National Rivers and Streams Assessment 2023/24 Laboratory Operations Manual Version 1.0 February 2023


United States Environmental Protection Agency
Office of Water
Washington, DC
EPA 841-B-22-008

National Rivers and Streams Assessment

2023/24

Laboratory Operations

Manual

Version 1.0

xvEPA

February 2023

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NOTICE

The intention of the National Rivers and Streams Assessment 2023/24 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 2023 - 2024: Quality Assurance Project Plan EPA-841-B-22-004

National Rivers and Streams Assessment 2023 - 2024: Site Evaluation Guidelines EPA-841-B-22-005

National Rivers and Streams Assessment 2023 - 2024: Wadeable Field Operations Manual EPA-841-B-
22-006

National Rivers and Streams Assessment 2023 - 2024: Non-Wadeable Field Operations Manual EPA-841-
B-22-007

National Rivers and Streams Assessment 2023 - 2024: Laboratory Operations Manual EPA-841-B-22-008

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 2023/24. 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. This document and associated QAPP ensure compliance with the EPA policies
Assuring the Competency of EPA Laboratories (2004) and Policy to Assure Competency of Laboratories,
Field Sampling, and Other Organizations Generating Environmental Measurement Data under Agency-
Fun ded A cguisitions (2011).

The suggested citation for this document is:

USEPA. 2022. National Rivers and Streams Assessment 2023 - 2024: Laboratory Operations Manual. EPA-
841-B-22-008. U.S. Environmental Protection Agency, Office of Water, Washington, DC.

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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

2.2	Roles and Contact Information	10

2.3	Sample Tracking	11

2.4	Reporting	11

3.0 ALGAL TOXIN IMMUNOASSAY PROCEDURE: CYLINDROSPERMOPSIN	12

3.1	Summary of Method	12

3.2	Health and Safety Warnings	12

3.3	Definitions and Required Resources (Personnel Qualifications, Laboratories, and Equipment)	12

3.3.1 Definitions	12

3.4	General Requirements for Laboratories	14

3.4.1	Expertise.	14

3.4.2	Quality assurance and quality control requirements	14

3.4.3	Personnel	15

3.4.4	Equipment/Materials	15

3.5	Sample Receipt	15

3.6	Procedure	16

3.6.1	Sample Preparation	16

3.6.2	Kit Preparation	17

3.6.3	Insertion of Contents into Wells	18

3.6.4	Dilutions (if needed)	23

3.7	Pertinent QA/QC Procedures	23

3.7.1	QC Samples	24

3.7.2	Summary of QA/QC Requirements	24

3.8	Sample and Record Retention	26

4.0 ALGAL TOXIN IMMUNOASSAY PROCEDURE: MICROCYSTIN	27

4.1	Summary of Method	27

4.2	Health and Safety Warnings	27

4.3	Definitions and Required Resources (Personnel, Laboratories, and Equipment)	28

4.3.1 Definitions	28

4.4	General Requirements for Laboratories	29

4.4.1	Expertise	29

4.4.2	Quality assurance and quality control requirements	29

4.4.3	Personnel	29

l/l

4.4.4	Equipment/Materials	29	H

4.5	Sample Receipt	30	ljj

4.6	Procedure	31	2

4.6.1	Sample Preparation	31	u

4.6.2	Kit Preparation	32	q

4.6.3	Insertion of Contents into Wells.	33	lu

4.6.4	Dilutions (if needed)	38	^

iii

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4.7	Quality Measures	38

4.7.1	Assistance Visits	38

4.7.2	QC Samples	39

4.7.3	Summary ofQA/QC Requirements	39

4.8	Sample and Record Retention	41

5.0 BENTHIC MACROINVERTEBRATES	42

5.1	Introduction	42

5.2	Summary of Method	42

5.3	Health and Safety Warnings	42

5.4	Definitions and Required Resources (Laboratory, Personnel, and Equipment)	43

5.4.1	Definitions.	43

5.4.2	Laboratory	45

5.4.3	Personnel.	45

5.4.4	Equipment/Materials	46

5.4.4.1	Sample Preparation (Subsampling] and Sorting Equipment/Materials	46

5.4.4.2	Taxonomy Identification Equipment/Materials	47

5.5	Sample Receipt	47

5.6	Subsampling	48

5.7	Sorting	50

5.8	Taxonomy Identification	53

5.9	Data Entry	58

5.10	Sample and Record Retention	58

5.11	External Taxonomic Quality Control	58

5.12	Quality Assurance/Quality Control (QA/QC)	61

5.13	References	63

6.0 FECAL INDICATOR: ENTEROCOCCI	64

6.1	Scope & Application	64

6.2	Summary of Method	64

6.3	Definitions of Method	64

6.4	Interferences	65

6.5	Health & Safety Warnings	65

6.6	Personnel Qualifications	66

6.7	Equipment and Supplies	66

6.8	Reagents & Standards	66

6.9	Preparations Prior to DNA Extraction & Analysis	66

6.10	Procedures for Processing & qPCR Analysis of Sample Concentrates	68

6.10.1	Sample Processing (DNA Extraction)	68

6.10.2	Sample Analysis by Enterococcus qPCR	69

6.10.2.1 Preparation of qPCR assay mix	69

6.10.3	Sample analysis sequence for SmartCycler.	71

6.11	Storage & Timing of Processing/Analysis of Filter Concentrates	71

6.12	Chain of Custody	71

6.13	Quality Assurance/Quality Control (QA/QC) Procedures	71

6.14	Method Performance	72

6.15	Record Keeping & Data Management	72	h

6.16	Waste Management & Pollution Prevention	72	Ei

6.17	Literature Cited	73	z

6.18	Tables, Diagrams, Flowcharts, Checklists, and Validation Data	73	8

6.18.1	Enterococcus qPCR Analysis Decision Tree (ADT)	76	^

6.18.2	"Modified" MagNA Pure LC DNA Purification Kit III Protocol	76	lu

7.0 FISH VOUCHER SPECIMENS	79	<

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7.1	Summary of Procedures	79

7.2	Health and Safety Warnings	79

7.3	Definitions and Required Resources (Personnel, Taxonomy Laboratories, and Equipment)	80

7.3.1	Definitions.	80

7.3.2	General Requirements for Taxonomists and Taxonomy Laboratories	81

7.3.3	Personnel.	81

7.3.4	Equipment/Materials.	82

7.4	Sample Receipt	82

7.5	QC Identification	84

7.6	Assistance Visits	88

in Sample and Record Retention	88

7.8	Summary of QC Requirements for Fish Voucher Specimens	88

7.9	References	89

8.0 FISH TISSUE PLUG	91

9.0 DIATOMS	93

9.1	Summary of Procedure	93

9.2	Health and Safety Warnings	93

9.3	Required Equipment	93

9.3.1	Laboratory	93

9.3.2	Equipment/Materials.	94

9.3.2.1	Subsampling Equipment/Materials	94

9.3.2.2	Diatoms	94

9.3.2.2.1	Preparation of Diatom Slides	94

9.3.2.2.2	Analysis of Diatoms	95

9.4	Sample Receipt	95

9.5	Sample Preparation	97

9.6	Diatom Cleaning and Mounting	97

9.7	Analysis of Diatoms	103

9.7.1	Creation ofPre-Count Regional Voucher Flora and Develop Diatom Analyst Schedule	103

9.7.2	Diatom Sample Count	106

9.7.3	Internal Quality Control	107

9.8	Data Entry	109

9.9	Sample and Record Retention	109

10.0 PERIPHYTON biomass	110

11.0 PERIPHYTON META-GENOMICS and Antimicroibal Analysis (Research Effort)	112

12.0 WATER CHEMISTRY and CHLOROPHYLL A	113

12.1	Analytical Parameters	113

12.2	Sample Processing and Preservation	114

12.2.1	Water Chemistry Samples	114

12.2.2	Chlorophyll-a Samples	115

12.3	Performance-based Methods	115

12.4	Pertinent QA/QC Procedures	117

12.4.1	Laboratory Performance Requirements	118

12.4.2	Laboratory Quality Control Samples	118

12.4.3	Data Reporting, Review, and Management	123

12.5	Literature Cited	125

APPENDIX A: LABORATORY REMOTE EVALUATION FORMS	126	_

APPENDIX B: SAMPLE LABORATORY FORMS	133	2

	i

CO

Benthic Macroinvertebrate: Sorting Bench Sheet	134	<

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u

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Benthic Macroinvertebrates: Taxonomy Bench Sheet (optional)	135

Enterrococci (EPA Method 1606) Laboratory Bench Sheet	136

Fish Voucher: Taxonomy Bench Sheet (optional)	137

APPENDIX c: OTHER PERTINENT ATTACHMENTS	138

APPENDIX D: REPORTING TEMPLATES	166

APPENDIX E: EXAMPLE SOP FOR ASH FREE DRY MASS ANALYSIS OF PERIPHYTON BIOMASS	167

APPENDIX F: EXAMPLE SOPS FOR MERCURY IN FISH TISSUE PLUG ANALYSIS	184

LIST OF TABLES

Table 2.1 Contact information	10

Table 3.1 Cylindrospermopsin login: required data elements	16

Table 3.2: Cylindrospermopsin: required data elements- data submission	21

Table 3.3: Cylindrospermopsin: quality control- sample analysis	24

Table 4.1 Microcystis required data elements - login	31

Table 4.2 Microcystis required data elements-datasubmission	36

Table 4.3 Microcystis quality control-sample analysis	39

Table 5.1 Benthic macroinvertebrate: required data elements-login	48

Table 5.2 Benthic macroinvertebrate: list of taxa that are notto be sorted/counted	51

Table 5.3 Benthic macroinvertebrate: required data elements-sorting	52

Table 5.4 Benthic macroinvertebrate: target level of taxonomic identification-benthics commonly found in

FRESHWATER	55

Table 5.5 Benthic macroinvertebrate: target Level of taxonomic identification -chironomidae	56

Table 5.6 Benthic macroinvertebrate: required data elements -taxonomic identification	57

Table 5.7 Benthic macroinvertebrate: measurement data quality objectives	61

Table 5.8 Benthic macroinvertebrate: quality control - laboratory	62

Table 6.1 Enterococci: PCR assay mix composition (according to draft EPA method 1606)	73

Table 6.2 Enterococci: batch calibrator & enterococcus standards PCR run -7 samples	73

Table 6.3 Enterococci: sub batch test sample PCR run-26 samples & 1 method blank	73

Table 6.4 Enterococci: laboratory methods	74

Table 6.5 Enterococci: parameter measurement data quality objectives	75

Table 6.6 Enterococci: laboratory QC procedures-enterococci DNA sequences	75

Table 7.1 Fish voucher: required data elements-login	83

Table 7.2 Fish voucher: required data elements-data submission worksheet	87

Table 7.3 Fish voucher: measurement data quality objectives	89

Table 7.4 Fish Voucher: quality control-taxonomic identification	89

Table 7.5 Fish voucher: data validation	89

Table 8.1 Fish tissue plug: measurement data quality objectives	91

Table 8.2 Fish tissue plug: quality control	91

Table 9.1 Required data elements for diatom sample login	96

Table 9.2 Required data elements for diatom subsampling	97

Table 9.3 Required data elements for diatom cleaning & mounting	102

Table 9.4 Diatom sample analysis required data elements	108

Table 10.1 Quality assurance measures for AFDM	110	£2

	i

Table 10.2 Data field requirements from labs for AFDM	Ill	co

Table 12.1 Water chemistry: NRSA 2023-2024 parameters	113	H

Ll_

Table 12.2 Water chemistry: acid preservatives added for various analytes	114	o

Table 12.3 Water chemistry: NRSA 2023-2024 analytical methods (Central Laboratory, EPAORD-Corvallis)	116	^

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Table 12.4 Water chemistry & chlorophyll-a: laboratory method performance requirements	119

Table 12.5 Water chemistry: quality control- laboratory samples	121

Table 12.6 Water chemistry: quality control- data validation	123

Table 12.7 Water chemistry: data reporting criteria	123

Table 12.8 Water chemistry: constants for converting major ion concentration from mg/Lto heq/L	124

Table 12.9 Water chemistry: factors to calculate equivalent conductivities of major ions	125

Table D.0.1 Fish identification: standard common and scientific names	139

LIST OF FIGURES

Figure 3.1 Cylindrospermopsin: sample template	19

Figure4.1 Microcystin: sample template	34

Figure 6.1 Enterococci: qPCR analysis decision tree (ADT)	76

Figure 7.1 Fish voucher: example QC fish taxonomy bench sheet (optional)	90

Figure 9.1 Example of light micrograph image after rotation, histogram, and scale bar processing	104

Figure 9.2 Example plate of a regional voucher flora collected before diatom sampling counting has begun	105

Figure 12.1 Water chemistry: sample processing procedures	115

LIST OF ACRONYMS

ASTM American Society for Testing and Materials	GEQ genomic equivalent

LFM lab fortified matrices

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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
NO2 nitrite
NO3 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

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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

SO4	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

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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:

1	Assess the condition of the Nation's rivers and streams

2	Establish a baseline to compare future rivers and streams surveys for trends assessments

3	Evaluate changes in condition from the 2004 Wadeable Streams Assessment, the NRSA 2008/09,
NRSA 2013/14 and NRSA 2018/19

4	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:

1.	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?

2.	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 1028 new
unique sites selected to estimate current satus and 790 unique sites that are resamples from a previous
NRSA survey. 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.

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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 2023/24 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

Title

Name

Contact Information

EPA HQ NRSA Project Lead

Richard Mitchell, OW

mitchell.richard(® eoa.gov
202-566-0644

EPA HQ NRSA Project QA Lead

Sarah Lehmann, OW

lehmann.sarah(® eoa.gov
202-566-1379

EPA HQ Logistics Lead

Brian Hasty, OW

Hasty.brian@epa.gov
202-564-2236

EPA HQ NRSA Laboratory Review
Manager

Kendra Forde, OW

kendra.forde(® eoa.gov
202-564-0417

Information Management Center
Coordinator (NARS IM)

Michelle Gover, GDIT

Gover. michelle(® eoa.gov
541-754-4793

Contractor Logistics Coordinator

Chris Turner, GLEC

cturnerPglec.com
715-829-3737

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2.3 Sample Tracking

Samples are collected by a large number of different field crews during the index period (May through
September). The actual number of rivers and streams sampled on a given day will vary widely during this
time. Field crews will submit electronic forms when they have shipped samples and the NARS IM Center
will input each sample into the NARS IM database. Laboratories can track sample shipment from field
crews by accessing the NARS IM database. Participating laboratories will be given access to the NARS IM
system, where they can acquire tracking numbers and information on samples that have been shipped
to them by field crews (either by overnight shipment for perishable samples or batch shipments for
preserved samples). Upon sample receipt, the laboratory must immediately log in to the database and
confirm that samples have arrived. Overnight samples may not be loaded into the database prior to
sample arrival but should be tracked by the laboratory and receipt information inputted into the
database when sample information is loaded. Each lab will make arrangements with the NARS IM
Coordinator, listed above, to ensure access is granted.

