 150 of 185
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
380
Scorpaeniformes
Cottidae
Cottus princeps
klamath lake sculpin
381
Petromyzontiformes
Petromyzontidae
Lampetro similis
klamath lamprey
382
Cypriniformes
Catostomidae
Cotostomus snyderi
klamath largescale sucker
383
Cypriniformes
Catostomidae
Cotostomus rimiculus
klamath smallscale sucker
384
Elopiformes
Elopidae
Elops sourus
ladyfish
385
Cypriniformes
Cyprinidae
Richordsonius egregius
lahontan redside
386
Cypriniformes
Cyprinidae
Couesius plumbeus
lake chub
387
Cypriniformes
Catostomidae
Erimyzon sucetto
lake chubsucker
388
Acipenseriformes
Acipenseridae
Acipenser ful vescens
lake sturgeon
389
Salmoniformes
Salmonidae
Solvelinus nomoycush
lake trout
390
Salmoniformes
Salmonidae
Coregonus clupeaformis
lake whitefish
391
Perciformes
Centrarchidae
Micropterus solmoides
largemouth bass
392
Cypriniformes
Cyprinidae
Compostomo oligolepis
largescale stoneroller
393
Cypriniformes
Catostomidae
Cotostomus macrocheilus
largescale sucker
394
Perciformes
Eleotridae
Eleotris amblyopsis
largescaled spinycheek
395
Cyprinodontiformes
Poeciliidae
Gambusia geiseri
largespring gambusia
396
Cypriniformes
Cyprinidae
Rhinichthys deoconi
las vegas dace
397
Cypriniformes
Cyprinidae
Ph oxin us soylori
laurel dace
398
Petromyzontiformes
Petromyzontidae
Lampetro aepyptera
least brook lamprey
399
Cypriniformes
Cyprinidae
lotichthys phlegethontis
least chub
400
Salmoniformes
Salmonidae
Coregonus sardinella
least cisco
401
Perciformes
Percidae
Etheostoma microperca
least darter
402
Cyprinodontiformes
Poeciliidae
Heteran dria formosa
least killifish
403
Siluriformes
Ictaluridae
Noturus hildebrandi
least madtom
404
Cypriniformes
Cyprinidae
Snyderichthys copei
leatherside chub
405
Cyprinodontiformes
Cyprinodontidae
Cyprinodon bovinus
leon springs pupfish
406
Cypriniformes
Cyprinidae
Rhinich thys falcatus
leopard dace
407
Perciformes
Percidae
Percina panther in a
leopard darter
408
Cypriniformes
Cyprinidae
Hybopsis lineapunctata
lined chub
409
Cyprinodontiformes
Fundulidae
Fundulus lineolatus
lined topminnow
410
Perciformes
Percidae
Etheostoma chuckwachatte
lipstick darter
411
Cypriniformes
Cyprinidae
Lepidomeda vittata
little Colorado spinedace
412
Cypriniformes
Cyprinidae
Rhinichthys cobitis
loach minnow
413
Perciformes
Percidae
Percina caprodes
logperch
414
Perciformes
Percidae
Etheostoma neopterum
lollypop darter
415
Perciformes
Centrarchidae
Lepomis megalotis
longear sunfish
416
Cypriniformes
Cyprinidae
Agosia chrysogaster
longfin dace
417
Perciformes
Percidae
Etheostoma longimanum
longfin darter
-------
2018/19 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.1, June 2018 Page 151 of 185
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
418
Salmoniformes
Osmeridae
Spirinchus tholeichthys
longfin smelt
419
Perciformes
Percidae
Percino macrocephoto
longhead darter
420
Perciformes
Gobiidae
Gillichthys mirobill's
longjaw mudsucker
421
Cypriniformes
Cyprinidae
Rhinichthys cotoroctoe
longnose dace
422
Perciformes
Percidae
Percino nosuto
longnose darter
423
Lepisosteiformes
Lepisosteidae
Lepisosteus osseus
longnose gar
424
Cypriniformes
Cyprinidae
Notropis longirostris
longnose shiner
425
Cypriniformes
Catostomidae
Cotostomus cotostomus
longnose sucker
426
Cypriniformes
Catostomidae
Deltistes luxotus
lost river sucker
427
Elopiformes
Elopidae
Elops offinis
machete
428
Scorpaeniformes
Cottidae
Cottus bend ire i
malheur sculpin
429
Cypriniformes
Cyprinidae
Dionda orgentoso
manantial roundnose minnow
430
Cyprinodontiformes
Poeciliidae
Gombusio rhizophoroe
mangrove gambusia
431
Cyprinodontiformes
Aplocheilidae
Rivulus mormorotus
mangrove rivulus
432
Scorpaeniformes
Cottidae
Cottus klomothensis
marbled sculpin
433
Siluriformes
Ictaluridae
Noturus in sign is
margined madtom
434
Scorpaeniformes
Cottidae
Cottus marginatus
margined sculpin
435
Cyprinodontiformes
Fundulidae
Fundulus confluentus
marsh killifish
436
Perciformes
Percidae
Etheostomo sell ore
maryland darter
437
Perciformes
Cichlidae
Cichlosomo urophtholmus
mayan cichlid
438
Perciformes
Gobiidae
Ctenogobius cloytonii
mexican goby
439
Cyprinodontiformes
Poeciliidae
Poecilio sphenops
mexican molly
440
Cypriniformes
Catostomidae
Moxostomo oustrinum
mexican redhorse
441
Cypriniformes
Cyprinidae
Compostomo ornotum
mexican stoneroller
442
Characiformes
Characidae
Astyonox mexiconus
mexican tetra
443
Perciformes
Cichlidae
Cichlosomo citrinellum
midas cichlid
444
Petromyzontiformes
Petromyzontidae
Lompetro minima
miller lake lamprey
445
Cypriniformes
Cyprinidae
Notropis volucellus
mimic shiner
446
Cypriniformes
Cyprinidae
Notropis spectrunculus
mirror shiner
447
Atheriniformes
Atherinopsidae
Menidio oudens
mississippi silverside
448
Cypriniformes
Cyprinidae
Hybognothus nucha lis
mississippi silvery minnow
449
Perciformes
Percidae
Etheostomo tetrazonum
missouri saddled darter
450
Cypriniformes
Cyprinidae
Moapa coriacea
moapa dace
451
Perciformes
Percidae
Percino kothae
mobile logperch
452
Cypriniformes
Catostomidae
Cotostomus microps
modoc sucker
453
Hiodontiformes
Hiodontidae
Hiodon tergisus
mooneye
454
Scorpaeniformes
Cottidae
Cottus boirdii
mottled sculpin
455
Petromyzontiformes
Petromyzontidae
Ichthyomyzon greeleyi
mountain brook lamprey
-------
2018/19 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.1, June 2018 Page 152 of 185
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
456
Siluriformes
Ictaluridae
Noturus eleutherus
mountain madtom
457
Mugiliformes
Mugilidae
Agonostomus monticolo
mountain mullet
458
Cypriniformes
Cyprinidae
Ph oxin us oreos
mountain redbelly dace
459
Cypriniformes
Cyprinidae
Lythrurus lirus
mountain shiner
460
Cypriniformes
Catostomidae
Cotostomus plotyrhynch us
mountain sucker
461
Salmoniformes
Salmonidae
Prosopium williomsoni
mountain whitefish
462
Perciformes
Cichlidae
Oreochromis mossambicus
mozambique tilapia
463
Perciformes
Percidae
Etheostomo osprigene
mud darter
464
Perciformes
Centrarchidae
Aconthorchus pomotis
mud sunfish
465
Cypririodontiformes
Fundulidae
Fundulus heteroclitus
mummichog
466
Esociformes
Esocidae
Esox mosquinongy
muskellunge
467
Perciformes
Gobiidae
Gobiosoma bosc
naked goby
468
Perciformes
Percidae
Ammocrypto beonii
naked sand darter
469
Siluriformes
Ictaluridae
Noturus placid us
neosho madtom
470
Cypriniformes
Cyprinidae
Notropis scobriceps
new river shiner
471
Perciformes
Percidae
Etheostomo nionguoe
niangua darter
472
Perciformes
Cichlidae
Oreochromis niloticus
nile tilapia
473
Gasterosteiformes
Gasterosteidae
Pungitius pungitius
ninespine stickleback
474
Petromyzontiformes
Petromyzontidae
Ichthyomyzon fossor
northern brook lamprey
475
Percopsiformes
Amblyopsidae
Amblyopsis speloeo
northern cavefish
476
Cypriniformes
Catostomidae
Hypentelium nigricans