When the samples arrive from the field crews, laboratories should also receive tracking forms in the
shipment (refer to the NRSA 2023/24 FOM). These forms will list the samples that should be included in
the shipment. Laboratory personnel should cross check the forms with the samples received to verify
that there are not any inconsistencies. If any sample is missing or damaged, contact the NARS IM
Coordinator immediately.

2.4 Reporting

All labs must provide data analysis information to the HQ Project Management Team and the NARS IM
Center by March 1, 2024 for data collected in 2023; March 1st, 2025 for data collected in 2024; or as
stipulated in contractual agreements. These reports must include the following information:

2. Sample Type (indicator)

3. Site ID (ex: CAS9-0918)

4. Sample ID (ex: 999000)

5. Pertinent information to the indicator

6. Metadata for all fields

See Appendix C for reporting templates that labs will submit electronically.

The submitted file name must state the following:

7. Indicator name (ex: microcystin)

8. Date of files submission to NARS IM Center by year, month, and day (ex: 2024_11_01)

9. Lab name (ex: MyLab)

Combined, the file name would look as follows: WaterChemistry_2024_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.

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3.0 ALGAL TOXIN IMMUNOASSAY PROCEDURE: CYLINDROSPERMOPSIN

This chapter describes an enzyme-linked immunosorbent assay procedure that measures concentrations
of total cylindrospermopsin in water samples. The laboratory uses Eurofins Technologies (formerly
Abraxis) Cylindrospermopsin Test Kits ("kits") to conduct the analyses.

3.1 Summary of Method

Frozen cylindrospermopsin samples will be shipped on dry ice from the field crews to the contract
batching laboratory. The contract batching laboratory will maintain frozen samples and send the
batched samples to the analysis laboratory in coolers on ice. Samples will arrive in the analysis
laboratory frozen, and they can be held in a freezer for several weeks. Cylindrospermopsin analyses
laboratories will need to process the samples within the 90-day holding time and in accordance with
timeframes outlined in contractual agreements.

The procedure is an adaption of the instructions provided by Eurofins Technologies (formerly Abraxis)
for determining total cylindrospermopsin concentrations using its ELISA kits. For freshwater samples,
the procedure's reporting range is 0.1 ng/Lto 2.0 ng/L, although, theoretically, the procedure can
detect, not quantify, cylindrospermopsin concentrations as low as 0.05 ng/L. For samples with
concentrations higher than 2.0 ng/L of cylindrospermopsin, the procedure includes the necessary
dilution steps.

3.2 Health and Safety Warnings

The laboratory must require its staff to abide by appropriate health and safety precautions, because the
kit substrate solution contains tetramethylbenzidine (TMB) and the stop solution contains diluted
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.

1. Laboratory personnel must wear proper personal protection clothing and equipment (e.g., lab
coat, protective eyewear, gloves).

2. When working with potential hazardous chemicals (e.g., weak acid), laboratory personnel must
avoid inhalation, skin contact, eye contact, or ingestion. Laboratory personnel must avoid
contacting skin and mucous membranes with the TMB and stopping solution. If skin contact
occurs, remove clothing immediately. Wash and rinse the affected skin areas thoroughly with
large amounts of water.

3.3 Definitions and Required Resources (Personnel Qualifications,
Laboratories, and Equipment)

This section provides definitions and required resources for using the procedure.

3.3.1 Definitions
The following terms are used throughout the procedure:

Absorbance (A) is a measure of the amount of light that is absorbed in a sample. A standard statistical
curve is used to convert the absorbance value to the concentration value of cylindrospermopsin.

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Calibration Range is the assay range for which analysis results can be reported with confidence. For
undiluted samples, it ranges from the reporting limit of 0.1 ng/L to a maximum value of 2.0 ng/L. Please
note, NARS IM cannot accept characters within numeric fields. Values outside the range are handled as
follows. If the value is:

•	< 0.05 ng/L, then the laboratory reports the result as is (without characters) and flags the
sample as a non-detect (i.e. DATA_FLAG=ND).

•	Between 0.05 ng/L and the reporting limit of 0.1 ng/L (i.e., >0.05 ng/L and <0.1 ng/L), the
laboratory should record the value, but assign a Quality Control (QC) code to the value
indicating that the result is between the detection limit and the reporting limit (i.e.,
DATA_FLAG=J).

•	>2.0 ng/L, the result indicates that the sample value is outside of the calibration range and must
be diluted and re-run using another analytical run. Leave the CONC column blank and record 'HI'
in the DATA FLAG column.

Coefficient of Variation (CV): The precision for a sample is reported in terms of the percent CV of its
absorbance values. To calculate the %CV, first calculate 5 (standard deviation) as follows:

Equation 3.1 Standard deviation

1/2

1 V

S =

- A)2

n

i=i

where n is the number of replicate samples, A„ is the absorbance measured for the /'th replicate. Samples
are evaluated in duplicate (i=l or 2); controls are either evaluated in duplicate or triplicate (i=l, 2, 3). A
is the average absorbance of the replicates. Then, calculate %CV as:

%CV =

A

Dark or Dimly Lit: Away from sunlight, but under incandescent lighting is acceptable.

i/i
CL

O

Equation 3.2 Percent (%) coefficient of variation	oc

S

CL

X 100	O

Q

b

QC
3
Q

Duplicate samples (D): are defined as the second aliquot of an individual sample within a well plate.	^

Each sample including the standards are run in pairs and both results for the primary and duplicate	cc

CL

sample are reported in the result column of the lab deliverable.	^

i/i
i/i
<
O

Method Detection Limit (MDL) is the minimum concentration at which the analyte can be detected with	3

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 method detection limit is less than the reporting limit of 0.1	—

Hg/L, at which the measured value of the analyte can be reported with confidence. Also see "Sample-
Specific Detection Limit" below.

O

<

o

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13

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Primary samples (P): are defined as the first aliquot of a sample within a well plate. Each sample is
analyzed in pairs. The results of both this aliquot and the secondary, duplicate aliquot are reported in
the result column of the lab deliverable.

Relative Standard Deviation (RSD) is the same as the coefficient of variation (%CV). Because many of
the plate reader software programs provide the %CV in their outputs, the procedure presents the
quality control requirement in terms of %CV instead of RSD.

Reporting Limit (RL): For undiluted samples, the reporting limit is 0.1 ng/L. A reporting limit is the point
at which the measured value of the analyte can be reported with confidence.

Sample-Specific Detection Limit: Most samples will have a sample-specific detection limit equal to the
method detection limit of 0.05 ng/L. For diluted samples, the sample-specific detection limit will be the
product of the method detection limit of 0.05 ng/L and the dilution factor. Typical values for the dilution
factor will be 10 or 100.

Standard Deviation (S) shows variation from the average.

3.4 General Requirements for Laboratories

3.4.1 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.

3.4.2 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 2023 - 2024 QAPP Certification Page.

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3.4.3 Personnel

Laboratory Technician: This procedure may be used by any laboratory technician who is familiar with
the NRSA 2023 - 2024 QAPP, and this procedure in the NRSA 2023 - 2024 LOM. The laboratory
technician also must be familiar with the use of a multichannel pipette and plate readers.

External QC Coordinator is an EPA staff person who is responsible for selecting and managing the "QC
contractor." To eliminate the appearance of any inherent bias, the QC contractor must be dedicated to
QA/QC functions, and thus, must not be a primary laboratory or a field sampling contractor for NRSA QC
contractor is responsible for complying with instructions from the External QC Coordinator; coordinating
and paying for shipments of the performance samples to participating laboratories; comparing
immunoassay results from the laboratories; and preparing brief summary reports.

3.4.4 Equipment/Materials

The procedures require the following equipment and information:

• Eurofins Technologies Cylindrospermospin ELISA (Microtiter) Test Kit, Product # 522011.

• Adhesive Sealing Film (Parafilm) for Micro Plates: Used to cover plates during incubation.

• Data Template - See Figure 3.1

• Distilled or Deionized Water: For diluting samples when necessary.

• ELISA evaluation software.

• 2 glass scintillation vials (20 mL).

• Multichannel Pipette & Tips: An 8-channel pipette is used for this method. Proficient use of the
multichannel pipette is necessary to achieve reliable results. Practice with water if you have
never used this before.

• Norm-ject syringes (or equivalent).

• Paper Towels: For blotting the microtiter plates dry after washing.

• Permanent Marker (Sharpie Fine Point): For labeling samples, bottles, plates and covers.

• Plate Reader (such as Metertech, Model M965 AccuReader): Complete with Metertech PC Mate
software for operation of machine. This machine reads the microtiter plates.

• Project Quality Control Samples.

• Reagent Reservoirs (Costar Cat Number 4870): Plain plastic reservoir for reagents that
accommodate the use of a multi-channel pipette.

• Test tubes: For dilutions, if needed.

• Timer: For measuring incubation times.

• Vortex Genie: For mixing dilutions.

• Whatman Glass fiber syringe filter (25mm, GF 0.45 pim filter).

3.5 Sample Receipt

Cylindrospermopsin samples are kept on ice in the field, frozen as soon as possible and kept frozen until
they are shipped on dry ice to a central facility ("batching laboratory"). Periodically, the batching
laboratory will ship the frozen samples to the cylindrospermopsin laboratory. The batching and
cylindrospermopsin laboratory may retain the frozen samples for several weeks but samples must be
analyzed within the 90-day holding time.

Because USEPA initiates tracking procedures designed to recover any missing shipment, the laboratory
personnel responsible for tracking samples must start the following login steps within 24 clock hours of
receiving a delivery.

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1.	Report receipt of samples to the NARS IM Team by completing and emailing the sample tracking
spreadsheet with the sample login and sample condition information. (See Section 2.2 Roles
and Contact Information the manual for contact information).

2.	Inspect each sample THE SAME DAY THEY ARE RECEIVED:

o Verify that the sample IDs in the shipment match those recorded on the:

¦	Chain of custody forms when the batching laboratory sends the samples to the
cylindrospermopsin laboratory; or

¦	Sample tracking form if the field crew sends the shipment directly to the state.

o For each sample, record the date received and lab comment (including Condition
Code as described below) in the sample tracking spreadsheet with the appropriate
Site ID/ Sample ID for the NARS IM Team.

i.	OK: Sample is in good condition

ii.	C: Sample container was cracked

iii.	L: Sample container is leaking

iv.	ML: Sample label is missing

v.	NF: Sample is not frozen

o If any sample is damaged or missing, contact the USEPA HQ Laboratory Review
Coordinator to discuss whether the sample can be analyzed.

3.	Store samples in the freezer until sample preparation begins.

4.	Maintain the chain of custody or sample tracking forms with the samples.

Table 3.1 Cylindrospermopsin login: required data elements

FIELD

FORMAT

DESCRIPTION

LAB_NAME

Text

Name or abbreviation for QC laboratory

DATE_RECEIVED

MMDDYY

Date sample was received by laboratory

SITEJD

text

NRSA site ID as used on sample label

VISIT_NO

numeric

Sequential visits to site (1 or 2)

SAMPLEJD

numeric

Sample ID as used on field sheet (on sample label)

DATE_COL

MMDDYY

Date sample was collected

CONDITION_CODE

text

Condition codes describing the condition of the sample
upon arrival at the laboratory.





Flag

Definition





OK

Sample is in good condition





C

Sample container is cracked





L

Sample or container is leaking





ML

Sample label is missing





NF

Sample is not frozen





Q

Other quality concerns, not identified above

COND_COMMENT

text

Comments about the condition of the sample.

3.6 Procedure

The following sections describe the sample, kit preparation and analysis.

3.6.1 Sample Preparation

For each frozen sample (500 mL per sample), the laboratory technician runs it through a freeze-thaw
cycle three times to lyse the cells as follows:

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1. All cycles: Keep the samples in dark or dimly lit areas (i.e., away from sunlight, but under
incandescent lighting is acceptable).

2. First freeze-thaw cycle:

o Start with a frozen 500 ml sample.

o Thaw the sample to room temperature (approximately 25° C). Swirl the sample to check for

ice crystals. At this temperature, no ice crystals should be present in the sample,
o Shake well to homogenize the sample, then transfer 10 mL to an appropriately labeled clean
20 mL glass vial.

3. Second freeze-thaw cycle:
o Freeze the vial.

o Keep the large sample bottle (from the 500 mL initial sample) frozen for future use.
o Thaw the sample vial contents to room temperature.

4. Third freeze-thaw cycle:
o Freeze the vial.

o Thaw the vial contents to room temperature.

o Filter the vial contents through a new, syringe filter (0.45 pim) into a new, labeled 20 mL
glass scintillation vial. Norm-ject syringes and Whatman Glass fiber syringe filters (25mm, GF
0.45 pirn filter) or similar alternatives are acceptable. One new syringe and filter should be
used per sample.

3.6.2 Kit Preparation

The technician prepares the kits using the following instructions:

Check the expiration date on the kit box and verify that it has not expired. If the kit has
expired, discard and select a kit that is still within its marked shelf life. (Optional: Instead of
discarding the kit clearly mark all expired components as expired and consider keeping it for
training activities.)

1. 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 ng/L,
o S3: 0.25 Hg/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

• Cylindrospermospin-HRP conjugate Solution (vortex before use)

• Antibody solution (rabbit anti-Cylindrospermopsin)

• Wash Solution 5X Concentrate

• Substrate (Color) Solution

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• Stop Solution

• Dilutant

• Foil bag with 12 microtiter plate strips

2. If any bottles are missing or damaged, discard the kit. This step is important because Abraxis has
calibrated the standards and reagents separately for each kit.

3. Adjust the microtiter plate, samples, standards, and the reagents to room temperature.

4. Remove 12 microtiter plate strips (each for 8 wells) from the foil bag for each kit. The plates
contain 12 strips of 8 wells. If running less than a whole plate, remove unneeded strips from the
strip holder and store in the foil bag, ziplocked closed, and store in the refrigerator (4-8 °C).

5. Prepare a negative control (NC) using distilled water.

6. The standards, controls, antibody solution, enzyme conjugate, color solution, and stop solutions
are ready to use and do not require any further dilutions.

7. Dilute the wash solution with deionized water. (The wash solution is a 5X concentrated
solution.) In a 1L container, dilute the 5X solution 1:5 (i.e., 100 mL of the 5X wash solution plus
400 mL of deionized water). Mix thoroughly. Set aside the diluted solution to wash the
microtiter wells later.

8. Handle the stop solution containing diluted H2S04 with care.

3.6.3 Insertion of Contents into Wells

This section describes the steps for placing the different solutions into the 96 wells. Because of the

potential for cross contamination using a shaker table, the following steps specify manual shaking of the ^

kits instead mechanized shaking. O

LU
Q_

9. While preparing the samples and kit, turn the plate reader on so it can warm up. The plate o
reader needs a minimum of 30 minutes to warm up. g

10. Turn on the computer so that it can control and access the plate reader.

11. Print the template (Figure 3.1) to use as reference when loading the standards, controls, and

samples as described in the next step. Templates contain rows, labeled with a marking pen, of 3

strips of 8 wells that snap into the blank frame. If the laboratory wishes to use a different

template, provide a copy to the USEPA HQ Laboratory Review Coordinator for approval prior to §

first use. >

12. Using the lOO-piL pipette, add 50 piL, each, of the standards, controls, and samples to the $
appropriate wells in the plate. Place all seven standards (0.00, 0.05, 0.10, 0.25, 0.50, 1.0 and 2.0 O
Hg/L), the kit control (0.75 piL), and negative control, in pairs (duplicate), starting in the well in ^
the upper left-hand corner of the kit as shown in Figure 3.1. Verify that the software displays the ^
same template or make any necessary corrections. Laboratories with access to an autopipetter i=
may use said machinery after proper documentation of set up, training and calibration has been O
provided and approved by EPA HQ Laboratory Review Coordinator prior to first use. <

12345 6 7 8 9

A SO S4 NC P4 P8 P12 P16 P20 P24

P28

P32

P36

q
m*

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D12

D16

D20

D24

D28

D32

D36

P13

P17

P21

P25

P29

P33

P37

D13

D17

D21

D25

D29

D33

D37

P10

P14

P18

P22

P26

P30

P34

P38

D10

D14

D18

D22

D26

D30

D34

D38

Pll

P15

P19

P23

P27

P31

P35

P39

Dll

D15

D19

D23

D27

D31

D35

D39

Figure 3.1 Cylindrospermopsin: sample template

Key: S0-S6 = Standards;

KC = Control supplied with Kit (i.e., Kit Control);

NC = Negative Control (Laboratory Reagent Blank);

P = Primary run for each unknown sample collected by field crew;
D= "DUPLICATE" run for each matching unknown Primary sample

13. 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.

14. Place the sealing Parafilm over the wells.

15. Manually mix the contents by moving the strip holder in a rapid circular motion on the benchtop
for 30 seconds. Be careful not to spill the contents.

16. Place the plate in an area away from light for 45 minutes.

17. After 45 minutes, carefully remove the Parafilm.

18. 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. §

19. Add 100 piL of substrate/ color solution to all wells using the multi-channel pipettor. ^

20. Cover the wells with Parafilm.

21. 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. u

22. Place the strip holder in an area away from light for 30-45 minutes. ^

23. After 30-45 minutes, remove the Parafilm, add 100 piL of stop solution to the wells using the ^

multi-channel pipette and reagent reservoir in the same sequence as the substrate solution. ^

24. Use a microplate ELISA photometer (plate reader) to determine the absorbance at 450 nm. The §
software (i.e., commercial ELISA evaluation program) calculates the absorbance and ^
concentration values of the samples from the calibration curve and the average values for each ^
pair. Use a 4-parameter standard curve fit to determine the concentrations. ^

25. Dispose of solution in plates in a laboratory sink. Rinse plates and sink with water to dilute the h

weak acid present. <

26. Perform QC evaluations of the data as follows:

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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 3.7.4 Dilutions .

ii. The %CV > 15% between the duplicate absorbance values for a sample.

27. Record the results, even if the data failed the quality control requirements in #26b, for each well
in the USEPA's data template (Table 3.2). The required entries are for the following columns:

a. SAM_CODE should be one of the following codes: S0-S6 for standards; KC or NC, for
controls; QC for quality control samples; P for primary run of unknown samples, D for
duplicate/secondary run of unknown samples within a well plate.

b. CONC contains the numeric concentration value. Two special cases:

i. Non-detected concentrations: If the sample is non-detected, provide the result within
CONC column, record the data as 'ND' in the DATA FLAG column and provide the
sample-specific detection limit in the method detection limit column (MDL). See Section
3.3 for calculating the sample-specific detection limit for a diluted sample.

ii. If the result shows that it is "HIGH," this indicates that the sample value is outside of the
calibration range and must be diluted and re-run using another analytical run. Leave the
CONC column blank and record 'HI' in the DATA FLAG column.3

c. DATA FLAGS have codes for the following special cases:

i. ND if the sample was non-detected;

ii. J if the value is detected but at a level below the reporting limit of 0.1 ng/L (for
undiluted samples);

a EPA compares the cylindrospermopsin data values to 15 |ig/L. which is the magnitude of the EPA criteria for
recreational waterbodies in Recommended Human Health Recreational Ambient Water Quality Criteria or
Swimming Advisories for Microcystins and Cylindrospermopsin. 2019. EPA 822-R-19-001. Retrieved June 5, 2019.
https://www.epa.gov/sites/production/files/2019-05/documents/hh-rec-criteria-habs-document-2019.pdf

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iii.	H if the sample did not meet the holding time and was not analyzed within 90
days.

iv.	HI if the concentration value registers as HIGH (exceeds the calibration range),
d. QUALITY FLAGS have codes for the following special cases:

i.	QCF if there is a QC failure per step 26 above. The QCF code must be used for all
failures to facilitate data analysis.

ii.	Qfor any other quality issue (describe in COMMENTS)

e.	DILUTION FACTOR is only required if the sample was diluted.

f.	AVG_CONC and CV_ABSORB are required for all duplicate runs (use all three values if the
controls are used in triplicate).

Table 3.2: Cylindrospermopsin: required data elements- data submission

FIELD	COLUMN HEADING FORMAT	DESCRIPTION

LABORATORY ID

LAB ID

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

COND COMMENT

Text

Any comment based on the condition code flags

COMMENT









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

CO
Q_

o

C£L

LU
Q_

CO

o

CC

o

b

QC
3
Q

LU

U

o

QC
CL

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FIELD	COLUMN HEADING FORMAT	DESCRIPTION







Code

Definition







KC

Kit Control







NC

Negative Control







SO, SI, S2, S3, S4,

Standard







S5, S6









QC

Quality Control

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)

PRIMARY OR

PRIM_DUP

Text

Regular samples are listed as "P" for Primary/first

DUPLICATE





run or "D" for second run (see Figure 3.1)

CONCENTRATION

CONC

Numeric

Concentration or sample-specific detection limit of
contents of well in ng/L. Sample-specific detection
limit should be 0.1 ng/L if the sample hasn't been







diluted.



UNITS

UNITS

Text

The units of the concentration of the CONC column

MDL*

MDL

Numeric

Method 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 for all

ABSORBANCE





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 for a

CONCENTRATION





sample. Substitute 0.1 ng/Lfor any result recorded







as <0.1 ng/L



OA FLAG (if

QA_FLAG

Text

Data qualifier codes associated with specific

appropriate)





identifications of voucher samples. These codes
provide more information than those used when
reporting receipt of samples. A technician may use
alternative or additional qualifiers if definitions are
provided as part of the submitted data package
(e.g., as a separate worksheet page of the data







submission file).









Flag

Definition







ND

Concentration below
detection. Unless the sample
was diluted, the concentration
will be 0.05 ng/L







H

Sample did not meet the
holding time and was not
analyzed within 90 days.

CO
Q_

o

C£L

LU
Q_

CO

o

CC

Q

b

cc
3
Q

LU

U

o

cc

CL

<
LT)
LT)
<
O

X

o

<

o

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22

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FIELD COLUMN HEADING FORMAT DESCRIPTION

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

QC failure

Other quality concerns, not
identified above

LABORATORY
COMMENT

LAB_COMMENT

Text

Explanation for data flag(s) (if needed) or other
comments.

*ln the event for sample dilution is necessary to overcome the matrix effect, please notify EPA Laboratory Coordinator

3.6.4 Dilutions (if needed)

Dilutions if needed are prepared as follows (using clean glass tubes):

• 1:10 dilution

a. Add 900 piL of distilled or deionized water to a clean 20 mL vial. (Note: Dilutions may also be
made using the kit's diluent rather than distilled or deionized water.)

b. Pipette 100 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.) o.

c. Mix by vortexing. ^

d. Multiply final concentration and Abraxis' method detection limit of 0.05 ng/L by 10 to obtain £

i/i

the sample-specific detection limit of .5 ng/L. O

• 1:100 dilution E

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 q

water.) ^

QC
CL

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.
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3.7.1 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, which may include no action, flagging the data, or excluding some or all
the laboratory's data.

3.7.2 Summary of QA/QC Requirements

Table 3.3 provides a summary of the quality control requirements. For cylindrospermopsin, the
precision for a sample is reported in terms of the percent coefficient of variation (%CV) of its absorbance
values. Relative Standard Deviation (RSD) is the same as the %CV. Because many of the plate reader
software programs provides the CV in their outputs, the procedure presents the quality control
requirement in terms of %CV instead of RSD. Accuracy is calculated by comparing the average
concentration of the kit control with the required range (0.75 +/- 0.15).

Table 3.3: Cylindrospermopsin: quality control- sample analysis

Quality Control
Activity

Description and Requirements

Corrective Action

Kit - Shelf Life

Is within its expiration date listed on kit box.

If kit has expired, then discard or set
aside for training activities.

Kit - Contents

All required contents must be present and in
acceptable condition. This is important because
Abraxis has calibrated the standards and reagents
separately for each kit.

If any bottles are missing or damaged,
discard the kit.

Calibration

All of the following must be met:

o Standard curve must have a correlation

coefficient of >0.99;
o Average absorbance value, Ao, for SO must

be >0.80; and
o Standards S0-S6 must have decreasing
average absorbance values. That is, if Ai is
the average of the absorbance values for
Si, then the absorbance average values
must be: Ao > Ai > A2 > A3 > A4 >As>A6

If any requirement fails:

Results from the analytical run are not
reported.

All samples in the analytical run are
reanalyzed until calibration provides
acceptable results. At its discretion,
the laboratory may consult with
USEPA for guidance on persistent
difficulties with calibration.

Kit Control

The average concentration value of the duplicates
(or triplicate) must be within the range of 0.75 +/-

If either requirement fails:

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Quality Control

Description and Requirements

Corrective Action

Activity

0.15 ng/L. That is, results must be between 0.60
and 0.90.

• Results from the analytical run
are not reported

• The laboratory evaluates its
processes, and if appropriate,
modifies its processes to correct
possible contamination or other
problems.

• The laboratory reanalyzes all
samples in the analytical run until
the controls meet the
requirements.

Negative Control

The values for the negative control replicates must
meet the following requirements:

o All concentration values must be < 0.1

Hg/L (i.e., the reporting limit); and
o One or more concentration results must
be nondetectable (i.e., <0.05 ng/L)

Sample
Evaluations

All samples are run in duplicate. Each duplicate pair
must have %CV<15% between its absorbance
values.

If %CV of the absorbance for the
sample>15%, then:

• Record the results for both
duplicates using different start
dates and/or start times to
distinguish between the runs.

• Report the data for both
duplicate results using Quality
Control Failure flag "QCF"; and

• Re-analyze the sample in a new
analytical run. No samples are to
be run more than twice without
EPA approval.

If the second run passes, then the
data analyst will exclude the data
from the first run (which will have
been flagged with "QCF"). If both runs
fail, the data analyst will determine if
either value should be used in the
analysis (e.g., it might be acceptable
to use data if the CV is just slightly
over 15%).

ResuIts Within
Calibration Range

All samples are run in duplicate. If both of the
values are less than the upper calibration range
(i.e., 2.0 ng/Lfor undiluted samples), then the
requirement is met.

If a result registers as "HIGH", then
record the result with a data flag of
"HI." If one or both duplicates register
as 'HIGH/ then the sample must be
diluted and re-run. No samples are to
be run more than twice without EPA
approval. 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

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Quality Control

Description and Requirements

Corrective Action

Activity

laboratory practices that might
account for differences between the
laboratory and others. With this
additional information, the External
QC Coordinator will determine an
appropriate course of action,
including no action, flagging the data,
or excluding some or all of the
laboratory's data.

3.8 Sample and Record Retention

The laboratory shall retain:

1. The sample materials, including vials, for a minimum of 3 years from the date the EPA publishes
the final report. During this time, the laboratory shall freeze the materials. The laboratory shall
periodically check the sample materials for degradation.

2. Original records, including laboratory notebooks and the reference library, for a minimum of 10
years from the date that EPA publishes the final report.

After the stated time periods, the laboratory shall follow its internal protocols for disposal.

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4.0 ALGAL TOXIN IMMUNOASSAY PROCEDURE: MICROCYSTIN

This chapter describes an immunoassay procedure that measures concentrations of total microcystins in
water samples using Eurofins Technologies (formerly Abraxis) Microcystins-ADDA Test Kits ("kits")b.

Each kit is an enzyme-linked immunosorbent assay (ELISA) for the determination of microcystins and
nodularins in water samples. Microcystins refers to the entire group of toxins, all of the different
congeners, rather than just one congener. Algae can produce one or many different congeners at any
one time, including Microcystin-LR (used in the kit's calibration standards), Microcystin-LA, and
Microcystin-RR. The different letters on the end signify the chemical structure (each one is slightly
different) which makes each congener different.

4.1 Summary of Method

Frozen microcystin samples will be shipped on dry ice from the field crews to the contract batching
laboratory. The contract batching laboratory will send the batched frozen samples to the analysis
laboratory in coolers on ice where they can be held in a freezer until ready for analysis. Microcystin
analyses laboratories will need to process the samples within the 90-day holding time and in accordance
with timeframes outlined in contractual agreements.

The procedure is an adaption of the instructions provided by Abraxis for determining total microcystins
concentrations using its ELISA-ADDA kits.c For freshwater samples, the procedure's reporting range is
0.15 ng/Lto 5.0 ng/L, although, theoretically, the procedure can detect, not quantify, 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 o

sulfuric acid. In addition to the laboratory's usual requirements such as a Chemical Hygiene Plan, the y

laboratory must adhere to the following health and safety procedures: ^

1. Laboratory facilities must properly store and dispose of solutions of weak acid.

2. Laboratory personnel must wear proper personal protection clothing and equipment (e.g., lab
coat, protective eyewear, gloves). O

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 o

QC
3
Q

b Eurofins Technologies, "Microcystins-ADDA ELISA (Microtiter Plate): Product No. 520011." Retrieved on March ><

12, 2020 from https://www.eurofins-technologies.com/microcvstins-nodularins-adda-epa-etv-epa-method-546- h

elisa-96-tests.html <

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occurs, remove clothing immediately. Wash and rinse the affected skin areas thoroughly with
large amounts of water.

4.3 Definitions and Required Resources (Personnel, Laboratories, and
Equipment)

This section provides definitions and required resources for using the procedure.

4.3.1 Definitions
The following terms are used throughout the procedure:

Absorbance (A) is a measure of the amount of light in a sample. A standard statistical curve is used to
convert the absorbance value to the concentration value of microcystins.

Calibration Range is the assay range for which analysis results can be reported with confidence. For
undiluted samples, it ranges from the reporting limit of 0.15 ng/L to a maximum value of 5.0 ng/L.
Values outside the range are handled as follows. If the value is:

< 0.10 ng/L, then the laboratory reports the result as being non-detected ("<0.10 ng/L").
Between 0.10 ng/L and the reporting limit of 0.15 ng/L (i.e., >0.10 ng/L and <0.15 ng/L), the
laboratory should record the value, but assign a QC code to the value (i.e., DATA_FLAG=J).
5.0 ng/L, the laboratory must dilute and reanalyze the sample.