northern hog sucker
477
Siluriformes
Ictaluridae
Noturus stigmosus
northern madtom
478
Esociformes
Esocidae
Esox lucius
northern pike
479
Cypriniformes
Cyprinidae
Ptychocheilus oregonensis
northern pikeminnow
480
Cyprinodontiformes
Fundulidae
Fundulus konsoe
northern plains killifish
481
Cypriniformes
Cyprinidae
Phoxinus eos
northern redbelly dace
482
Cyprinodontiformes
Fundulidae
Fundulus cotenotus
northern studfish
483
Cypriniformes
Catostomidae
Moxostomo collopsum
notchlip redhorse
484
Cypriniformes
Cyprinidae
Diondo sere no
nueces roundnose minnow
485
Cypriniformes
Cyprinidae
Cyprinello collisemo
ocmulgee shiner
486
Petromyzontiformes
Petromyzontidae
Ichthyomyzon bdellium
ohio lamprey
487
Perciformes
Percidae
Etheostomo okaloosae
okaloosa darter
488
Perciformes
Elassomatidae
Elossomo okefenokee
okefenokee pygmy sunfish
489
Perciformes
Percidae
Percino squomoto
olive darter
490
Esociformes
Umbridae
Novumbro hubbsi
Olympic mudminnow
491
Gasterosteiformes
Syngnathidae
Micro phis brochyurus
opossum pipefish
492
Perciformes
Percidae
Etheostomo rodiosum
orangebelly darter
493
Perciformes
Percidae
Etheostomo bellum
orangefin darter
-------
2018/19 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.1, June 2018 Page 153 of 185
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
494
Siluriformes
Ictaluridae
Noturus gilberti
orangefin madtom
495
Cypriniformes
Cyprinidae
Notropis ommophilus
orangefin shiner
496
Perciformes
Sciaenidae
Cynoscion xonth ulus
orangemouth corvina
497
Perciformes
Centrarchidae
Lepomis humilis
orangespotted sunfish
498
Cypriniformes
Cyprinidae
Pteronotropis merlini
orangetail shiner
499
Perciformes
Percidae
Etheostomo spectobile
orangethroat darter
500
Cypriniformes
Cyprinidae
Oregonichthys cromeri
oregon chub
501
Cypriniformes
Cobitidae
Misgurnus onguillicoudotus
oriental weatherfish
502
Siluriformes
Loricariidae
Pterygoplichthys multirodiotus
Orinoco sailfin catfish
503
Perciformes
Cichlidae
Astronotus ocellotus
oscar
504
Siluriformes
Ictaluridae
Noturus lochneri
ouachita madtom
505
Cypriniformes
Cyprinidae
Lythrurus snelsoni
ouachita shiner
506
Cyprinodontiformes
Cyprinodontidae
Cyprinodon rodiosus
owens pupfish
507
Cypriniformes
Catostomidae
Cotostomus fumeiventris
owens sucker
508
Perciformes
Centrarchidae
Ambloplites constellotus
ozark bass
509
Percopsiformes
Amblyopsidae
Amblyopsis rosoe
ozark cavefish
510
Cypriniformes
Cyprinidae
Erimystox horryi
ozark chub
511
Perciformes
Percidae
Percino fulvitoenio
ozark logperch
512
Siluriformes
Ictaluridae
Noturus alboter
ozark madtom
513
Cypriniformes
Cyprinidae
Notropis nubilus
ozark minnow
514
Scorpaeniformes
Cottidae
Cottus hypselurus
ozark sculpin
515
Cypriniformes
Cyprinidae
Notropis ozarconus
ozark shiner
516
Petromyzontiformes
Petromyzontidae
Lampetro tridentoto
pacific lamprey
517
Scorpaeniformes
Cottidae
Leptocottus ormotus
pacific staghorn sculpin
518
Acipenseriformes
Polyodontidae
Polyodon spothulo
paddlefish
519
Cypriniformes
Cyprinidae
Lepidomedo oltivelis
pahranagat spinedace
520
Cyprinodontiformes
Goodeidae
Empetrichthys lotos
pahrump poolfish
521
Scorpaeniformes
Cottidae
Cottus beldingii
paiute sculpin
522
Perciformes
Percidae
Etheostomo pollididorsum
paleback darter
523
Cypriniformes
Cyprinidae
Notropis olbizonotus
palezone shiner
524
Cypriniformes
Cyprinidae
Hybopsis omnis
pallid shiner
525
Acipenseriformes
Acipenseridae
Scophirhynchus olbus
pallid sturgeon
526
Cypriniformes
Catostomidae
Moxostomo pisolobrum
pealip redhorse
527
Cypriniformes
Cyprinidae
Mylocheilus courinus
peamouth
528
Cypriniformes
Cyprinidae
Morgoriscus morgorito
pearl dace
529
Perciformes
Percidae
Percino ouroro
pearl darter
530
Cyprinodontiformes
Poeciliidae
Gombusio nobilis
pecos gambusia
531
Cyprinodontiformes
Cyprinodontidae
Cyprinodon pecosensis
pecos pupfish
-------
2018/19 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.1, June 2018 Page 154 of 185
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
532
Cypriniformes
Cyprinidae
Mocrhybopsis tetronema
peppered chub
533
Cypriniformes
Cyprinidae
Notropis per poll id us
peppered shiner
534
Cypriniformes
Cyprinidae
Notropis orca
phantom shiner
535
Perciformes
Percidae
Percino crosso
piedmont darter
536
Perciformes
Haemulidae
Orthopristis chrysoptero
pigfish
537
Cypririodontiformes
Poeciliidae
Belonesox belizonus
pike killifish
538
Perciformes
Percidae
Etheostomo morioe
pinewoods darter
539
Cypriniformes
Cyprinidae
Lythrurus matutinus
pinewoods shiner
540
Perciformes
Sparidae
Logodon rhomboides
pinfish
541
Salmoniformes
Salmonidae
Oncorhynchus gorbuscho
pink salmon
542
Percopsiformes
Aphredoderidae
Aphredoderus soyonus
pirate perch
543
Scorpaeniformes
Cottidae
Cottus pitensis
pit sculpin
544
Petromyzontiformes
Petromyzontidae
Lampetro lethophoga
pit-klamath brook lamprey
545
Cyprinodontiformes
Fundulidae
Fundul us zebrin us
plains killifish
546
Cypriniformes
Cyprinidae
Hybognothus plocitus
plains minnow
547
Cyprinodontiformes
Fundulidae
Fundul us sciodicus
plains topminnow
548
Cypriniformes
Cyprinidae
Cyprinello lepido
plateau shiner
549
Salmoniformes
Osmeridae
Hypomesus olid us
pond smelt
550
Cypriniformes
Cyprinidae
Notropis oriommus
popeye shiner
551
Cyprinodontiformes
Poeciliidae
Poeciliopsis gracilis
porthole livebearer
552
Scorpaeniformes
Cottidae
Cottus girordi
potomac sculpin
553
Cypriniformes
Cyprinidae
Mocrhybopsis oustrolis
prairie chub
554
Cypriniformes
Cyprinidae
Lythrurus bellus
pretty shiner
555
Scorpaeniformes
Cottidae
Cottus osper
prickly sculpin
556
Cypriniformes
Cyprinidae
Cyprinello proserpino
proserpine shiner
557
Cypriniformes
Cyprinidae
Opsopoeodus emilioe
pugnose minnow
558
Cypriniformes
Cyprinidae
Notropis a nog en us
pugnose shiner
559
Perciformes
Centrarchidae
Lepomis gibbosus
pumpkinseed
560
Cyprinodontiformes
Fundulidae
Leptoluconio ommoto
pygmy killifish
561
Siluriformes
Ictaluridae
Noturus stonouli
pygmy madtom
562
Scorpaeniformes
Cottidae
Cottus poulus
pygmy sculpin
563
Salmoniformes
Salmonidae
Prosopium coulterii
pygmy whitefish
564
Cypriniformes
Catostomidae
Corpiodes cyprinus
quill back
565
Cyprinodontiformes
Goodeidae
Crenichthys nevodoe
railroad valley springfish
566
Perciformes
Percidae
Etheostomo coeruleum
rainbow darter
567
Cypriniformes
Cyprinidae
Notropis chrosomus
rainbow shiner
568
Salmoniformes
Osmeridae
Osmerus mordox
rainbow smelt
569
Salmoniformes
Salmonidae
Oncorhynchus my kiss
rainbow trout
-------
2018/19 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.1, June 2018 Page 155 of 185
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
570
Cyprinodontiformes
Fundulidae
Luconio porva
rainwater killifish
571
Cypriniformes
Catostomidae
Xyrauchen texonus
razorback sucker
572
Perciformes
Sciaenidae
Sciaenops ocellotus