Coefficient of Variation (CV): The precision for a sample is reported in terms of the %CV of its
absorbance values. To calculate the %CV, first calculate 5 (standard deviation) as follows:

1/2

5 =

i=1

where n is the number of replicate samples, A„ is the absorbance measured for the /'th replicate. Samples

are evaluated in duplicate (i=l or 2); controls are either evaluated in duplicate or triplicate (i=l, 2, 3). A

is the average absorbance of the replicates. Then, calculate %CV as: ^

x 100

%cv =

IS)

Dark or Dimly Lit: Away from sunlight, but under incandescent lighting is acceptable. ge

Duplicate samples (D) are defined as the second aliquots of an individual sample within a well plate. £

Each sample, including the standards, are urn in pairs and both results for the primary (P) and duplicate O

aliquot are reported in the result column of the lab deliverable. £-

Method Detection Limit is the minimum concentration at which the analyte can be detected with ^

confidence. In other words, the outcome can be reported with confidence that it is greater than zero §

(i.e., present in the sample). The detection limit is less than the reporting limit of 0.15 ng/L at which the ^

measured value of the analyte can be reported with confidence. Also see "Sample-Specific Detection ^

Limit." i

Primary samples (P) are defined as the first aliquot of a sample within a well plate. Each sample is O

analyzed in pairs. The result of both the primary aliquot and secondary, duplicate aliquot are reported in ^

the result column of the lab deliverable.

o
<3-

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Relative Standard Deviation (RSD) is the same as the coefficient of variation (%CV). Because many of
the plate reader software programs provides the CV in their outputs, the procedure presents the quality
control requirement in terms of %CV instead of RSD.

Reporting Limit: For undiluted freshwater sample, the reporting limit is 0.15 ng/L. A reporting limit is
the point at which the measured value of the analyte can be reported with confidence.

Sample-Specific Detection Limit: Most samples will have a sample-specific detection equal to the
method's detection limit of 0.1 ng/L. For diluted samples, the sample-specific detection limit will be the
product of the method's detection limit of 0.1 ng/L and the dilution factor. Typical values for the dilution
factor will be 10 or 100.

Standard Deviation (S) shows variation from the average

4.4 General Requirements for Laboratories

4.4.1 Expertise

To demonstrate its expertise, the laboratory shall provide EPA with one or more of the following:

• Memorandum that identifies the relevant services that the laboratory provided for the National
Aquatic Resource Surveys in the past five years.

4.4.2 Quality assurance and quality control requirements

To demonstrate its ongoing commitment, the person in charge of quality issues for the organization
shall sign the NRSA 2023 - 2024 QAPP Certification Page.

4.4.3 Personnel

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:

• Eurofins Technologies (formerly Abraxis ADDA Test Kit, Product #520011

• Adhesive Sealing Film (Parafilm) for Micro Plates (such as Rainin, non-sterile, Cat. No. 96-SP-
100): Used to cover plates during incubation.

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• Data Template - See Error! Reference source not found.2

• Distilled or Deionized Water: For diluting samples when necessary.

• ELISA evaluation software

• Glass scintillation, LC, vials (two vials of 2 mL each)

• Glass vials with Teflon-lined caps of size:

o 20 mL

o 4 mL (for dilutions)

• Multichannel Pipette & Tips: A single-channel and an 8-channel pipette are used for this
method.

• Norm-ject syringes (or equivalent)

• Paper Towels: For blotting the microtiter plates dry after washing.

• Permanent Marker (Sharpie Fine Point): For labeling samples, bottles, plates and covers.

• Plate Reader (e.g., Metertech Model M965 AccuReader; ChroMate®; or equivalent readers with
software to read the microtiter plates and measure absorbances).

• Reagent Reservoirs (e.g., Costar Cat Number 4870): Plain plastic reservoir for reagents that
accommodate the use of a multi-channel pipette.

• Test tubes: For dilutions, if needed.

• Timer: For measuring incubation times.

• Vortex Genie: For mixing dilutions.

• Whatman Glass fiber syringe filter (25mm, GF 0.45 pim filter)

4.5 Sample Receipt

Microcystins samples are kept on ice while in the field, frozen as soon as possible and kept frozen until
they are shipped on dry ice to a central facility ("batching laboratory") or the State's laboratory.
Periodically, the batching laboratory ships the frozen samples to the microcystins laboratory. The
batching and microcystins laboratory may retain the frozen samples for several weeks but samples must
be analyzed within the 90-day holding time.

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 (Table 4.1 Microcystin: required data elements - login.

1. Report receipt of samples in the NARS IM sample tracking system (within 24 clock hours).

2. Inspect each sample THE SAME DAY THEY ARE RECEIVED:

a. Verify that the sample IDs in the shipment match those recorded on the:

i. Chain of custody forms when the batching laboratory sends the samples to the
microcystins laboratory; or

ii. Sample tracking form if the field crew sends the shipment directly to the state
laboratory.

b. Record the information in into NARS IM, including the Condition Code for each sample:

i. OK: Sample is in good condition

ii. C: Sample container was cracked

iii. L: Sample container is leaking

iv. ML: Sample label is missing

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v. NF: Sample is not frozen

c. If any sample is damaged or missing, contact the EPA HQ Laboratory Review Coordinator to
discuss whether the sample can be analyzed.

3. Store samples in the freezer until sample preparation begins.

4. Maintain the chain of custody or sample tracking forms with the samples.

Table 4.1 Microcystin: required data elements - login

FIELD

FORMAT

DESCRIPTION

LAB ID

text

Name or abbreviation for QC laboratory

DATE RECEIVED

MMDDYY

Date sample was received by lab

SITE ID

text

LA site ID as used on sample label

VISIT NUMBER

numeric

Sequential visits to site (1 or 2)

SAMPLE ID

numeric

Sample id as used on field sheet (on sample label)

DATE COLLECTED

MMDDYY

Date sample was collected

CONDITION

text

Condition codes describing the condition of the sample upon arrival at

CODE

the laboratory.

Flag

Definition

Blank or N

Not a sample (Blank, standard or control)

Sample is in good condition

Sample container is cracked

Sample or container is leaking

Sample label is missing

Sample is not frozen

Other quality concerns, not identified above

CONDITION

text

Comments a

Dout the condition of the sample. Required for "Q".

COMMENT

Optional for others.

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 mL to an appropriately labeled clean
20 mL glass vial.

3. Second freeze-thaw cycle:

a. Freeze the vial.

b. Keep the large sample bottle (from the 500 mL initial sample) frozen for future use.

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c. Thaw the sample vial contents to room temperature.

4. Third freeze-thaw cycle:

a. Freeze the vial.

b. Thaw the vial contents to room temperature.

c. Filter the vial contents through a new, syringe filter (0.45 pim) into a new, labeled 20 mL
glass scintillation vial. Norm-ject syringes and Whatman Glass fiber syringe filters (25mm, GF
0.45 pirn filter) or similar alternative are acceptable. One new syringe and filter should be
used per sample.

4.6.2 Kit Preparation

The technician prepares the kits using the following instructions:

1. Check the expiration date on the kit box and verify that it has not expired. If the kit has expired,
discard and select a kit that is still within its marked shelf life. (Instead of discarding the kit,
consider keeping it for training activities.)

2. Verify that each kit contains all the required contents:

• Microtiter plate

• Standards (6) referenced in this procedure as follows with the associated concentration:
o SO:0 ng/L

o SI: 0.15 ng/L
o S2: 0.40 ng/L
o S3: 1.0 ng/L
o S4: 2.0 ng/L
o S5: 5.0 ng/L

• Kit Control (KC): 0.75 ng/L

• Antibody solution

• Anti-Sheep-HRP Conjugate

• Wash Solution 5X Concentrate

• Color Solution

• Stop Solution

• Diluent

• Foil bag with 12 microtiter plate strips

3. If any bottles are missing or damaged, discard the kit. This step is important because Abraxis has
calibrated the standards and reagents separately for each kit.

4. Adjust the microtiter plate, samples, standards, and the reagents to room temperature.

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.

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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

This section describes the steps for placing the different solutions into the 96 wells. Because of the
potential for cross contamination using a shaker table, the following steps specify manual shaking of the
kits instead mechanized shaking.

11. While preparing the samples and kit, turn the plate reader on so it can warm up. The plate
reader needs a minimum of 30 minutes to warm up.

12. Turn on the computer so that it can control and access the plate reader.

13. Print the template (Figure 4.1 Microcystin: sample template) to use as reference when loading
the standards, controls, and samples as described in the next step. Templates contain rows,
labeled with a marking pen, of strips of 8 wells that snap into the blank frame. (If the laboratory
wishes to use a different template, provide a copy to the EPA HQ Laboratory Review
Coordinator for approval prior to first use.

14. Using the lOO-piL pipette, add 50 piL, each, of the standards, controls, and samples to the
appropriate wells in the plate. Place all six standards (0.00, 0.15, 0.40, 1.00, 2.0 and 5.0 ng/L),
the kit control (0.75 piL), and negative control, in pairs, starting in the well in the upper left-hand
corner of the kit as shown in Figure 4.1. Verify that the software displays the same template or
make any necessary corrections. Laboratories with access to an autopipetter may use said
machinery after proper documentation of set up, training and calibration has been provided and
approved by EPA HQ Laboratory Review Coordinator prior to first use.

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P13

P17

P21

P25

P29

P33

P37

D13

D17

D21

D25

D29

D33

D37

P10

P14

P18

P22

P26

P30

P34

P38

D10

D14

D18

D22

D26

D30

D34

D38

Pll

P15

P19

P23

P27

P31

P35

P39

Dll

D15

D19

D23

D27

D31

D35

D39

P12

P16

P20

P24

P28

P32

P36

P40

D12

D16

D20

D24

D28

D32

D36

D40

Figure 4.1 Microcystin: sample template

Key: S0-S5 = Standards; KC = Control supplied with Kit (i.e., Kit Control);

NC = Negative Control (Laboratory Reagent Blank);

P = Primary aliquot for each unknown sample collected by field crew;

D= "DUPLICATE" aliquot for each matching unknown Primary sample.

15. Add 50 piL of the pink antibody solution to each well using the multi-channel pipettor and a
reagent reservoir. Use dedicated reagent reservoirs for each reagent to avoid contamination
from one reagent to another.

16. Place the sealing Parafilm over the wells.

17. Manually mix the contents by moving the strip holder in a rapid circular motion on the benchtop
for 30 seconds. Be careful not to spill the contents.

18. Place the plate in an area away from light for 90 minutes.

19. After 90 minutes, carefully remove the Parafilm. 5

20. Empty the contents of the plate into the sink, pat inverted plate dry on a stack of paper towels, >
and then wash the wells of the plate three times with 250 piL of washing solution using the O
multi-channel pipette. After adding the washing solution each time, empty the solution into the ^
sink and use the paper towels as before. ^

21. Add 100 liL of enzyme conjugate solution to all wells using the multi-channel pipettor. 3

22. Cover the wells with Parafilm.

23. Manually mix the contents by moving the strip holder in a rapid circular motion on the benchtop §

for 30 seconds. Be careful not to spill the contents. ^

24. Place the strip holder in an area away from light for 30 minutes. $

25. After 30 minutes, remove the Parafilm, decant, and rinse the wells three times again with 250 O
piL of washing solution as described in step 10. ^

26. 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. ?

27. Cover the wells with Parafilm. ^

28. 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. ^

29. Place the plate in an area away from light for 20 minutes. o

<3-

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30. After 20 minutes, remove the Parafilm and add 50 piL of stopping solution to the wells in the
same sequence as for the color solution. This will turn the contents a bright yellow color. After
adding the stopping solution, read the plate within 15 minutes.

31. Within 15 minutes of adding the stopping solution, use the microplate ELISA photometer (plate
reader) to determine the absorbance at 450 nm. The software (i.e., commercial ELISA evaluation
program) calculates the absorbance and concentration values of the samples from the
calibration curve and the average values for each pair. Use a 4-parameter standard curve fit to
determine the concentrations.

32. Dispose of solution in plates in a lab sink. Rinse plates and sink with water to dilute the weak
acid present.

33. Perform QC evaluations of the data as follows:

a. If the following failures occur, then the laboratory must reanalyze all samples in the
analytical run:

i. Standard curve with a correlation coefficient of less than 0.99 (i.e., R<0.99)

ii. Standards S0-S5 must have decreasing absorbance values. First, calculate the average
values for each standard. That is, if A, is the absorbance average for S,, then the
absorbance averages must be:

Ao > Ai > A2 > A3 > A4 >As

iii. The average absorbance of the standard SO less than 0.8 (i.e., A0< 0.8).

iv. Two or more negative control samples with detectable concentrations of microcystins
(i.e., values > 0.1 ng/L). If this occurs, then evaluate possible causes (e.g., cross-
contamination between samples), and if appropriate, modify laboratory processes
before the next analytical run.

v. Results for control samples of outside the acceptable range of 0.75 +/- 0.185 ppb. That
is, results must be between 0.565 and 0.935.

b. If either, or both, of the following failures occur, then the sample must be reanalyzed
(maximum of two analyses, consisting of the original analysis and, if necessary, one
reanalysis):

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.

34. Record the results, even if the data failed the quality control requirements in #23b, for each well
in EPA's data template (see Table 4.2for required elements). The required entries are for the
following columns:

a. TYPE should be one of the following codes: S0-S5 for standards; KC, NC, or SC for controls; P
(primary) or D (for duplicate) of unknown sample.

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 with a salinity <3.5
ppt. See Section 4.3.1 for calculating the sample-specific detection limit for a diluted
sample.

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ii. If the result shows that it is HI'/' this indicates that the sample value is outside of the
calibration range and must be diluted and re-run using another analytical run. Leave the
CONC column blank and record 'HI' in the DATA FLAG column.

c. DATA FLAGS have codes for the following special cases:

i. ND if the sample was non-detected;

ii. J if the value is detected but at a level below the reporting limit of 0.15 ng/L (for
undiluted samples);

iii. H if sample did not meet the holding time and was not analyzed within 90 days.

iv. HI if the concentration value registers as HIGH (exceeds the calibration range).

d. QUALITY FLAGS have codes for the following special cases:

i. QCF if there is a QC failure per step 23 above. The QCF code must be used for all failures
to facilitate data analysis.

ii. Qfor any other quality issue (describe in COMMENTS)

e. DILUTION FACTOR is only required if the sample was diluted.

f. DUP AVG and DUP CV are required for duplicate samples and control samples (use all three

values if the controls are used in triplicate).

Table 4.2 Microcystin: required data elements - data submission

STAGE 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 (blank if standard or control)

VISIT NUMBER

numeric

Sequential visits to site (1 or 2) (blank if standard or control)

SAMPLE ID

numeric

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

DATE

COLLECTED

MMDDYY

Date sample was collected (blank if standard or control)

CONDITION
CODE

text

Sample condition upon arrival at the laboratory (blank if
standard or control)

Flag

Definition

Blank or
N

Not a sample (blank, standard, or control)

Sample is in good condition

Sample container is cracked

Sample or container is leaking

Sample label is missing

Sample is not frozen

Other quality concerns, not identified above

CONDITION
COMMENT

text

Comments about the condition of the sample.

i/i
>
u
O

QC
3
Q

QC
CL

<
LT)
LT)
<
O

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STAGE

FIELD

FORMAT

DESCRIPTION

ANALYSIS

BATCH ID

Numeric

Batch identification code, assigned by lab

TECHNICIAN

text

Name or initials of technician performing the procedure

KIT EXPIRE DATE

MMDDYY

Expiration date on kit box

KIT ID

text

Kit identification code. If one does not exist, assign a unique
code to each kit.

numeric

R2 from curve fit to the average absorbance values for the
standards. Value is between 0 and 1.

TYPE

text

Type of solution being tested in the well

Code

Definition

Kit Control

Negative Control

SO, SI, S2, S3, S4,
S5

Standard

Quality control sample

Sample of unknown concentration

LOCATION

text

Location of well in the kit (e.g., B5 would be the fifth well
from the left in the second row B)

PRIM_DUP

text

Primary or duplicate run of a sample of unknown
concentration (see Figure 4.1)

CONC

numeric

Concentration or sample-specific detection limit of contents of
well in ng/L. Sample-specific detection limit should be 0.1 ng/L
for a sample with salinity <3.5 ppt which hasn't been diluted.

UNITS

text

The units of the concentration of the CONC column

MDL

numeric

Minimum detection limit in the same units as the CONC
column

numeric

Reporting Limit in same units as the CONC column

ABSORBANCE

numeric

Absorbance value

DILUTION
FACTOR

numeric

10, 100, etc for number of times the sample was diluted. If
not diluted, leave blank or record 1

CV_ABSORB

numeric

Calculated %CV of duplicate values of absorbance for a
sample. Only calculated for TYPE=U, KC, or NC. Enter %CV.
Value is between 0 and 100%.

AVG_ABSORB

numeric

Calculated average of absorbance values for a sample. Only
provided for TYPE=U, KC, NC, or SC. Average value of the
original sample and its duplicate (or replicates for KC and
NC).

AVG_CONC

numeric

Calculated average of concentration values for a sample.

OA FLAG (if
appropriate)

text

Data qualifier codes associated with specific identifications
of voucher samples. These codes provide more information
that those used when reporting receipt of samples. A
technician may use alternative or additional qualifiers if
definitions are provided as part of the submitted data
package (e.g., as a separate worksheet page of the data
submission file).

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STAGE

FIELD

FORMAT

DESCRIPTION

Flag

Definition

Concentration below detection. Unless the sample
was diluted, the concentration will be 0.1 ng/L

Sample did not meet the holding time and was not
analyzed within 90 days.

Result indicated that a high concentration (i.e.,
outside calibration range)

Concentration above detection but below reporting
limit.

QCF

QC failure

Other quality concerns, not identified above

LAB COMMENTS

text

Explanation for data flag(s) (if needed) or other comments.

4.6.4 Dilutions (if needed)

Dilutions if needed are prepared as follows (using clean glass tubes):

1:10 dilution

a. Add 900 piL of distilled water to a clean vial. (Note: Dilutions may also be made using the kit's
diluent rather than distilled water.)

b. Pipette 100 piL from the sample into the vial. (To provide more accurate dilutions and less
chance of contaminating the diluent, the diluent should be added to the vial before the sample.)

c. Mix by vortexing.

d. Multiply final concentration and Abraxis' detection limit of 0.1 ng/L by 10 to obtain the sample-

specific detection limit of 1.0 ng/L. >

• 1:100 dilution ^

a. Add 3.96 mL of distilled water to a clean, appropriately labeled glass vial. (Note: Dilutions may ^
also be made using the kit's diluent rather than distilled water.)

b. Vortex the sample to mix thoroughly, then pipette 40 piL from the sample and add to the water
(or diluent) in the appropriate labeled vial. Vortex. §

c. Multiply the final concentration and Abraxis' detection limit of 0.1 ng/L by 100 to obtain the

l/l

QC
3
Q

QC
CL

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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

Once or twice during the survey, sets of five identical Performance Evaluation (PE) samples will be
provided to all participating laboratories by an External QC Coordinator. As determine by the External
QC Coordinator, the PE samples may be synthetic; aliquots of additional samples collected at NRSA sites;
or reference samples obtained from an organization such as the National Institute of Standards and
Technology or Eurofins Technologies (formerly Abraxis). Each laboratory shall analyze the PE samples
following the same procedures used for the other samples analyzed. The External QC Coordinator will
compare the results to the expected value. The results of the comparisons shall be made available to the
EPA QC Coordinator for review. Based upon the evaluation, the External QC Coordinator may request
additional information from one or more laboratories about any unique laboratory practices that might
account for differences and may convene a conference call with all participating labs to identify causes
of and if possible, reconcile those differences. With this additional information, the External QC
Coordinator shall determine an appropriate course of action, including no action, re-running samples,
flagging the data, or excluding some or all the laboratory's data.

4.7.3 Summary of QA/QC Requirements

Table 4.3 provides a summary of the quality control requirements described in Sections 4.5 and 4.6.

Table 4.3 Microcystin: quality control - sample analysis

Quality Control

Description and Requirements

Corrective Action

Activity

Kit - Shelf Life

Is within its expiration date listed on kit box.

If kit has expired, then discard or
set aside for training activities.

Kit - Contents

All required contents must be present and in
acceptable condition. This is important
because Abraxis has calibrated the standards
and reagents separately for each kit.

If any bottles are missing or
damaged, discard the kit.

Calibration

All of the following must be met:
o Standard curve must have a

correlation coefficient of >0.99;
o Average absorbance value, A0, for SO
must be >0.80; and
Standards S0-S5 must have decreasing average
absorbance values. That is, if A, is the average
of the absorbance values for Si, then the
absorbance average values must be: A0> Ai>
A2 > A3 > A4 >As

If any requirement fails:
• Results from the analytical run

are flagged QCF
All samples in the analytical run
are reanalyzed until calibration
provides acceptable results.

Kit Control

The average concentration value of the
duplicates (or triplicate) must be within the
range of 0.75 +/- 0.185 ng/L. That is, results
must be between 0.565 and 0.935.

If either requirement fails:
• Results from the analytical run
are not reported

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Quality Control

Description and Requirements

Corrective Action

Activity

Negative Control

The values for the negative control replicates
must meet the following requirements:
o All concentration values must be <
0.15 ng/L (i.e., the reporting limit);
and

One or more concentration results must be
nondetectable (i.e., <0.10 ng/L)

• The lab evaluates its

processes, and if appropriate,
modifies its processes to
correct possible
contamination or other
problems.

The lab reanalyzes all samples in
the analytical run until the
controls meet the requirements.

Sample
Evaluations

All samples are run in duplicate. Each
duplicate pair must have %CV<15% between
its absorbance values.

If %CV of the absorbances for the
sample>15%, then:

10. Record the results for both
duplicates.

11. Report the data for both
duplicate results as Quality
Control Failure "QCF"; and

12. Re-analyze the sample in a
new analytical run. No samples
are to be run more than twice
without prior approval from
EPA.

If the second run passes, then the
data analyst will exclude the data
from the first run. If both runs fail,
the data analyst will determine if
either value should be used in the
analysis (e.g., it might be
acceptable to use data if the CV is
just slightly over 15%).

Results Within

Calibration

Range

All samples are run in duplicate. If both of the
values are less than the upper calibration
range (i.e., 5.0 ng/Lfor undiluted samples),
then the requirement is met.

If a result registers as "HIGH", then
record the result with a data flag
of "HI." If one or both duplicates
register as 'HIGH,' then the sample
must be diluted and re-run. No
samples are to be run more than
twice without prior approval from
EPA. 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

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Quality Control

Description and Requirements

Corrective Action

Activity

Method or unique laboratory
practices that might account for
differences between the
laboratory and others. With this
additional information, the
External QC Coordinator will
determine an appropriate course
of action, including no action,
flagging the data, or excluding
some or all of the laboratory's
data.

4.8 Sample and Record Retention

The laboratory shall retain:

2. Original records, including laboratory notebooks and the reference library, for a minimum of 10
years from the date that EPA publishes the final report.

After the stated time periods, the laboratory shall follow its internal protocols for disposal.

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5.0 BENTHIC 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:

• 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 (benthic organisms and sediments) avoid inhalation, skin contact, eye
contact, or ingestion. If skin contact occurs remove clothing immediately and wash /

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rinse thoroughly. Wash the affected skin areas thoroughly with large amounts of
soap and water.

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= A x 100

A + B (i)

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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.

\n- —

pde = *— — x 100

H\ ^ 2 (2)

Percent taxonomic disagreement (PTD): measure of taxonomic precision comparing the number of
agreements (positive comparisons, compp0s) of the primary taxonomist and internal or external QC
taxnomists. In the following equation, N is the total number of organisms in the larger of the two counts.

PTD =

comPpos

100

(3)

Pickate: Sort residue from all grids originally sorted. This is the remaining material left from the grid,
after the sorter has removed all benthic macroinvertebrates. This could include small stones, sticks or
leaves, etc.

Quarter: A subsection of a grid. If a sorter finds many organisms within a grid, they will evenly divide the
grid into four "quarters".

Sorting Bench Sheet: Form used by the laboratory to record information about the sample during the
sample preparation and sorting procedure.

Primary laboratory: The laboratory that 1) sorts the sample; and 2) provides the first identification of
benthic macroinvertebrates in the sample.

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.)

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5.4.2 Laboratory

The procedure may be used by any laboratory that has expertise in each of the following areas:

3. 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.

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• 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.

• 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 overtime. 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

1.) U.S. 35 sieve (500 pim)

2.) Round buckets

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3.) Standard gridded screen (370-nm mesh screen, 30 squares, each 6 cm2)
with white plastic holding tray (Caton tray, 30cm x 36cm, 4cm deep)

4.) 6-cm scoop

5.) 6-cm2 metal dividing frame ("cookie cutter")

6.) White plastic or enamel pan (6" x 9") for sorting

7.) Scissors

8.) Teaspoon

9.) Permanent ink pen (e.g Pigma Micron® pen)

10.) Dropper

11.) Fine-tipped forceps (watchmaker type, straight and curved)

12.)Vials with caps or stoppers

13.)Sample labels for vials

14.)70-80% ethanol (denatured or non-denatured)

15.)Sorting Bench Sheet or other Sheet or database approved by EPA's External
QC Coordinator

16.)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.

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• 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.

4. Inspect each jar THE SAME DAY THEY ARE RECEIVED:

a. Refill them with 70-80% ethanol if necessary.

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.

5. After refilling the sample containers, store them at room temperature until sorting begins.

6. 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).

7. 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

Not enough preservative used

Sample shipping problem (explain in QA_COMMENTS
field)

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.

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3. Transfer the homogenized sample material to the gridded screen portion of the grid (use more than
one subsampling device if necessary). Wash the sample thoroughly by running tap water over it to
remove any fine material. If there is more than one jar for any particular sample, empty and wash
each jar onto the Caton-type grid one at a time, making sure to spread each jar's contents evenly
across the tray. Multiple jars from the same sample should all be emptied onto the same Caton grid
(or suitable alternative subsampling tray). If the amount of leaf litter or other detrital material
exceeds that which fills 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.

• 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.

1) 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.

2) 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.

3) 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

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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.

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

At least 10% of the tray or three grids in the case of a Caton tray (assuming 30 grids) would be h

randomly selected. oe

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 q

(quartered) being sorted in successive order (compositing of the first three grids is not done). Q

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. ^

LU
CO

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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).

a) 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.

b) 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.

c) 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.

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)

2Strict water column taxa are those that do not have at least one life stage that is benthic (i.e., bottom-
dwelling).

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Trichoptera (adults)

Ostracoda

BRYOZOA

NEMATODA

d) 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.

3. 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.

4. 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.

5. 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".

6. 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.

7. 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. qq

i—

QC
LU
>

u
<

T.
I—

¦z.

LU
CO

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

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VISIT NUMBER
SAMPLE ID

DATE COLLECTED
DATE SORTED

PROPORTION
SORTED

CORRECTION
FACTOR

OA FLAG (if
appropriate)

QA.COMMENTS
LAB COMMENTS

numeric

MMDDYY

numeric

text

text
text

sequential visits to site (1 or 2, if specified on label)
sample number as used on field sheet (on sample label)
date sample was taken

date that the sorter started working on the sample
proportion of sample sorted based upon number of grids and
quarters selected and available

numeric subsampling correction factor

QA/QC flag (lab may use its own flags, if defined in QA_COMMENTS
field or provided to NARS IM team)

Flag
C

Definition

Organism in poor condition or fragments
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
explanation for QA FLAG (if needed)

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).

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8. Copy block of taxa names to a text file.

1.1 Save the text file

1.2 Go to the ITIStaxa match screen (http://www.itis.usda.gov/taxmatch_ftp.html)

1.3 Follow the onscreen instructions to upload the file. Use all of the current defaults.

2. Finish with two lists, one of matches with TSNs and one with non-matches. Check the non-
matches for the following common problems.

5 Abbreviations

6 Extra information identifiers (e.g., sp., spp.,, nr., cf., genus 1, w/ hair chaete)

7 Extra character (e.g., "?", "Acentrella Pturbida", blank space)

8 The word "probably" or "prob" (e.g., "Microcylloepus prob. similis")

9 I Ding to a lower level than is in ITIS (e.g, to species rather than genus)

10 Double names (e.g., Callibaetis callibaetis)

11 Common misspellings

12 Tribes/Subfamilies/Subgenus sometimes do not appear in ITIS

13 Species w/ incorrect Genus (Hydatopsyche betteni)

14 Split level taxonomy (e.g., Cricotopus/Orthocladius)

15 Invalid name (e.g., taxonomic change, synonym; Sphaeriidae vs. Pisiidae)

16 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

7. If early instar or juvenile specimens can be identified, they are counted:

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(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
sheet.

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.

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

Genus

ANNELIDA

(any, except

Hirudinea,

Polychaeta)

Hirudinea

Oligochaeta

(any, including
Tubificinae, except as
noted below)

Enchytraeidae

Lumbriculidae

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Target Level of

TAXON

Identification

Polychaeta

ARTHROPODA

Hydrachnidia

Insecta

(any, except as noted)

Coleoptera

Diptera (any, except as
noted)

Ceratopogoninae

subfamily

Chironomidae

see Table
4

Dolichopodidae

Empididae

Ephydiridae

Muscidae

Phoridae

Scathophagidae

Sciomyzidae

Stratiomyidae

Syrphidae

Tabanidae

Ephemeroptera

Hemiptera (Heteroptera)

Lepidoptera

Megaloptera

Odon at a

Plecoptera

Trichoptera

Malacostraca

(any)

Amphipoda

Decapoda

Isopoda

Mysidacea

COELENTERATAt

MOLLUSCA

(any, except
Hydrobiidae)

Bivalvia

Gastropoda

(any, except
Hydrobiidae)

Hydrobiidae

NEMERTEA

PLA TYHELMINTHES

Turbellaria

anyt

TColelenterata need only be identified to the phylum level. z

t Turbellaria need only be identified to the class level. O

Table 5.5 Benthic macroinvertebrate: target Level of taxonomic identification -chironomidae <

i—

¦z.