red drum
573
Cyprinodontiformes
Cyprinodontidae
Cyprinodon rubrofluviatilis
red river pupfish
574
Cypriniformes
Cyprinidae
Notropis boirdi
red river shiner
575
Cypriniformes
Cyprinidae
Cyprinello lutrensis
red shiner
576
Perciformes
Percidae
Etheostomo luteovinctum
redband darter
577
Perciformes
Cichlidae
Tilopio zillii
redbelly tilapia
578
Perciformes
Centrarchidae
Lepomis ouritus
redbreast sunfish
579
Perciformes
Centrarchidae
Lepomis microlophus
redear sunfish
580
Perciformes
Centrarchidae
Micropterus coosoe
redeye bass
581
Cypriniformes
Cyprinidae
Notropis horperi
redeye chub
582
Cyprinodontiformes
Fundulidae
Fundulus rubrifrons
redface topminnow
583
Perciformes
Percidae
Etheostomo whipplei
redfin darter
584
Esociformes
Esocidae
Esox omericonus
redfin pickerel
585
Cypriniformes
Cyprinidae
Lythrurus umbrotilis
redfin shiner
586
Perciformes
Percidae
Etheostomo rufilineotum
redline darter
587
Cypriniformes
Cyprinidae
Notropis chiliticus
redlip shiner
588
Cypriniformes
Cyprinidae
Clinostomus elongotus
redside dace
589
Cypriniformes
Cyprinidae
Richordsonius bolteotus
redside shiner
590
Cypriniformes
Cyprinidae
Nocomis osper
redspot chub
591
Perciformes
Percidae
Etheostomo artesiae
redspot darter
592
Perciformes
Centrarchidae
Lepomis miniatus
redspotted sunfish
593
Perciformes
Cichlidae
Geophogus surinamensis
redstriped eartheater
594
Cypriniformes
Cyprinidae
Nocomis effusus
redtail chub
595
Cypriniformes
Cyprinidae
Relictus solitarius
relict dace
596
Perciformes
Percidae
Etheostomo chienense
relict darter
597
Scorpaeniformes
Cottidae
Cottus perplexus
reticulate sculpin
598
Cypriniformes
Cyprinidae
Lythrurus fumeus
ribbon shiner
599
Cypriniformes
Cyprinidae
Phenacobius cotostomus
riffle minnow
600
Scorpaeniformes
Cottidae
Cottus gulosus
riffle sculpin
601
Cypriniformes
Cyprinidae
Gilo pondoro
rio grande chub
602
Perciformes
Cichlidae
Cichlosomo cyonoguttotum
rio grande cichlid
603
Perciformes
Percidae
Etheostomo grah ami
rio grande darter
604
Cypriniformes
Cyprinidae
Notropis jemezanus
rio grande shiner
605
Cypriniformes
Cyprinidae
Hybognathus a mar us
rio grande silvery minnow
606
Cypriniformes
Catostomidae
Catostomus plebeius
rio grande sucker
607
Cypriniformes
Catostomidae
Carpiodes carpio
river carpsucker
-------
2018/19 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.1, June 2018 Page 156 of 185
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
608
Cypriniformes
Cyprinidae
Nocomis micropogon
river chub
609
Perciformes
Percidae
Percino shumordi
river darter
610
Perciformes
Gobiidae
Awaous bo no no
river goby
611
Petromyzontiformes
Petromyzontidae
Lompetro oyresii
river lamprey
612
Cypriniformes
Catostomidae
Moxostomo corinotum
river redhorse
613
Cypriniformes
Cyprinidae
Notropis blennius
river shiner
614
Perciformes
Percidae
Etheostomo podostemone
riverweed darter
615
Perciformes
Centrarchidae
Ambloplites covifrons
roanoke bass
616
Perciformes
Percidae
Percino roonoko
roanoke darter
617
Cypriniformes
Catostomidae
Hypentelium roonokense
roanoke hog sucker
618
Perciformes
Percidae
Percino rex
roanoke logperch
619
Cypriniformes
Catostomidae
Moxostomo robustum
robust redhorse
620
Perciformes
Centrarchidae
Ambloplites rupestris
rock bass
621
Perciformes
Percidae
Etheostomo ru pest re
rock darter
622
Cypriniformes
Cyprinidae
Notropis suttkusi
rocky shiner
623
Cypriniformes
Cyprinidae
Lythrurus ordens
rosefin shiner
624
Cypriniformes
Cyprinidae
Hybopsis rubrifrons
rosyface chub
625
Cypriniformes
Cyprinidae
Notropis rubellus
rosyface shiner
626
Cypriniformes
Cyprinidae
Clinostom us fun d uloides
rosyside dace
627
Scorpaeniformes
Cottidae
Cottus osperrimus
rough sculpin
628
Cypriniformes
Cyprinidae
Notropis boileyi
rough shiner
629
Atheriniformes
Atherinopsidae
Membros mortinico
rough silverside
630
Cypriniformes
Cyprinidae
Notropis semperosper
roughhead shiner
631
Perciformes
Gobiidae
Neogobius melonostomus
round goby
632
Salmoniformes
Salmonidae
Prosopium cylindroceum
round whitefish
633
Cypriniformes
Cyprinidae
Diondo episcopo
roundnose minnow
634
Cypriniformes
Cyprinidae
Gilo robusto
roundtail chub
635
Cypriniformes
Cyprinidae
Scordinius erythrophtholmus
rudd
636
Perciformes
Percidae
Gymnocepholus cernuus
ruffe
637
Perciformes
Percidae
Etheostomo phytophilum
rush darter
638
Cyprinodontiformes
Fundulidae
Fundulus escombioe
russetfin topminnow
639
Cypriniformes
Catostomidae
Thoburnio homiltoni
rustyside sucker
640
Cypriniformes
Cyprinidae
Notropis sobinoe
sabine shiner
641
Cypriniformes
Cyprinidae
Orthodon microlepidotus
sacramento blackfish
642
Perciformes
Centrarchidae
Archoplites interruptus
sacramento perch
643
Cypriniformes
Cyprinidae
Ptychocheilus grand is
sacramento pikeminnow
644
Cypriniformes
Catostomidae
Cotostom us occiden talis
sacramento sucker
645
Perciformes
Percidae
Percino vigil
saddleback darter
-------
2018/19 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.1, June 2018 Page 157 of 185
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
646
Perciformes
Percidae
Etheostomo flovum
saffron darter
647
Cypriniformes
Cyprinidae
Notropis rubricroceus
saffron shiner
648
Cyprinodontiformes
Poeciliidae
Poecilia la tip in no
sailfin molly
649
Cypriniformes
Cyprinidae
Pteronotropis hypselopterus
sailfin shiner
650
Cyprinodontiformes
Cyprinodontidae
Cyprinodon sol in us
salt creek pupfish
651
Cyprinodontiformes
Fundulidae
Fundulus jenkinsi
saltmarsh topminnow
652
Cyprinodontiformes
Poeciliidae
Gombusio georgei
san marcos gambusia
653
Percopsiformes
Percopsidae
Percopsis tronsmontono
sand roller
654
Cypriniformes
Cyprinidae
Notropis st ro mine us
sand shiner
655
Cypriniformes
Cyprinidae
Notropis scepticus
sandbar shiner
656
Cypriniformes
Cyprinidae
Semotilus lumbee
sandhills chub
657
Cypriniformes
Catostomidae
Cotostomus sontoonoe
santa ana sucker
658
Cyprinodontiformes
Cyprinodontidae
Cyprinodon orcuotus
santa cruz pupfish
659
Cypriniformes
Cyprinidae
Cyprinello zonemo
santee chub
660
Cypriniformes
Cyprinidae
Cyprinello onolostono
satinfin shiner
661
Perciformes
Percidae
Sander canadensis
sauger
662
Perciformes
Percidae
Etheostoma fricksium
savannah darter
663
Perciformes
Percidae
Etheostoma serrifer
sawcheek darter
664
Clupeiformes
Clupeidae
Harengula jaguana
scaled sardine
665
Perciformes
Percidae
Ammocrypta vivax
scaly sand darter
666
Cypriniformes
Cyprinidae
Lythrurus fasciolaris
scarlet shiner
667
Siluriformes
Ictaluridae
Noturus trautmani
scioto madtom
668
Petromyzontiformes
Petromyzontidae
Petromyzon mar in us
sea lamprey
669
Perciformes
Percidae
Etheostoma thalassinum
seagreen darter
670
Cyprinodontiformes
Fundulidae
Fundulus seminolis
seminole killifish
671
Perciformes
Centrarchidae
Ambloplites ariommus
shadow bass
672
Cypriniformes
Catostomidae
Erimyzon tenuis