FAMILY GENUS

REQUIRED LEVEL OF IDENTIFICATION

Chironomidae (except as noted
below)

Genus

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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

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

Damaged Organism, poor condition or fragments

Immature

Indeterminate (explain in QA_COMMENTS field)

<
cc

O
cc
u
<

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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.

Other quality concerns, not identified above

QA_COMMENTS text explanation for QA FLAG (if needed)

LAB COMMENTS text general laboratory analysis comments

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 ci/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:

5. 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

i/i

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

I—
QC

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 q

u
<

from three laboratories into one batch at a convenient time for the QC contractor). u

c. To the extent practicable, the External QC Coordinator and QC contractor will schedule
batch evaluations evenly throughout the project period.

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8. The QC contractor coordinates and pays for delivery of the selected samples, bench sheets,
and chain-of-custody forms.

9. 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
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.

10. 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.

11. 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.

12. The QCtaxonomist:

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.

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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."

13. The External QC Coordinator will compare the taxonomic results (counts AND
identifications) generated by the primary and secondary laboratories. If either laboratory
identified the organisms to a lower level than required by Table 5.4 and Table 5.5 or by
nomenclature 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:

a. Enumeration as measured by PDE defined in Section 5.4.1; and

b. Taxonomy as measured by PTD defined in Section 5.4.1. Table 3 of Stribling (2003) provides
examples of what would be considered agreements.

14. 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.

15. 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.

16. 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.

17. 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:

28. Confirm the sorting and identification steps are properly implemented.

29. Assist with questions from laboratory personnel.

30. Suggest corrections if any errors are made.

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5.12 Quality Assurance/Quality Control (QA/QC)

Equation 5.1 Percent sorting efficiency (PSE).

Number of organisms recovered by the sorter compared to the combined (total) number of recoveries
by the sorter (A) and QC Officer (B) for a sample. PSE should be >90%.

PSE =

x 100

A +B

Equation 5.2 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. PDE should be <5%.

PDE =

: 100

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 =

rcompposA

x 100

Table 5.7 Benthic macroinvertebrate: measurement data quality objectives

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)

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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

cc
u
<

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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.

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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.

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Field Filter Blank: A volume of sterile PBS, free of target organisms (i.e. Enterococcus) filtered through a
sterile filter and processed in parallel with all other samples to serve as a sentinel for detection of
reagent contamination or contamination transferred between samples by processing and analysis.

Field Replicates: Samples collected from rivers and streams that are collected at the same sampling site
one right after the other with only slight temporal variation. They are not "splits" of the same sample
volume.

Filtrate: Sample liquid or buffer rinsate passing through the filter into the vacuum flask.

Laboratory Quality Samples: Mock samples created in the lab such as lab blanks, lab-fortified blanks
(LFBs), and Lab-Fortified Matrices (LFMs) used to assure lack of sample contamination and to measure
analytical recovery during performance of sample processing and analysis methods.

Performance Testing (PT) / Performance Evaluation Sample (PES): Calibrator samples (filters spiked
with E. faecalis grown in Brain Heart Infusion Broth) and Laboratory Fortified Blanks (Phosphate
Buffered Saline; PBS) spiked with Enterococcus faecalis cells from BHI Broth suspension) will be assayed
by EPA Method 1600 and Draft EPA Method 1606 to ascertain method performance. Ball-T Bioballs®
which contain a specified number of E. faecalis cells may also be acquired to determine the performance
of the Relative Quantitation Method. Purified E. faecalis DNA acquired from the American Type Culture
Collection and TIB Mol Biol Inc. is used to test the performance of the Absolute Quantitation Method.

Retentate: The sample residue retained by the filter after the sample is vacuum-filtered. The retentate
contains particulates, microbiota, and macrobiota from which the DNA is extracted into buffer by bead-
beating for subsequent qPCR analysis.

Rinsate: The volume of phosphate buffered saline (PBS) applied to a sample's filter retentate in order to
"wash" any residual fine particles, smaller than the filter's nominal pore size, through the retentate and
the filter.

Sample Processing Control (SPC): A surrogate homologue analyte (e.g. Salmon DNA) spiked into each
sample to determine the recovery of target analyte and/or detect assay inhibition caused by matrix
effects.

Standards: Known amounts or numbers of copies of Enterococcus genomic DNA analyzed by the
Enterococcus qPCR assay to generate a Standard Curve (Log Copy Number vs. Crossing Point Value) in
order to determine Enterococcus genomic copy numbers in "Unknown" test sample extracts by Absolute
Quantitation Method.

6.4 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

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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

1) Clean powderless latex or vinyl gloves

2) Goggles or Face Shield

3) Roche MagNA Lyser

4) Roche MagNA Pure LC (automated nucleic acid isolation and purification platform)

5) High Speed Microfuge

6) Micropipettors

7) 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

8) 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.

9) Roche MagNA Lyser Rotor Cooling Block

10) 2-mL tube racks

11) Permanent marking pens (fine point and regular point) for labeling tubes

12) 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

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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.

6. 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.

18. 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).

19. 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 /uL) times the total number of samples to be analyzed
including controls, water samples, and calibrator samples. For example, for 18 samples, prepare
enough Salmon/DNA extraction buffer for 24 extraction tubes (18 ) / 6 = 3, therefore, 3 extra
tubes for water sample filtration blanks (method blanks) and 3 extra tubes for calibrator
samples). Note that the number of samples is divided by 6 because you should conduct one
method blank for every 6 samples analyzed. Additionally, prepare excess volume to allow for
accurate dispensing of 600 /iL per tube, generally 1 extra tube. Thus, in this example, prepare
sufficient Salmon DNA Extraction Buffer for 24 tubes plus one extra. The total volume SAE
needed per sample is 600 iiL. Hence for the SAE volume for 25 sample tubes is equal to 15,000
jiL. Dilute the Salmon DNA working stock 1:50, for a total volume needed (15,000 juL) 50 = 300 jiL
of 10 /ug/mL Salmon DNA working stock. TheAE buffer needed is the difference between the
total volume and the Salmon testes DNA working stock. For this example, 15,000 iiL - 300 iiL =
14,700 jiL AE buffer needed.

20. 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.

21. Enterococcus faecalis DNA for Standards.

22. Frozen Reference Stock (20-nL) at 2.89 x 10s GEQs per piL.

23. Dilute 10-piL of the Frozen Reference stock 363-fold to a final volume of 3,630 piL AE buffer.
Aliquot 20-piL volumes into many 200-nL microfuge tubes and store frozen at -20 2C. The net
concentration of Enterococcus GEQs is 8,000 / piL. Each week perform a series of 10-fold and
4-fold dilutions from one thawed tube of the 8,000 GEQ/^L standard solution to create 800
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:

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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.

24. The calibrator sample filters are spotted with 104 or 10s Enterococcus faecalis cells and this
number is incorporated into the Relative Quantitation EXCEL spreadsheet.

25. 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.

6. 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.

26. 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.

27. 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.

28. 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

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extracted 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

d. 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.

29. 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 -202C.

30. 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.

31. 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.

32. 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.

33. 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.

34. Set 32 Smart tubes in Cepheid Racks in PCR cabinet along with micro-pippetors and expose
to germicidal UV lamp for 15-min.

35. 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.

36. 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.

37. Check to make sure each Smart tube is properly labeled and identifiable by sample number
or l-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.

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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

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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.

6.10.3 Sample analysis sequence for SmartCycler

Example: For analyses on a single 16-position SmartCycler, calibrator samples and water samples are
analyzed in separate runs and a maximum of 6 water samples (or 2 replicates of 3 samples) are analyzed
per run, as described in Table 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 2023/24 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.

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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 2023/24 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.

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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

1.5 nL

Bovine Serum Albumen (20 mg/mL)

2.5 nL

TaqMan® master mix

12.5 nL

Primer/probe working stock solution

3.5 nL*

Note: This will give a final concentration of 1 nM of each primer and 80 nM of probe in the reactions. Prepare
sufficient quantity of assay mix for the number of samples to be analyzed per day including calibrators and negative
controls plus at least two extra samples. It is strongly recommended that preparation of assay mixes be performed
each day before handling of DNA samples.

Table 6.2 Enterococci: batch calibrator & enterococcus standards PCR run - 7 samples

Sample Description*

Quantity
Samples

PCR Assay Master Mix

Quantity PCR
Reactions

3 Calibrators (5- and/or 25-fold dilution)

Salmon DNA (Sketa)

3 Calibrators (5- and/or 25-fold dilution)

Enterococcus

4 Enterococcus faecalis DNA Standards

Enterococcus

No template control (reagent blank)

Enterococcus

* 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)

Enterococcus

Method blank or Sample PCR Reaction Duplicate,
(1- or 5-fold dilution)

Enterococcus

Non-diluted SAE Buffer

Enterococcus

Water samples, (1- or 5-fold dilution)

Salmon DNA

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Method blank or Sample PCR Reaction Duplicate,

Salmon DNA

(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
Measurement

OA
Class

Expected
Range and/
or Units

Summary of Method

References

Sample
Collection

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

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

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

-40Cto+40 C

Batches of sample tubes shipped on dry ice
to lab for analysis.

NRSA Laboratory
Methods Manual
2008

DNA Extraction
(Recovery)

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)

<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.

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Table 6.5 Enterococci: parameter measurement data quality objectives

Expected

Variable or QA Range and/or

Measurement Class Units Summary of Method References

DNA Extraction
(Recovery)

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

<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

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Use single stock of
E. faecalis
calibrator

For all qPCR
calibrator
samples for
quantitation

All calibrator sample Cp (Ct)
must have an RSD <30%

If calibrator Cp (Ct) values exceed
an RSD value of 30% a batch's
calibrator samples shall be re-
analyzed and replaced with new
calibrators to be processed and
analyzed if RSD not back within
range.

DATA PROCESSING & REVIEW

100% verification
and review of
qPCR data

All qPCR
amplification
traces, raw and
processed data
sheets

All final data will be checked
against raw data, exported
data, and calculated data
printouts before entry into
LI MS and upload to Corvallis,
OR database.

Second tier review by contractor
and third tier review by EPA.

6.18,1 Enterococcus qPCR Analysis Decision Tree (ADT

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

e. 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
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(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.

38. 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.

39. 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.

40. 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.

41. 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.

42. 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.

43. 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.

44. 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.

45. 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.

46. 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.

47. 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.

48. Repeat steps 9 to 13 for second batch of 16 samples (lysates). Re-cover sample cartridge
with adhesive film for storage. Centrifuge sample cartridge opposite a balance cartridge for
75-sec (1-min, 15-sec) at 2800 rpm in IEC centrifuge (or equivalent) with rotor adaptors for

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microtiter plates in place. Insert the film-covered sample cartridge in MagNA Pure LC
platform.

49. 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 ng/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.

50. 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.

51. 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.

52. Prepare Elution Buffer Control from 9.3ng/mL Salmon DNA Stock by diluting a small volume
to 37.2pg/1000nL (1-mL). This control sample is only analyzed by the Sketa qPCR assay. The
Ct value obtained represents that value expected in Sketa qPCR assays of each MagNA Pure
LC purified sample if 100% of the Salmon DNA was recovered and detected. Vortex to mix
on low speed briefly prior qPCR analysis. Centrifuge for 1.5-min to coalesce droplets.
Remove film to aliquot sub-samples and re-place with new film cover to restore at cool
temperatures.

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7.0 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.

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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.

7.3.1 Definitions

The following terms are used throughout the procedure:

Bench Sheet: Optional form used by the QC taxonomist to record information about the voucher
specimen. See Figure 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.

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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.

53. 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.

54. 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).

55. 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

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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.

1. Fine-tipped forceps (watchmaker type, straight and curved)

2. Dissecting microscope (3.5-12X magnification)

3. Dividers and/or calipers for obtaining measurement and proportional measurement data
from specimens

4. Scalpel

5. List of standard common and scientific names used in NRSA (Appendix D)

6. Taxonomic keys or published descriptions in up-to-date, regional faunal works including
online resources

7. Keys or descriptions for the most recent, accepted taxonomic revisions of individual groups

8. Descriptions in other taxonomic studies, especially original descriptions (preferably
published).

9. Voucher samples

a. Sample tracking form that accompanies each shipment of samples

b. Site location

c. Physical specimens or image file

d. Access to additional site information upon request to EPA

10. 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.

• Report receipt of QC voucher samples (including digital images) in the NARS IM sample
tracking system (within 24 clock hours).

56. 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.

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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.

57. Store preserved samples at room temperature until identification begins.

58. Maintain the chain of custody or sample tracking forms with the samples.

59. 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.

60. 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

Sample is in good condition

Sample container is cracked

Sample or container is leaking

Sample label is missing

Sample is not or insufficiently preserved

Sample shipping problem (explain in QA_COMMENTS field)

Other quality concerns, not identified above

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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:

• Process the QC voucher sample by voucher tag.

61. 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.

62. 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.

63. 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.

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64. 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.

65. If identifications are problematic or difficult (e.g., specimens or images do not contain fully
diagnostic characters), even after consulting the references in Step 4, consider the following
aspects:

a. Mature males in breeding condition present colors and/or tuberculation that can be used to
identify the taxon. Assume that their taxon identification applies to non-breeding male and
female specimens with similar characters.

b. Mature specimens that have a more developed morphology can be used to identify the
taxon. Assume that their taxon identification applies to less developed specimens with
similar characters.

c. Location can be used as the basis of identification if the taxon is well-studied or allopatric
(e.g., speciation that occurs when populations of the same species become isolated from
each other such that they do not occur in any one place together.)

d. Hybrids are typically detected by presence of diagnostic characters of two closely related
taxa.

e. Commonly-stocked hybrids, such as muskellunge x northern pike (tiger muskie), or striped
bass x white bass (wiper), are diagnosed in many keys.

f. Backcrosses (i.e., progeny of crosses of hybrids and a parent species) may not be
distinguishable from the parent species on the basis of visual and partially dissected
characters. Because the evidence is consistent with non-hybrid characters, assume that the
specimen is not a hybrid.

66. 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.

67. 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.

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68. If a tagged lot from the field turns out to include multiple taxa (assuming multiple
individuals were provided):

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

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.

69. 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

70. 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.

71. Select another bag and repeat Steps 1-10.

72. 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

because:

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NRSA 2023/24/Data Templates folder. Table 7.2 identifies the contents and formats used by
EPA's file.

73. Save the submission worksheet file using the following convention: Taxonomist
name+affliation-abbreviation+submission date (yyyymmdd).xlsx (e.g.,
JSmith_ABCfirm_20180615.xlsx).

74. 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

text

Unique common name from NRSA master taxa list (Appendix D). For

SCIENTIFIC NAME

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

numeric

The number of individuals of taxa noted

INDIVIDUALS

CONDITION CODE

text

Condition codes associated with the condition of the sample upon
arrival at the laboratory.

Flag

Definition

Sample is in good condition

Sample container is cracked

Sample or container is leaking

Sample label is missing

Sample is not or insufficiently preserved

Sample shipping problem (explain in QA_COMMENTS field)

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

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Q Other quality concerns, not identified above

( DATA FLAG (if text Data qualifier codes associated with specific identifications of voucher

appropriate) samples. These codes provide more information that those used when

reporting receipt of samples. A QC taxonomist may use alternative or
, additional qualifiers if definitions are provided to the external QC
| Coordinator as part of the submitted data package (e.g., as a separate
worksheet page of the data submission file).

Flag 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:

• 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.

75. 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%.

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PTD =

compr

: 100

Table 7.3 Fish voucher: measurement data quality objectives

Variable or Measurement

Precision

Accuracy

Completeness

Identification

85% a

99%

a As measured by (100%-PTD)

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

All data sheets

Genera known to occur in

Data qualifiers on data that fail

"reasonable-ness"

given rivers/streams or

reasonableness check. No

checks

geographic area

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.

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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.

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

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8.0 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 8.1 Fish tissue plug: measurement data quality objectives

Variable or Measurement

MDL

Quantitation Limit

Mercury

0.47 ng/g

5.0 ng/g

Table 8.2 Fish tissue plug: quality control

Activity

Evaluation/Acceptance Criteria

Corrective Action

Demonstrate competency
for analyzing fish samples to
meet the performance
measu res

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

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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.

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9.0 DIATOMS

This procedure is applicable to the analysis of the diatom samples collected for the National Rivers and
Streams Assessment (NRSA) 2023/24. 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

9.1 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.

9.2 Health and Safety Warnings

In addition to the laboratory's usual requirements, personnel must implement the following health and
safety procedures for this procedure:

1. Wear or apply proper personal protection clothing and equipment (e.g. lab
coat, protective eyewear, gloves).

2. When working with potential hazardous chemicals (e.g. a mounting medium
with a high refractive index such as ™) 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.

9.3 Required Equipment
9.3.1 Laboratory

To participate in NRSA, each laboratory first must demonstrate to EPA that it has appropriate
qualifications as follows:

3. Analytical work. To demonstrate its expertise, the laboratory shall provide EPA
with one or more of the following:

samples.

76. Memorandum that identifies the relevant services that the laboratory provided for the
National Aquatic Resource Surveys in the past five years.

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77. Memorandum that describes the laboratory's participation in relevant round robin studies
and/or performance studies.

78. Report of relevant findings from an on-site technical assessment or audit.

79. Quality procedures.

80. 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).

81. To demonstrate its ongoing commitment, the person in charge of quality issues for the
laboratory shall sign the NRSA QAPP Certification Page.

82. Reporting standardized data.

83. 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.

84. To demonstrate its ongoing commitment to use LIMS, the memorandum also shall confirm
that the laboratory will use LIMS to record and report results from the procedure.

9.3.2 Equipment/Materials

The procedure requires the following equipment and materials for sample preparation (subsampling)
and taxonomic identifications:

9.3.2.1 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

9.3.2.2 Diatoms

9.3.2.2.1 Preparation of Diatom Slides

1. Corning ceramic-top hot plate with temperature control

2. Positive-draw chemical hood

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3. 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)

4. Glass microscope slides (1x3 inches; 2.5 x 7.5 cm)

5. 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.

6. Mounting medium with a high refractive (1.7) index (e.g., Naphrax™)

7. Diamond scribe

8. Disposable plastic pipettes

9. Adjustable pipettor (0 - 250^1); adjustable pipettor (200 -1000^1)

10. Pipette tips for adjustable pipettors

11. Round-style tooth picks

12. Forceps

13. Polished, rounded wooden splints

14. Wash bottle filled with distilled (DW) or reverse osmosis (RO) water

15. Single-edged razor blades

16. Ethanol, 70%

17. Acetone

18. Tissues (e.g., Kimwipe®)

19. Slide labels

20. Wax (the kind commonly used for candle making and canning foods)

21. 10% HCI

9.3.2.2.2 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

9.4 Sample Receipt

Under U.S. regulations, shippers must classify samples preserved in formalin as "Dangerous Goods" and
handle according to hazardous material shipping requirements. At the laboratory, only personnel
certified to handle hazardous material can receive the hazardous samples. Because EPA initiates tracking
procedures designed to recover any missing shipment, the laboratory personnel start the following login
steps within 24 clock hours of receiving a delivery.

4. 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.

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85. Inspect each jar THE SAME DAY THEY ARE RECEIVED:

a. Verify that the site identification and sample number on the label also appear on the chain
of custody form in the shipment.

b. Notify the NARS IM team if any jars were broken and/or if there are discrepancies between
the custody form and the 50 mL bottles.

c. Verify that the bottles contain enough formalin. If not, then:

i. Add 2 mL of a 10% formalin solution

ii. Continue to add, if necessary, increments of 2 mL of the solution. Check after each
addition to determine if additional solution is required.

iii. Record the amount of solution added to the bottle in the Comments_Arrival field of
the Login spreadsheet (Table 9.1).

86. 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).

87. Verify that the login information includes the required data elements in Table 9.1. After
completing all required elements, provide the information to the data entry personnel.

Table 9.1 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

CON DITION_CODE

text

Condition codes describing the condition of the sample upon
arrival at the laboratory.

CODE

Definition

Sample is in good condition

Sample container is cracked

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

Sample or container is leaking

Too little sample left for the procedure

Sample label is missing

Preservative added, add amount in comments field

Other quality concerns, not identified above

FORMALIN ADDED

numeric

If the la

Doratory 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.

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9.5 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 9.2 after
completing all required elements, provide the information to the data entry personnel.

Table 9.2 Required data elements for diatom subsampling

FIELD

FORMAT

DESCRIPTION

LAB ID

text

Name or abbreviation for QC laboratory

SITE J D

text

NRSA site id as used on sample label

VISIT_NUMBER

numeric

sequential visits to site (1 or 2)

SAMPLEJD

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

9.6 Diatom Cleaning and Mounting

The diatom cleaning steps remove both extracellular and intracellular organic material by digesting it
with nitric acid. After removing the organic matter, all details of diatom structures essential to
taxonomic identification should be clearly visible. To clean the diatoms, the laboratory technician may
follow the cleaning steps in ANS Protocol P-13-42 "Diatom Cleaning by Nitric Acid Digestion with a
Microwave Apparatus" (http://diatom.ansp.org/nawqa/pdfs/P-13-42.pdf) or the following nitric acid
cleaning method:

1. Place the subsample of material for diatoms in a 250 mL beaker.

2. Place the beaker in a positive draw hood. Carefully add 50 mL of concentrated nitric acid.

3. Simmer on a hot plate or warmer for about 3 hours or until organic matter has dissolved. If small
bits of coarser debris remain after acid-cleaning, they can be rinsed and removed at a later time
with forceps.

4. Rinse the diatoms in the cleaned sample repeatedly to remove the nitric acid by repeatedly
adding deionized water to the cleaned diatom sample to fill the beaker, letting the diatoms
settle to the bottom, and then siphoning the water from the beaker before refilling again. This
method is similar to instructions by ANSP, but draw from center of the water column, not the
top. Also gently spin beakers and sprinkler water on the surface with a squirt bottle about
midway through settling to dislodge diatoms adsorbed onto the water surface and sides of
beakers (remember surface tension). We recommend using 1200 mLtall beakers.

5. Let samples in these beakers settle for a total of 8 hours or more.

6. After settling and no additional disturbance of the diatoms on the bottom of the beaker, slowly
and carefully siphon water with a fine tip glass pipette, like a Pasteur pipette. Usually, 20 mL
should be left above the settled diatoms in a 1200 mL tall beaker to minimize siphoning and

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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:

• Abide by the following Safety Precautions:

• Wear safety glasses, protective gloves and lab coats at all times when handling liquid and/or
solidified mounting medium. For example, Naphraxd is a hazardous substance because it
contains toluene, which volatilizes readily when heated.

• Use a positive-draw fume hood at all times for heating of the mounting medium.

• 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.

• 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:

• Keep the laboratory room and bench surfaces as clean as possible and free of debris.

• Use disposable pipettes, stirrers, etc. where feasible.

• 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.)

• Dry all equipment before storing it. This step prevents growth of algae and fungi.

• Verify that all necessary equipment is available. See Section 9.3.2 for recommended supplies.

• Determine if the vial's contents will produce satisfactory slides. Consider the following:

• 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).

• Generally, between 10 and 20 diatom valves orfrustules should be present in a single high
power microscope field (1000X).

• The number of specimens per field will need to be reduced if samples contain considerable
amounts of silt.

d Naphrax™ is a commercially-available toluene-based mounting medium with high refractive index.

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• 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).

• 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:

1. 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:

1. Allow the vial to settle for approximately 4 hours.

2. 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.

3. Allow the vial to settle for approximately 4 more hours (total of approximately 8 hours)

4. Use a micropipettor to remove the required amount of water from the vial of material. Keep
track of volume removed from the vial.

5. Record the SUBSAMPLE_DCF (i.e, subsample dilution/concentration factor).

2. If a concentration of cleaned material greater than two to five times is required, then re-
subsample the original sample following the steps in Section 9.5. Use the entire remaining
sample only if necessary. Digest the subsample as described previously (i.e., using ANS Protocol
P-13-42).

3. 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.

4. 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:

1. Scan the slide in its entirety under lOOOx magnification, and estimate the total number of
individuals on the slide.

2. Determine if the slide is countable (i.e., 100 specimens or more can be counted within 4 hours),
considering factors such as:

6.0 Density of diatoms.

7.0 Evidence of dissolution

8.0 Amount of debris (silt, clay, broken remains of diatoms and other siliceous organisms) that
would make it difficult to identify specimens accurately.

3. 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:

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1) Drip a known portion of the untreated sample onto a coverslip.

2) Dry the coverslip at room temperature.

3) 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:

1. Use forceps to remove single coverslips from the storage container.

2. Carefully clean each by wiping with a tissue (e.g., Kimwipe®e).

3. Place each coverslip on a marked space. (Be sure this space is clean and dry to avoid
cross-contamination.)

4. 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:

¦ 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:

• 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.

• Agitate the sample vial to homogenize the diatom suspension.

• Using an adjustable pipettor, quickly withdraw the required amount from near the central
portion of the sample.

• Eject this material smoothly and carefully into the layer of distilled water already on the
coverslip.

• Repeat until achieving a homogeneous suspension on the coverslip.

• Record the subsample amount ejected.

¦ 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).

¦ If the coverslip overflows, discard the coverslip, wipe the drying area, and repeat the procedure
with a freshly cleaned coverslip.

¦ Discard the pipette tip when finished with each vial.

5. Once the marked space is loaded with coverslip preparations, do not disturb until the
coverslips are dry.

6. Dry the coverslips as follows:

• Leave them undisturbed at room temperature for several hours;

• Use gentle heat (warm to the touch only) to hasten evaporation using a:

• Crook-neck lamp with incandescent light bulb placed 15 - 30 cm over the coverslips;

e Kimwipe® is a commercially-available product.

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• Slide maker that provides heat;

• 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.

• Once completely dry, put the coverslips on the hot plate preheated to 250 to 3002F.

• Leave coverslips on hot plate for 3-5 minutes. Verify that:

• Heat has driven nearly all water from the material on the coverslips; and

• Diatom frustules adhere to the surface of the glass.

• Remove the coverslips from the hotplate and inspect them. Consider:

• If the pattern of diatoms distributed on any coverslip is not even and smooth, discard and
prepare a new coverslip.

• If coverslip distributions are still unsatisfactory after repeated attempts, consult an algal analyst.

• Mount coverslip on slide in a POSITIVE-DRAW FUME HOOD sing the following steps.

• 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.

• Remove the appropriate coverslip from the aluminum plate with forceps, being careful to
handle the coverslip only at the extreme corners.

• 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.

• 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.

• Remove the slide from the hot plate.

• Gently position the coverslip using the rounded toothpicks.

• Bring the edges of the coverslip parallel to the edges of the microscope slide.

• 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.

• Set aside the mount to finish cooling.

• 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.

• 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.

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• Wipe the mount clean with a tissue (e.g., Kimwipe®).

• Add a paper label to slides before analyzing the slides.

• Mark the sample ID number on the side of the slide without a label with a diamond pencil.

• 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.

• Preserve and store cleaned material.

• Record the data elements described in Table 9.3. Comments should include information such as:

2. Observations about any interfering materials (sand, silt, etc.)

3. Rationale for using a sparsely populated slide.

4. Information on all burn mount attempts, successful or unsuccessful.

Table 9.3 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)

VIALJD

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

QC_TAXONOMIST

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.

EJECT_AMOUNT

numeric

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

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9.7 Analysis of Diatoms

The taxonomist should count 600 valves for any properly concentrated and prepared sample as
described in Section 9.6. 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.

9.7.1 Creation of Pre-Count Regional Voucher Flora and Develop Diatom Analyst
Schedule

The following section explains the process for creating a pre-count regional voucher flora. These
regional flora were developed during NRSA 2018-2019 and will be used for NRSA 2023/24 and future
NRSA surveys. This section can help understand how those voucher flora were developed and how
new taxa could be added to the existing regional voucher flora. New regional voucher flora do not
need to be Created for NRSA. This section also contains information on how to develop sample
analysis schedules for diatom analysts.

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:

5. The project lead taxonomist obtains light micrographs (LM) images from a minimum of
80% of the project prepared slides.

For common taxa images should be limited to 1-2, for example, Navicula rhynchocephala, Cocconeis
placentula.

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.

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.

Image filename should include slide number, date, and sequential number (e.g.
"15565a_12092016_01").

6. Once all images are captured, process images as a group using GIMP, Photoshop
or other image software. Rotate, crop and add a 10 |jm scale bar to each raw
image, then export as a jpeg file (Fig. 9.1)

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.

7. 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 100x, which allows users to measure cells directly on the
digital plate.

Use a template with the dimensions 17x25cm, 600ppi, greyscale, white background.

Note that it is easiest to import all the jpeg images at once, each as a layer labelled with their sample
identification information. Layers can be immediately locked together and scaled to lOOx. Large cells
may need to be scaled separately, for example, we capture some valves using the 60x objective.

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8. Sort images into their "best" morphological groups, or genera.

9. Provide a list of additional sequential provisional names on the plate (Fig. 9.2)

10. Once the regional voucher flora documents have been developed, then the
project lead taxonomist will develop an analysts schedule.

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).

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.