sharpfin chubsucker
673
Perciformes
Percidae
Etheostoma acuticeps
sharphead darter
674
Perciformes
Percidae
Percina oxyrhynchus
sharpnose darter
675
Scorpaeniformes
Cottidae
Clinocottus acuticeps
sharpnose sculpin
676
Cypriniformes
Cyprinidae
Notropis oxyrhynchus
sharpnose shiner
677
Perciformes
Percidae
Etheostoma tecumsehi
shawnee darter
678
Perciformes
Sparidae
Archosargus probatocephalus
sheepshead
679
Cyprinodontiformes
Cyprinodontidae
Cyprinodon variegatus
sheepshead minnow
680
Perciformes
Percidae
Percina peltata
shield darter
681
Perciformes
Gobiidae
Tridentiger bifasciatus
shimofuri goby
682
Perciformes
Embiotocidae
Cymatogaster aggregata
shiner perch
683
Perciformes
Centrarchidae
Micropterus cataractae
shoal bass
-------
2018/19 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.1, June 2018 Page 158 of 185
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
684
Cypriniformes
Cyprinidae
Mocrhybopsis hyostomo
shoal chub
685
Perciformes
Gobiidae
Tridentiger borbotus
shokihaze goby
686
Cyprinodontiformes
Poeciliidae
Poecilia mexicana
shortfin molly
687
Cypriniformes
Catostomidae
Moxostomo macrolepidotum
shorthead redhorse
688
Scorpaeniformes
Cottidae
Cottus confusus
shorthead sculpin
689
Salmoniformes
Salmonidae
Coregonus zenithicus
shortjaw cisco
690
Salmoniformes
Salmonidae
Coregonus reighordi
shortnose cisco
691
Lepisosteiformes
Lepisosteidae
Lepisosteus plotostomus
shortnose gar
692
Acipenseriformes
Acipenseridae
Acipenser brevirostrum
shortnose sturgeon
693
Cypriniformes
Catostomidae
Chosmistes brevirostris
shortnose sucker
694
Scorpaeniformes
Cottidae
Cottus greenei
shoshone sculpin
695
Acipenseriformes
Acipenseridae
Scophirhynchus plotorynchus
shovelnose sturgeon
696
Cypriniformes
Cyprinidae
Mocrhybopsis meeki
sicklefin chub
697
Cypriniformes
Cyprinidae
Hypophthoimichthys molitrix
silver carp
698
Cypriniformes
Cyprinidae
Mocrhybopsis storeriana
silver chub
699
Petromyzontiformes
Petromyzontidae
Ichthyomyzon unicuspis
silver lamprey
700
Perciformes
Sciaenidae
Bairdiella chrysouro
silver perch
701
Cypriniformes
Catostomidae
Moxostomo anisurum
silver redhorse
702
Cypriniformes
Cyprinidae
Notropis photogenis
silver shiner
703
Cypriniformes
Cyprinidae
Notropis shumardi
silverband shiner
704
Cypriniformes
Cyprinidae
Notropis buccotus
silverjaw minnow
705
Cypriniformes
Cyprinidae
Notropis con did us
silverside shiner
706
Cypriniformes
Cyprinidae
Notropis stilbius
silverstripe shiner
707
Clupeiformes
Clupeidae
Aloso chrysochloris
skipjack herring
708
Cypriniformes
Cyprinidae
Notropis uronoscopus
skygazer shiner
709
Perciformes
Percidae
Etheostomo smithi
slabrock darter
710
Perciformes
Percidae
Etheostomo boschungi
slackwater darter
711
Perciformes
Gobiidae
Ctenogobius pseudofasciatus
slashcheek goby
712
Cypriniformes
Cyprinidae
Erimystox cahni
slender chub
713
Siluriformes
Ictaluridae
Noturus exilis
slender madtom
714
Scorpaeniformes
Cottidae
Cottus tenuis
slender sculpin
715
Perciformes
Percidae
Percino phoxocephoto
slenderhead darter
716
Cypriniformes
Cyprinidae
Pimepholes tenellus
slim minnow
717
Scorpaeniformes
Cottidae
Cottus cognotus
slimy sculpin
718
Perciformes
Percidae
Etheostomo grocile
slough darter
719
Cypriniformes
Cyprinidae
Notropis bucculo
smalleye shiner
720
Perciformes
Centrarchidae
Micropterus dolomieu
smallmouth bass
721
Cypriniformes
Catostomidae
Ictiobus bubolus
smallmouth buffalo
-------
2018/19 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.1, June 2018 Page 159 of 185
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
722
Cypriniformes
Catostomidae
Moxostomo breviceps
smallmouth redhorse
723
Perciformes
Percidae
Etheostomo microlepidum
smallscale darter
724
Perciformes
Centropomidae
Cen tropom us parallel us
smallscale fat snook
725
Perciformes
Eleotridae
Eleotris perniger
smallscaled spinycheek
726
Pristiformes
Pristidae
Prist is pectin at a
smalltooth sawfish
727
Siluriformes
Ictaluridae
Noturus baileyi
smoky madtom
728
Siluriformes
Ictaluridae
Ameiurus brunneus
snail bullhead
729
Perciformes
Percidae
Percina tanasi
snail darter
730
Cypriniformes
Catostomidae
Chasmistes muriei
snake river sucker
731
Perciformes
Percidae
Etheostoma si mote rum
snubnose darter
732
Salmoniformes
Salmonidae
Oncorhynchus nerka
sockeye salmon
733
Cypriniformes
Cyprinidae
Gila ditaenia
sonora chub
734
Cypriniformes
Catostomidae
Catostomus insignis
sonora sucker
735
Cyprinodontiformes
Cyprinodontidae
Cyprinodon erem us
sonoyta pupfish
736
Perciformes
Percidae
Etheostoma olivaceum
sooty darter
737
Cypriniformes
Catostomidae
Cycleptus meridionalis
southeastern blue sucker
738
Petromyzontiformes
Petromyzontidae
Ichthyomyzon gagei
southern brook lamprey
739
Percopsiformes
Amblyopsidae
Typhlichthys subterraneus
southern cavefish
740
Pleuronectiformes
Paralichthyidae
Paralichthys lethostigma
southern flounder
741
Perciformes
Percidae
Percina austroperca
southern logperch
742
Cyprinodontiformes
Poeciliidae
Xiphophorus maculatus
southern platyfish
743
Cypriniformes
Cyprinidae
Ph ox in us erythrog aster
southern redbelly dace
744
Siluriformes
Loricariidae
Pterygoplichthys anisitsi
southern sailfin catfish
745
Perciformes
Percidae
Ammocrypta meridiana
southern sand darter
746
Siluriformes
Doradidae
Platydoras armatulus
southern striped raphael
747
Cyprinodontiformes
Fundulidae
Fundulus stel lifer
southern studfish
748
Cypriniformes
Cyprinidae
Macrhybopsis aestivalis
speckled chub
749
Cypriniformes
Cyprinidae
Rhinichthys osculus
speckled dace
750
Perciformes
Percidae
Etheostoma stigmaeum
speckled darter
751
Cyprinodontiformes
Fundulidae
Fundulus rath bum'
speckled killifish
752
Siluriformes
Ictaluridae
Noturus leptacanthus
speckled madtom
753
Cypriniformes
Cyprinidae
Meda fulgida
spikedace
754
Perciformes
Percidae
Etheostoma barrenense
splendid darter
755
Cypriniformes
Cyprinidae
Pogonichthys macrolepidotus
splittail
756
Scorpaeniformes
Cottidae
Cottus ricei
spoonhead sculpin
757
Perciformes
Sciaenidae
Leiostom us xan th urus
spot
758
Cypriniformes
Cyprinidae
Erimonax monachus
spotfin chub
759
Cyprinodontiformes
Fundulidae
Fundulus luciae
spotfin killifish
-------
2018/19 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.1, June 2018 Page 160 of 185
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
760
Cypriniformes
Cyprinidae
Cyprinello spiloptero
spotfin shiner
761
Perciformes
Percidae
Etheostomo squomiceps
spottail darter
762
Cypriniformes
Cyprinidae
Notropis hudsonius
spottail shiner
763
Perciformes
Centrarchidae
Micropterus punctulotus
spotted bass
764
Siluriformes
Ictaluridae
Ameiurus serroconthus
spotted bullhead
765
Perciformes
Percidae
Etheostomo moculatum
spotted darter
766
Lepisosteiformes
Lepisosteidae
Lepisosteus oculotus
spotted gar
767
Perciformes
Sciaenidae
Cynoscion nebulosus
spotted seatrout
768
Perciformes
Eleotridae
Eleotris picto
spotted sleeper
769
Cypriniformes
Catostomidae
Minytremo melonops
spotted sucker
770
Perciformes
Centrarchidae
Lepomis punctotus
spotted sunfish
771
Perciformes
Cichlidae
Tilopio morioe
spotted tilapia
772
Percopsiformes
Amblyopsidae
Forbesichthys ogossizii
spring cavefish
773
Perciformes
Elassomatidae
Elossomo olobomoe
spring pygmy sunfish
774
Perciformes
Percidae
Percino uronideo
stargazing darter
775
Cypriniformes
Cyprinidae
Phenacobius uronops
stargazing minnow
776
Cyprinodontiformes
Fundulidae
Fundulus dispor
starhead topminnow
111
Pleuronectiformes
Pleuronectidae
Platichthys stel lotus
starry flounder
ns
Cypriniformes
Cyprinidae
Cyprinello whipplei
steelcolor shiner
779
Perciformes
Percidae
Etheostomo punctulatum
stippled darter
780
Cyprinodontiformes
Fundulidae
Fundulus bifox
stippled studfish
781
Perciformes
Percidae
Etheostomo derivotivum
stone darter
782
Siluriformes
Ictaluridae
Noturus flovus
stonecat
783
Perciformes
Percidae
Etheostomo frogi
strawberry darter
784
Cypriniformes
Cyprinidae
Erimystox dissimilis
streamline chub
785
Perciformes
Percidae
Etheostomo striotulum
striated darter
786
Perciformes
Percidae
Percino notogrommo
stripeback darter
787
Perciformes
Moronidae
Morone soxotilis
striped bass
788
Perciformes
Percidae
Etheostomo virgotum
striped darter
789
Cypriniformes
Catostomidae
Moxostomo rupiscortes
striped jumprock
790
Perciformes
Gerreidae
Eugerres plumieri
striped mojarra
791
Mugiliformes
Mugilidae
Mugil cepholus
striped mullet
792
Cypriniformes
Cyprinidae
Luxilus chrysocepholus
striped shiner
793
Perciformes
Percidae
Etheostomo kennicotti
stripetail darter
794
Cypriniformes
Cyprinidae
Mocrhybopsis gelido
sturgeon chub
795
Siluriformes
Loricariidae
Hypostomus plecostomus
suckermouth catfish
796
Cypriniformes
Cyprinidae
Phenacobius mirobilis
suckermouth minnow
797
Salmoniformes
Osmeridae
Hypomesus pretiosus
surf smelt
-------
2018/19 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.1, June 2018 Page 161 of 185
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
798
Perciformes
Centrarchidae
Micropterus notius
suwannee bass
799
Cypriniformes
Cyprinidae
Notropis procne
swallowtail shiner
800
Perciformes
Percidae
Etheostomo fusiform e
swamp darter
801
Percopsiformes
Amblyopsidae
Chologoster cornuto
swampfish
802
Perciformes
Percidae
Etheostomo swonnonoo
swannanoa darter
803
Perciformes
Centropomidae
Centropomus ensiferus
swordspine snook
804
Siluriformes
Ictaluridae
Noturus gyrinus
tadpole madtom
805
Cypriniformes
Catostomidae
Cotostomus to hoe n sis
tahoe sucker
806
Cypriniformes
Cyprinidae
Notropis moculotus
taillight shiner
807
Perciformes
Percidae
Etheostomo tallapoosae
tallapoosa darter
808
Cypriniformes
Cyprinidae
Cyprinella gibbsi
tallapoosa shiner
809
Cypriniformes
Cyprinidae
Notropis braytoni
tamaulipas shiner
810
Perciformes
Percidae
Percino aurantiaca
tangerine darter
811
Elopiformes
Megalopidae
M ego lops atlanticus
tarpon
812
Perciformes
Centropomidae
Centropomus pectinatus
tarpon snook
813
Perciformes
Percidae
Etheostomo borbouri
teardrop darter
814
Cypriniformes
Cyprinidae
Notropis telescopus
telescope shiner
815
Cypriniformes
Cyprinidae
Tinco tinco
tench
816
Cypriniformes
Cyprinidae
Phoxinus tennesseensis
tennessee dace
817
Cypriniformes
Cyprinidae
Notropis leuciodus
tennessee shiner
818
Perciformes
Percidae
Etheostomo olmstedi
tessellated darter
819
Perciformes
Percidae
Percino carbonaria
texas logperch
820
Cypriniformes
Cyprinidae
Notropis omobilis
texas shiner
821
Cyprinodontiformes
Poeciliidae
Gambusia speciosa
tex-mex gambusia
822
Cypriniformes
Cyprinidae
Cyprinella lobroso
thicklip chub
823
Cypriniformes
Cyprinidae
Gilo crassicoudo
thicktail chub
824
Clupeiformes
Clupeidae
Dorosoma petenense
threadfin shad
825
Perciformes
Gobiidae
Eucyclogobius newberryi
tidewater goby
826
Perciformes
Gerreidae
Eucinostomus horengulus
tidewater mojarra
827
Perciformes
Percidae
Etheostomo tippeconoe
tippecanoe darter
828
Perciformes
Percidae
Etheostomo lochneri
tombigbee darter
829
Cypriniformes
Cyprinidae
Exoglossum louroe
tonguetied minnow
830
Siluriformes
Ictaluridae
Trogloglonis pattersoni
toothless blindcat
831
Cypriniformes
Cyprinidae
Notropis topeka
topeka shiner
832
Scorpaeniformes
Cottidae
Cottus rhotheus
torrent sculpin
833
Cypriniformes
Catostomidae
Thoburnia rhothoeca
torrent sucker
834
Cypriniformes
Cyprinidae
Cyprinella trichroistia
tricolor shiner
835
Perciformes
Percidae
Etheostomo trisella
trispot darter
-------
2018/19 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.1, June 2018 Page 162 of 185
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
836
Percopsiformes
Percopsidae
Percopsis omiscomoycus
trout-perch
837
Perciformes
Gobiidae
Proterorhinus mormorotus
tubenose goby
838
Perciformes
Percidae
Etheostomo gutselli
tuckasegee darter
839
Cypriniformes
Cyprinidae
Gilo bicolor
tui chub
840
Perciformes
Embiotocidae
Hysterocorpus troskii
tule perch
841
Perciformes
Percidae
Etheostomo inscriptum
turquoise darter
842
Perciformes
Percidae
Etheostomo tuscumbia
tuscumbia darter
843
Perciformes
Percidae
Etheostomo douglasi
tuskaloosa darter
844
Cypriniformes
Cyprinidae
Rhinichthys umatilla
umatilla dace
845
Cypriniformes
Cyprinidae
Oregonichthys kalawatseti
umpqua chub
846
Cypriniformes
Cyprinidae
Rhinichthys evermonni
umpqua dace
847
Cypriniformes
Cyprinidae
Ptychocheilus umpquoe
umpqua pikeminnow
848
Cypriniformes
Cyprinidae
Gilo atraria
Utah chub
849
Scorpaeniformes
Cottidae
Cottus echinotus
Utah lake sculpin
850
Cypriniformes
Catostomidae
Cotostomus ardens
Utah sucker
851
Cyprinodontiformes
Poeciliidae
Xiphophorus variatus
variable platyfish
852
Perciformes
Percidae
Etheostomo variatum
variegate darter
853
Siluriformes
Loricariidae
Pterygoplichthys disjunctivus
vermiculated sailfin catfish
854
Perciformes
Percidae
Etheostomo chermocki
vermilion darter
855
Perciformes
Gobiidae
Gobioides broussonetii
violet goby
856
Cypriniformes
Cyprinidae
Gilo seminudo
virgin chub
857
Cypriniformes
Cyprinidae
Lepidomeda mollispinis
virgin spinedace
858
Cypriniformes
Catostomidae
Moxostomo pappillosum
v-lip redhorse
859
Perciformes
Percidae
Etheostomo perlongum
waccamaw darter
860
Cyprinodontiformes
Fundulidae
Fundulus waccamensis
waccamaw killifish
861
Atheriniformes
Atherinopsidae
Menidia extenso
waccamaw silverside
862
Salmoniformes
Osmeridae
Hypomesus nipponensis
wakasagi
863
Siluriformes
Clariidae
Ciarias botrochus
walking catfish
864
Perciformes
Percidae
Sonder vitreus
walleye
865
Perciformes
Cichlidae
Oreochromis urolepis
wami tilapia
866
Perciformes
Centrarchidae
Lepomis gulosus
warmouth
867
Cypriniformes
Catostomidae
Cotostomus warnerensis
warner sucker
868
Cypriniformes
Cyprinidae
Luxilus coccogenis
warpaint shiner
869
Perciformes
Percidae
Etheostomo bellator
warrior darter
870
Cypriniformes
Cyprinidae
Lythrurus olegnotus
warrior shiner
871
Perciformes
Percidae
Etheostomo nuchole
watercress darter
872
Cypriniformes
Cyprinidae
Notropis greenei
wedgespot shiner
873
Cypriniformes
Cyprinidae
Notropis texonus
weed shiner
-------
2018/19 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.1, June 2018 Page 163 of 185
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
874
Cypriniformes
Cyprinidae
Rhinichthys obtusus
western blacknose dace
875
Petromyzontiformes
Petromyzontidae
Lampetro richordsoni
western brook lamprey
876
Cypririodontiformes
Poeciliidae
Gombusio of finis
western mosquitofish
877
Perciformes
Percidae
Ammocrypto cloro
western sand darter
878
Cypriniformes
Cyprinidae
Hybognothus orgyritis
western silvery minnow
879
Cypririodontiformes
Fundulidae
Fundulus btoirae
western starhead topminnow
880
Perciformes
Moronidae
Morone chrysops
white bass
881
Siluriformes
Ictaluridae
Ameiurus cotus
white catfish
882
Perciformes
Centrarchidae
Pomoxis annularis
white crappie
883
Mugiliformes
Mugilidae
Mugil curema
white mullet
884
Perciformes
Moronidae
Morone americana
white perch
885
Cypriniformes
Cyprinidae
Lepidomeda albivallis
white river spinedace
886
Cyprinodontiformes
Goodeidae
Crenichthys baileyi
white river springfish
887
Cyprinodontiformes
Cyprinodontidae
Cyprinodon tularosa
white sands pupfish
888
Cypriniformes
Cyprinidae
Luxilus albeolus
white shiner
889
Acipenseriformes
Acipenseridae
Acipenser transmontanus
white sturgeon
890
Cypriniformes
Catostomidae
Catostomus commersonii
white sucker
891
Cypriniformes
Cyprinidae
Cyprinella nivea
whitefin shiner
892
Cyprinodontiformes
Fundulidae
Fundulus albolineatus
whiteline topminnow
893
Cypriniformes
Cyprinidae
Notropis alborus
whitemouth shiner
894
Cypriniformes
Cyprinidae
Cyprinella galactura
whitetail shiner
895
Siluriformes
Ictaluridae
Satan eurystomus
widemouth blindcat
896
Scorpaeniformes
Cottidae
Cottus leiopomus
wood river sculpin
897
Perciformes
Percidae
Etheostoma vulneratum
wounded darter
898
Cypriniformes
Cyprinidae
Plagopterus argentissimus
woundfin
899
Siluriformes
Ictaluridae
Ictalurus pricei
yaqui catfish
900
Cypriniformes
Cyprinidae
Gila purpurea
yaqui chub
901
Cypriniformes
Catostomidae
Catostomus bernardini
yaqui sucker
902
Perciformes
Percidae
Etheostoma raneyi
yazoo darter
903
Cypriniformes
Cyprinidae
Notropis rafinesquei
yazoo shiner
904
Perciformes
Moronidae
Morone mississippiensis
yellow bass
905
Siluriformes
Ictaluridae
Ameiurus natalis
yellow bullhead
906
Perciformes
Percidae
Perca flavescens
yellow perch
907
Perciformes
Cichlidae
Cichlasoma salvini
yellowbelly cichlid
908
Perciformes
Percidae
Etheostoma moorei
yellowcheek darter
909
Perciformes
Gobiidae
Acanthogobius flaviman us
yellowfin goby
910
Siluriformes
Ictaluridae
Noturus flavipinnis
yellowfin madtom
911
Cypriniformes
Cyprinidae
Notropis lutipinnis
yellowfin shiner
-------
2018/19 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.1, June 2018 Page 164 of 185
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
912
Perciformes
Percidae
Etheostomo julioe
yoke darter
913
Perciformes
Percidae
Sander lucioperco
zander
914
Cypriniformes
Catostomidae
Moxostomo cf. poecilurum
apalachicola redhorse
915
Salmoniformes
Salmonidae
Solmo solor
atlantic salmon juvenile
916
Atheriniformes
Atherinopsidae
Menidio menidia
atlantic silverside
917
Cypriniformes
Cyprinidae
Hypophtholmichthys nobilis
bighead carp
918
Cypriniformes
Cyprinidae
Mylophoryngodon piceus
black carp
919
Perciformes
Pomatomidae
Pomotomus saltatrix
bluefish
920
Perciformes
Percidae
Etheostomo meodioe
bluespar darter
921
Salmoniformes
Salmonidae
Oncorhynchus clorkii utoh
bonneville cutthroat trout
922
Cypriniformes
Catostomidae
Moxostomo cf. lochneri
brassy jumprock
923
Anura
Ranidae
Rana cotesbeiono
bullfrog
924
Anura
Ranidae
Rana cotesbeiono
bullfrog tadpole
925
Scorpaeniformes
Cottidae
Cottus chottohoochee
Chattahoochee sculpin
926
Salmoniformes
Salmonidae
Oncorhynchus tshowytscho
chinook salmon (yoy)
927
Scorpaeniformes
Cottidae
Cottus cf. broadband sculpin
clinch sculpin
928
Salmoniformes
Salmonidae
Oncorhynchus clarkii clarkii
coastal cutthroat trout
929
Salmoniformes
Salmonidae
Oncorhynchus clarkii pleuriticus
Colorado river cutthroat trout
930
Anguilliformes
Congridae
Conger oceanicus
conger eel
931
Cypriniformes
Cyprinidae
Macrhybopsis cf. aestivalis
coosa chub
932
Cypriniformes
Cyprinidae
Semotilus X Luxilus atromaculatus x
chrysocephalus
creek chub x striped shiner
933
Salmoniformes
Salmonidae
Oncorhynchus clarkii x my kiss
cutbow
934
Perciformes
Percidae
Etheostomo planasaxatile
duck darter
935
Perciformes
Percidae
Etheostomo orientale
eastrim darter
936
Perciformes
Centropomidae
Cen tropom us parallel us
fat snook
937
Esociformes
Esocidae
Esox americanus vermiculatus
grass pickerel
938
Clupeiformes
Clupeidae
Brevoortia patron us
gulf menhaden
939
Scorpaeniformes
Cottidae
Cottus kanawhae
kanawha sculpin
940
Salmoniformes
Salmonidae
Oncorhynchus clarkii henshawi
lahontan cutthroat trout
941
Cypriniformes
Catostomidae
Pantosteus lahontan
lahontan sucker
942
Perciformes
Centrarchidae
Micropterus salmoides
largemouth bass (yoy)
943
Perciformes
Eleotridae
Eleotris amblyopsis
largescaled spinycheek sleeper
944
Cypriniformes
Catostomidae
Catostomus cf. latipinnis
little Colorado river sucker
945
Cypriniformes
Cyprinidae
Notropis amplamala
longjaw minnow
946
Cypriniformes
Cyprinidae
Pteronotropis stonei
lowland shiner
947
Cypriniformes
Cyprinidae
Cyprinus carpio
mirror carp
948
Caudata
Ambystomatidae
Dicamptodon tenebrosus
pacific giant salamander
949
Salmoniformes
Salmonidae
Oncorhynchus my kiss
rainbow trout (steelhead)
-------
2018/19 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.1, June 2018 Page 165 of 185
LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME
950
Anura
Ranidae
Rana ouroro
red-legged frog
951
Salmoniformes
Salmonidae
Oncorhynchus my kiss goirdneri
redband rainbow trout
952
Caudata
Salamandridae
Taricho granulosa
rough-skinned newt
953
Siluriformes
Ictaluridae
Noturus fasciatus
saddled madtom
954
Perciformes
Percidae
Sander canadensis x vitreus
saugeye
955
Cypriniformes
Cyprinidae
Notropis cf. spectrunculus
sawfin shiner
956
Cypriniformes
Cyprinidae
Hypophthaimichthys molitrix
silver carp
957
Cypriniformes
Catostomidae
Moxostoma robustum
smallfin redhorse
958
Perciformes
Channidae
Channa argus
snakehead
959
Anguilliformes
Ophichthidae
Myrophis punctatus
speckled worm eel
960
Anura
Ascaphidae
Ascaphus truei
tailed frog
961
Anura
Ascaphidae
Ascaphus truei
tailed frog (tadpole)
962
Scorpaeniformes
Cottidae
Cottus tallapoosae
tallapoosa sculpin
963
Perciformes
Percidae
Etheostoma tennesseense
tennessee darter
964
Cypriniformes
Cyprinidae
Cyprinella cf. zanema
thinlip chub
965
Gasterosteiformes
Gasterosteidae
Gasterosteus aculeatus
threespine stickleback
966
Esociformes
Esocidae
Esox lucius x masquinongy
tiger muskellunge
967
Salmoniformes
Salmonidae
Sal mo X Salvelinus trutta xfontinalis
tiger trout
968
Anura
Bufonidae
Bufo boreas
western toad
969
Perciformes
Percidae
Etheostoma occidentale
westrim darter
970
Salmoniformes
Salmonidae
Oncorhynchus clarkii lewisi
westslope cutthroat trout
971
Perciformes
Moronidae
Morone na
wiper
972
Perciformes
Gerreidae
Gerres cinereus
yellowfin mojarra
973
Perciformes
Percidae
Percina burtoni
blotchside darter
974
Anura
Pipidae
Xenopus laevis
african clawed frog
975
Salmoniformes
Salmonidae
Oncorhynchus apache x my kiss
apache x rainbow trout
976
Anura
Bufonidae
Bufo microscaphus
arizona toad
977
Anura
Hylidae
Pseudacris maculata
boreal chorus frog
978
Caudata
Ambystomatidae
Dicamptodon ensatus
California giant salamander
979
Caudata
Salamandridae
Taricha torosa
California newt
980
Anura
Hylidae
Pseudacris cadaverina
California treefrog
981
Anura
Bufonidae
Bufo hemiophrys
Canadian toad
982
Anura
Hylidae
Hyla arenicolor
canyon treefrog
983
Anura
Ranidae
Rana cascadae
cascade frog
984
Anura
Ranidae
Rana luteiventris
Columbia spotted frog
985
Caudata
Rhyacotritonidae
Rhyacotriton kezeri
Columbia torrent salamander
986
Anura
Ranidae
Rana boylii
foothill yellow-legged frog
987
Salmoniformes
Salmonidae
Oncorhynchus aguabonita
golden trout
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 166 of 185
LINE NO.
ORDER
FAMILY
SCIENTIFIC NAME
COMMON NAME
988
Anura
Ranidae
Rono clomitons
green frog
989
Caudata
Ambystomatidae
Dicamptodon oterrimus
idaho giant salamander
990
Petromyzontiformes
Petromyzontidae
Lampetro similis
klamath river lamprey
991
Anura
Ranidae
Lithobotes pipiens
leopard frog
992
Caudata
Ambystomatidae
Ambystomo mocrodoctylum
longtoed salamander
993
Anura
Ranidae
Rana yovopoiensis
lowland leopard frog
994
Anura
Ranidae
Ran a muscoso
mountain yellow-legged frog
995
Anura
Hylidae
Pseudocris regillo
pacific tree frog
996
Anura
Ranidae
Lithobotes bloiri
plains leopard frog
997
Salmoniformes
Salmonidae
Oncorhynchus my kiss x oguobonito
rainbow x golden trout
998
Anura
Bufonidae
Bufo punctotus
red-spotted toad
999
Cypriniformes
Cyprinidae
Richordsonius X Rhinichthys bolteotus x osculus
redside shiner x speckled dace
1000
Anura
Ranidae
Rono pretioso
spotted frog
1001
Anura
Ranidae
Rono sylvotico
wood frog
1002
Anura
Bufonidae
Bufo woodhousii
woodhouse's toad
1003
Anura
Ranidae
Lithobotes chiricohuensis
chiricahua leopard frog
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
APPENDIX E: REPORTING TEMPLATES
Laboratory Operations Manual
Page 167 of 185
Templates will be provided on the NARS SharePoint.
-------
2018/19 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.1, June 2018 Page 168 of 185
APPENDIX F: EXAMPLE SOP FOR ASH FREE DRY MASS ANALYSIS OF
PERIPHYTON BIOMASS
-------
S> APPENDIX F: EXAMPLE SOP FOR ASH FREE DRY MASS ANALYSIS OF PERIPHYTON BIOMASS
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 170 of 185
DyumK/WBDUEP-l}-!1-027
AMXfWKS 7314.5
October 2013
Page 2 «f IS
SOP
FOR
The Detennitiatioo: of Ash Free Dry Mass
WR5 73A.5
Biennial. Reviews*
Date
EPA Reviewer (WAtf. if
W'ACOR)
Date entered
bv.
QA Manager

*listed EPA reviewer has personally verified, or has been assured directly or
imdmsetly by those currently using this SOP. that it is being used, essentially
as written and that bo revisions are necessary.
-------
2018/19 National Rivers & Streams Assessment Laboratory Operations Manual
Version 1.1, June 2018 Page 171 of 185
Standard Operating Procedure for the
Determination of Ash Free Dry Mass
WRS 73A.5
Willamette Research Station
Analytical Laboratory
200 SW 35tb Street
Corvallis, Oregon
Prepared by Kai eu Baxter
December 2002
Revised bv Dianne Mever
April 2012
Contract Ep-D-11-027
Ityouunuc Corporation
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 172 of 185
Standard Operating Procedure for the
Determination Qf \j], Free Dry Mass
YVRS 73A.5
list of Animus Cantributixig
to this SOP
(Mfpnalj pnepami by Karen Baxter, 2002
Reiisiansi
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 173 of 185
DyumK/WBDUEP-l}-!1-027
AMXfWKS 7314.5
October 2013
Page 5 of 16
Stanilnrd Operating Procedure for the
Determination of Ash Free Dry Mass
WRS "3A.5
Table of Contents
I.0 Scope and Application 6
2.0 Steamy of Mefeod 5
3.0 Defcnliffliis ...6
41 Health md Safety Warnings 7
j 0 &ni(KB 7
7.0 Equipment mi Snjfifas 8
8.0 Reagents ant! Stmdudt I
9.0 Sample Hancflmg mi Storage 9
10.0 Quality Control 9
II.0 Calibration and StaadndBadna 9
1-0 Procedure 9
U .0 Data. Analysis and Calculations 1 §
140 MeflioiPafaniJHiKe 11
15.0 Polhitian PRrrcmbQa II
16.0 Waste Management II
17.0 Personnel Qualifications 11
18.0 References 12
19.0 Tables, Diagrams, Flowcharts,, and Validation Data 14
20.0 AjpenfeA 15
-------
y»
M
©
e s g>
K

If > w
|Q 8
I ^ ,§
II f
i
K'
SB
I
I 111
I
p
fit
a
c I

m
n
11
JS B *!
jri o I
& r 5"
-J £" w
gts- s
i s.
« I'
•3 *
1 r
APPENDIX F: EXAMPLE SOP FOR ASH FREE DRY MASS ANALYSIS OF PERIPHYTON BIOMASS
-------
APPENDIX F: EXAMPLE SOP FOR ASH FREE DRY MASS ANALYSIS OF PERIPHYTON BIOMASS
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 176 of 185
DyumK/WBDUEP-l}-!1-027
AMXfWKS 7314.5
October 2013
Page 8 of IS
7 J Equipment and Smppies
Nmim: Trade names, suppliers and part numbers are for informational purposes onfy
Mo amiBFSCMMB is lapfasd. Equivalentpmfarmmmm may bs achieved using
agpamim emd matmats other than those specified kern, but Swmemtmtim of
equivalent perfiMnartce that meets the requirements of this method is the
rfsponnMSy of the tobomtory.
7.1 Analytical Mhx with reajliiion to 0.1 mg
7.2 Gits fiber filtas (GF/F or eqprivalenO
7.3 Forai-air oven, with over temperature control and set at» temperature of
#rc
7.4 Aluminum weigh-boats
7.5 Muffle finH.ee with temperature capacity of 550°C
7.6 Tongs
7 7 Safety glasses
7.1 Nitrite gloves
7.9 Lab coat
iJ Reagents ami Standards
5.1 lieagenf Water
1.2 NIST certified weight set (0.0010— 200 g) to check balance calibration.
1.3 QCS: A large volume (appiiiinistelj 5 gallons bulk sample) of
periphyfcoa rich water is collected from a local stream. Bitches of QCS
samples (filtas) aie prepared at one time: and ftozat fur future use in the
AFDM analyses. To prepare the QCS samples, 30-mL aliqoots are
filtered through GF/F filters pwiously baked in a b& fcnace at 550°C
foe 30 minutes, and the filters axe frozen {-20BCr) in aluminum foil. Two
previously frozen QCS samples (filter) are processed with each batch of
AFDM sample fitters.
-------
APPENDIX F: EXAMPLE SOP FOR ASH FREE DRY MASS ANALYSIS OF PERIPHYTON BIOMASS
-------
00 APPENDIX F: EXAMPLE SOP FOR ASH FREE DRY MASS ANALYSIS OF PERIPHYTON BIOMASS
-------
<
fD
NJ
O
i00
§ S
-j
b
ft
©
©

f
111!
I
l*r
I

n
n
in
8*
I
c
D
fD
NJ
O
O
D
<
fD
99
go
r+
fD
O)
3
fD
O)
D"
O
o
73
fD
era
fD
ID

id APPENDIX F: EXAMPLE SOP FOR ASH FREE DRY MASS ANALYSIS OF PERIPHYTON BIOMASS
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 180 of 185
DyumK/WBDUEP-l}-!1-027
AMXfWKS 7314.5
October 2013
isgaUoflS
will hive demonstrated fat they are afcle to perform all aspects of fine SOP
thoroughly awl JKiepenieiitly prior to collecting research data.
17.1 Training
17 11 Not required of anyone who prepared ox significantly contributed to
17.1.2 Personnel assigned to implement the tasks in this SOP site letprireflto
read and undeistaiid fihe pracednres benm The EPA Erincipfc
IniKti^itar or designee {or contractor Wade Han Manager),
will review lite contents of fins Ammeat will the EPA (or
canlracterjfetsennei asagued. to this SOP and oteerae its
imipIennmMiom fiam slut to firnith. If hmd&-an training appears to be
reqomsd, it will be; provided.
If this SOP is for an iiistiinnent,prDfidfm^ is demonstrated following
the applicable sections of the Demonstration of Analyst Proficiency
paragraph of OfflD policy PPM 13 4 Mmimron QA/QC Pnscaiires for
tie ORD Mwimtniies Caoducting Research.
17.2 Training Documentation
17.2.1 EPA, BM-cantact staff: when the trainer has detenmned a trainee on
successfully implement ins SOP m flse collection of research data,
he/she will provide to the Divukn QA Manager wifliin 30 days, the
SOP trainee, taahuar, and date of flue detaMaaatiifM, He QAM
wil add this irdcamsfum to fc SOP table in WED's QA database.
17.2.2 Contract staff: siieeessfiil training wil be JocamailBi oh the
Employee Training Record form and signed by the supervisor. A copy
of the Hmtractar Employee Acceptance Foan fix this SOP signed by
contract stiff, will be forwarded to the cmfact Program Quality
Assurance Manager. The Work Plan Manager is encouraged to inform
the WACO®, when staff is newly trained «n Urns SOP, providing names
of trainee and trainer, and applicable date. Tie WACOM. will pass this
infounatiou on to tie WED QAM.
18.0 References
tie preparation of tins SOP
APHA. Standard Methods for the Examination of Water and
Wastewater. 2# liiiiofL 1998. American Pofalic Health
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 181 of 185
DyumK/WBDUEP-l}-!1-027
AMXfWKS 7314.5
October 2013
isga 13«f IS
1015 Fifteenth Stmt, N.W, WasMagtam DC, 20005,
Method 10300 D.
A51M., American. Society far Testing mi Materials, t
Specifications for Reagent Water. D1 »3-f9el ASTM: Philadelphia, PA,
1999
, DJ. amlD.V. Feci. (Eds.). Emnnximeiila] Monitoring t
Assessment Program: Integrated Quality Assurance Project PI
SurfaceWateis Resource Group, 1994 Activities. EPA 60G/X-91/080,
few. 2.00. U.S. Ensiiamneatal Protection Agoicy, Las ¥eg», NV. 1994.
Peck, DV, lit Jjeoreiiai, and DJ Xlemm la press Western Pilot
Study Field Operations Manual for Wadeable Stream*. U.S
Environmental Protection Agency. Washington. DC. 2005
l/l
Quality Assurance Han, Willamette Research Station, Analytical <
Laboratofy. Dynaniac evaporation, Reinstall 4, May 2012. Corvsllis,
Oregon
O
CQ
U.S.. EPA. Western Ecology Division, Health and Safety Handbook, U.S. O
EmOTOBBMlil ftotectioa Agency, Nrional Health and EinrirDiimental >
Effects Research Laboratory, Canaflis, 01 97333, January 2010. J
0C
O
LO
CO
>
m
LD
<
>
en
Q
en
CO
<
cn
O
u_
D_
O
CO
<
X
X
Q
D_
O.
<
181
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 182 of 185
DrmK/WEO'EP D-11 -0?7
AFDMIW1S73A.S
Octabtr 2013
Page 14 of 16
If J Tables, Diagrams, Flowcharts* and Validation Data
19.1 Example of Bate Form
WRS Analytical Lfrtrafery
Mmfa Free Dry Mass
Afiai¥stfD ate
Ashling:
Oafeffiime In
DateiTime Qui
ea°c

550"C

mac

Initial Weight (B|
Final Weight (g|
[After ashing (£$55@*C,
«24Ars ©SCC?
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 183 of 185
DyumK/WBDUEP-l}-!1-027
AMXfWKS 7314.5
October 2013
Page 15 of 18
20.0 Appendix A
Procedure for Ash Free Dry Mass Problem Simples
Samples namtdjhm thefield that coniam mem them lOmLof water are ctmsMmki to
be problem samples due to die water was'Mng sample offof the fitter used to determine
ashfrm Ay mass. Samples are sabgeciml to extraJUt-stim in wafer to retrieve ike solid
material from the wafer in rtw tufc*
I. Remove the sample fimn freezer a&d record simple ID. sample ¥oiuHies, and
cofectioit -iate am Mmstory data sheets (see Section 19.0). In tie comments,
record the amount' of water ea A problem sample contains.
j than 10 mL of water to juQy ieftost before beginning
3. Assemble dean&i filter ajjpajiftES with new, pneirioBSlf baked, GF/F filter and
sjsplj vicuum.
4. Whenever possible, remove the sample filter Iran the oeobrifiige tube and place in
j weigh boat. Empty the water from the centrifuge tot* it to lie
i apparatus. Using reigsnt water, rinse the centrifuge tube to ensure aO
I is fihered.
5. Rinse Hue filter appratui with reagent water to maximize the reawanr of sample.
6. Continue suction for at least 30 seconds alter all visible water passes through the
filter.
7. Carefully remove the filter from Hie iippsnitys and place it in the same j 'rmum mn
weigh. boat as the original filler.
I. Cover samples with foil to pemni filters fram Morning ant and dry the Simple ia
forest-air oven at 60°C for 24 hour*
9. Place warm, dry samples m a desiccator to cool. Weigh. samples HI miim
weigh-boats and recent to the meanest 0.1 mg). TMs is the initial sample weight.
10. Place the samples ia a muffle furnace ami bring Hie tenqieiBtuie to 550°C. Ash
the samples in a mnffle itanace by holding them al 550CC for 30 annates
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 184 of 185
DyumK/WBDUEP-l}-!1-027
AMXfWKS 7314.5
October 2013
Page IS of 18
11. Allow the fianaoe to cool to qquraxhmfefy 20Q°C Remove the samples from the
muffle fiansce and place in a dsriccatar to finish cooing. After ashing, tie
pimplate should appear gray and crumbly
12 Saturate the cooled samples with reagent water to rehydrate the clap.
13. Cover samples with foil to present fillers {ran Mowing oat and diy the samples in
¦ finced-air oven at 60°C for 24 homs.
14. Store fte dry sanities in a desiccator to cool Weigh the samples in dnminum
weigh-boilx and record the weight oa the datasheet (see Section 19.0} to Ik
nearest 01 mg. "ITfais it the final (ash free) 'Simple weight.
-------
2018/19 National Rivers & Streams Assessment
Version 1.1, June 2018
Laboratory Operations Manual
Page 185 of 185
APPENDIX G: EXAMPLE SOPS FOR MERCURY IN FISH TISSUE PLUG
ANALYSIS
Please see separate attachement for Appendix G
-------