Figure 9.1 Example of light micrograph image after rotation, histogram, and scale bar processing. ^

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cri

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EUN07

r/i

^Gnu

E>fll3

E^N14

EUN16

EUN17

EUN18

EUN19

EUN20

EUN21

EUN22

EUN23

EUN24

EUN25

EUN26

EUN27

EUN28

EUN29

EUN30

EUN12 EUN13

Figure 9.2 Example plate of a regional voucher flora collected before diatom sampling counting has begun.

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9.7.2 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:

9. Verify that all necessary equipment is available. See Section 9.3.2 for recommended supplies.

10. Verify that the slides can be used for diatom analysis:

• Review comments from the subsampling, cleaning, and mounting steps for any deviations that
may affect the outcome of diatom analysis.

• Scan slides at low to medium magnification (lOOx to 450x) to confirm that diatoms are:
o Evenly distributed on the coverslip.

o Present at a density appropriate for efficient counting. At high magnification (lOOOx), each field

should have 5-10 diatoms,
o If diatoms on the slides are very sparse, refer to procedures in Section 9.6 for handling low-
density samples.

• 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).

11. Create the transect and count the diatoms as follows:

• 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.

e. If etching the slide before counting:

c. Etch at least three lines on the diatom slide.

d. 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.

f. If etching during the count:

• 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).

• Etch a circle around the last field counted in the first row and at the start and end of all other rows.

g. Verify that the etching is clearly visible so that another taxonomist can easily
locate the circles and lines.

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• Focus the first field under oil immersion.

• 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).

• For each species/taxon, count diatom valves along the transect using the following rules:

• One valve is:

• A complete valve; or

• 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).

• Avoid counting valves in any disrupted areas of the mount, particularly edges that have optical
aberrations.

• 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).

• Record the level of magnification used to view the diatoms.

• 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 9.7.1.

• On a bench sheet, record the transect end coordinates from the microscope stage.

12. Calculate the total transect length and width (units are mm) evaluated for the sample. Record as
TRANS_LENGTH and TRANS_WIDTH in the database.

13. Verify that the SUBSAMPLE_DCF value reflects all dilution and concentration of the subsample.
Make any necessary corrections to the value.

14. Record the required data elements in described in Section 9.7.3 (Table 9.4).

15. 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 2023/24. A current list of
diatom taxa can be found at the biodata website: http://aquatic.biodata.usgs.gov

9.7.3 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)

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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

Percent taxonomic disagreement (PTD): measure of taxonomic precision for diatoms comparing the
number of agreements (positive comparisons, compp0s) of the primary taxonomist and internal or
external QC taxonomists. In the following equation, N is the total number of organisms in the larger of
the two counts.

PTD =

comp

-*• F

: 100

Table 9.4 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.

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

O
I—
<
Q

108

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TRANS WIDTH

numeric

Width (mm) of transects counted

COUNT CENSORED

COUNT

text

numeric

Number of diatom valves

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

9.8 Data Entry

Table 9.2, and Table 9.3 identify the required data elements that the laboratories must provide to EPA.
Table 9.4 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 cv for each taxonomist is
submitted once to EPA's External QC Coordinator.

9.9 Sample and Record Retention

After the taxonomist analyzes the vials for a sample, retain the vials for possible selection into the
external quality control evaluation. When the external evaluation is complete, process the vials
containing the remaining acid-cleaned material for long-term storage. The lab technician shall:

11. Work under a fume hood to add two to four drops of 100% buffered formalin to
each vial.

88. Add glass beads to the vials to help preserve the diatoms.

89. Tightly cap the vials and seal them by immersing the top 1/3 of the vial in melted wax.

90. Transfer the vials to long-term storage.

The laboratory shall retain:

4 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.

91. 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.

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10.0 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 10.1). 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 10.1 Quality assurance measures for AFDM

Activity Evaluation/Acceptance Criteria Corrective Action

Demonstrate competency
for analyzing AFDM to meet
the performance measures

Demonstration of past
experience with AFDM samples
in applying the laboratory SOP in
achieving the method detection
limit

Check condition of sample
when it arrives.

Assign appropriate condition code identified in
Table 10.2.

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. If the
original test fails, then the retest
also must be conducted within
the holding time.

Perform test, but note reason for performing
test outside holding time. EPA expects that the
laboratory will exercise every effort to perform
tests before the holding time expires.

Maintain quality control
specifications from selected
method/SOP (that meets
the measurement data
quality objectives).

Data meet all QC specifications
in the selected method/SOP.

Maintain completeness

Completeness objective is 95%
for all parameters.

Contact the EPA Survey QA Lead immediately
if issues affect laboratory's ability to meet
completeness objective.

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Table 10.2 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

Sample is in good condition

Sample container is cracked

Sample or container is leaking

Sample label is missing

Sample not frozen

Other quality concerns, not identified
above

CONDITION
COMMENT

text

Comments about the condition of the sample.

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11.0 PERIPHYTON META-GENOMICS AND ANTIMICROIBAL ANALYSIS
(RESEARCH EFFORT)

Laboratory methods incorporated in an ORD Quality Assurance Project Plan.

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12.0 WATER CHEMISTRY AND CHLOROPHYLL A

12.1 Analytical Parameters

A total of 19 parameters are determined from each bulk water chemistry sample collected (Table 12.1).
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 12.1 Water chemistry: NRSA 2023 - 2024 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 (NO3-NO2)

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)

Hg/L

Sulfate (SO4)

mg/L

Chloride (CI)

mg/L

Nitrate (NOs)

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 (SiOz)

mg/L

Total Suspended Solids (TSS)

mg/L

True Color

PCU

Performance objective based on use of
visual estimation method

Chlorophyll a

]ug/L (in extract)

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12.2 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 Table 2.1).

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 NRSAteam 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.

12.2.1 Water Chemistry Samples

Figure 12. 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 12.2) to the sample in the aliquot container. Cap tightly and
invert the bottle several times to mix.

5. Store all aliquots except the cation aliquot (filtered, acidified with HN03) in a refrigerator at 4°C.

Table 12.2 Water chemistry: acid preservatives added for various analytes

H2SC>4

HNO3

DOC

NHs-N

Total N

Total P

NO2-NO3

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Sample Receipt

4 L Bulk Sample
Inspect samples and complete
tracking form

Process Sample

within 24 hours

Filtration

Not Filtered

HDPE

bottle
Acid
washed
Preserve
with HN00

i—.

HDPE bottle
Not acid
washed
Store at 4 °C
in darkness

* ~

HDPE bottle

Acid washed
Preserve
with H2SO4

Analyses

Calcium (180

days)

Magnesium

(180 days)

Sodium (180

days)

Potassium

(180 days)

Analyses

Chloride (28
days)

Nitrate (7 days)
Sulfate (28
days)

Silica (28 days)
Nitrate-Nitrite
(IC) (7 days)
True Color (3
days)

_T_.

HDPE

I HDPE

bottle

i bottle

Acid

I Not acid

washed

Preserve

Store at 4

with

J °C in

\ _ /

f-J

Analyses

Ammonia-N (28 days)
Dissolved Organic
Carbon (28 days)
Nitrate-Nitrite (with FIA)
(28 days)

Analyses

Total Phosphorus
(28 days)

Total Nitrogen (28
days)

Analyses

pH (3 days)
ANC (7 days)
Conductivity
(7 days)
TSS (7 days)
Turbidity (3
days)

Figure 12.1 Water chemistry: sample processing procedures

12.2.2 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.

12.3 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 is equal to or above 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

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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 in past surveys (EPA ORD-Corvallis) are summarized in Table 12.3. 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 12.4.
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 2023 - 2024 QA files by the EPA Laboratory Review Coordinator.

Table 12.3 Water chemistry: NRSA 2023 - 2024 analytical methods (PESD Analytical Laboratory, EPA ORD-
Corvallis)

Analyte

Summary of Method*

References®

PESD-AL SOPh

pH (lab)

Automated, using ManSci PC-Titrate w/Titra-Sip
autotitrator and Ross combination pH electrode.
Initial pH determination for ANC titration

EPA 150.1 (modified), APHA
4500H

PESD-AL 16A.2

Specific conductance
(conductivity) @ 25°C

Electrolytic, Man-Tech TitraSip automated analysis
OR manual analysis, electrolytic

EPA 120.1, APHA 2510

PESD-AL 16A.2
PESD-AL 11A.5

Acid neutralizing
capacity (ANC)

Automated acidimetric titration to pH<3.5, with
modified Gran plot analysis

EPA 310.1, APHA 2320

PESD-AL 16A.2

Turbidity

Nephelometric; Man-Tech TitraSip automated

analysis,

Manual analysis using Hach turbidimeter (high
turbidity samples)

EPA 180, APHA 2130

PESD-AL 16A.2
PESD-AL 13A.4

Total suspended solids
(TSS)

Gravimetric, dried at 104 °C

EPA 160.2; APHA 2540D

PESD-AL 14B.4

True color (Hach Kit)

Visual comparison to calibrated glass color disk.

EPA 110.2 (modified), APHA 2120

PESD-AL 15A.4

f FIA=Flow injection analysis. AAS=Atomic Absorption Spectrometry

g 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.

h PESD-AL = Pacific Ecological System Division - Analytical Laboratory. References are to laboratory SOP being used
at central laboratory. Available upon request, (contact the Project Lead)

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Analyte

Summary of Method*

References®

PESD-AL SOPh

Dissolved Organic
Carbon (DOC)'

UV promoted persulfate oxidation to CO2 with
infrared detection

APHA 5310-C, EPA 415.3

PESD-AL 21A.5

Nitrate+Nitrite, as N
(fresh waters)

Ion Chromatography
OR

FIA automated colorimetric (cadmium reduction)

EPA 300.1; SW-846 9056A; APHA
4110B

EPA 353.2

APHA 4500-N03-N-E
Lachat 10-107-04-1-C

PESD-AL 36A.2
PESD-AL 40A.7

Nitrate

Measured as part of Nitrate-Nitrite when using IC or
calculated after measuring Nitrate+Nitrite using FIA.

See above

Ammonia, as N (fresh
waters)

FIA automated colorimetric (salicylate,
dichloroisocyanurate)

Lachat 10-107-06-3-D

PESD-AL 30A.5

Silica, dissolved (SiOz)
Fresh waters

FIA automated colorimetric (molybdate, stannous
chloride)

EPA366.0, APHA 4500-SiO2 F
Lachat 10-114-27-1-B

PESD-AL 32A.6

Total nitrogen (TN)

Persulfate Digestion; FIA Automated Colorimetric
Analysis (Cadmium Reduction, sulfanilamide)

EPA353.2 (modified)

APHA 4500-N-C (modified)
ASTM WK31786

PESD-AL 34A.6

Total phosphorus (TP)

Persulfate Digestion; Automated Colorimetric
Analysis (molybdate, ascorbic acid)

EPA 365.1 (modified), APHA
4500-P-E
USGS 1-4650-03
Lachat 115-01-1-B (modified)

PESD-AL 34A.6

Major anions,
dissolved
chloride, nitrate,
nitrite, sulfate

Ion Chromatography

EPA 300.1; SW-846 9056A; APHA
4110B

PESD-AL 40A.7

Major cations,
dissolved
calcium, sodium,
potassium,
magnesium

Inductively-coupled Plasma Atomic Emission
Spectroscopy (ICP-AES)

Flame AAS

EPA 200.7; EPA 6010D

PESD-AL 52A.1

Chlorophyll-a
(Chl-a)

Extraction 90% acetone analysis by fluorometry

EPA 445.0, EPA 446.0

PESD-AL 71A.5

12.4 Pertinent QA/QC Procedures <

A single analytical laboratory and some state laboratories will analyze the water chemistry samples. The ^

specific quality control procedures used by each laboratory are implemented to ensure that: i

1. Objectives established for various data quality indicators being met §

2. Results are consistent and comparable among all participating labs. S

The analytical laboratory demonstrated in previous studies that it can meet the required LRL (USEPA -a

2004). QA/QC procedures outlined in this manual and the NRSA 2023 - 2024 QAPP will be followed to S

ensure these LRLs are met for the NRSA 2023 - 2024. ^

' For DOC, "dissolved" is defined as that portion passing through a 0.45 urn nominal pore size filter. For other ^

analytes, "dissolved" is defined as that portion passing through a 0.4 urn pore size filter (Nucleopore or 0

equivalent).

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12.4.1 Laboratory Performance Requirements

Table 12.4 summarizes the pertinent laboratory performance requirements for the water chemistry and
chlorophyll-a indicators.

12.4.2 Laboratory Quality Control Samples

Table 12.5 summarizes the pertinent laboratory quality control samples for the water chemistry and
chlorophyll-a indicators.

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Table 12.4 Water chemistry & chlorophyll-a: laboratory method performance requirements

Potential

Lower

Range of

Reporting

Transition

Precision

Bias

Analyte

Units

Samples'

Limitk

Value1

Objective"1

Objective"

Conductivity

mS/cm at 25°C

1 to 77,000

2.0

± 1 or ±10%

± 1 or 5%

pH (laboratory)

Std Units

3.5 to 10

N/A

5.75, 8.25

>5.75 or
<8.25= ±0.15
<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

± 1 or ±10%

±1 or ±10%

Acid

Neutralizing
Capacity (ANC)

M-eq/L
(1 mg/Las
CaCO3=20 neq/L

-300 to
+75,000
(-16 to
3,750 mg as
CaC03)

N/A

± 5 or ±10%

Dissolved
Organic Carbon
(DOC)

mg/L

0.1 to 109

0.20

±0.10 or
±10%

J 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.

kThe 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. d

1 Value at which performance objectives for precision and bias switch from absolute (< transition value) to relative S

> 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. 9

m 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 range. For standard samples, precision is estimated as the standard deviation of repeated ^

measurements across batches at the lower concentration range, and as percent relative standard deviation of ^

repeated measurements across batches at the higher concentration range. ^

For pH precision, the looser criteria apply to more highly alkaline samples. For NRSA, that is less of a concern than jj

the ability to measure acidic samples accurately and precisely. g

I—

" 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 0
sample batches, and as the percent difference at the higher concentration range.

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Potential

Lower

Range of

Reporting

Transition

Precision

Bias

Analyte

Units

Samples*

Limitk

Value1

Objective"1

Objective"

Ammonia as
N(NHb-N)

mg/L

Oto 17

0.02

0.10

±0.01 or
±10%

Nitrate-Nitrite
(NO3-NO2)

mg/L

0 to 360 (as
nitrate)

0.02

0.10

±0.01 or
±10%

Total Nitrogen
(TN)

mg/L

0.1 to 90

0.02

0.10

±0.01 or
±10%

Total

Phosphorus
(TP)

Hg/L

0 to 22,000

± 2 or±10%

± 2 or ±10%

Sulfate (SO4)

mg/L

0 to 33,000

0.50

2.5

±0.25 or
±10%

Chloride (CI)

mg/L

0 to 19,000

0.20

±0.10 or
±10%

Nitrate (NOs)

mg/L

0 to 360

0.02

0.1

±0.01 or
±10%

± 0.01 ±10%

Calcium (Ca)

mg/L

0.04 to
5,000

0.10

0.5

± 0.05 or
±10%

±0.05 or
±10%

Magnesium
(Mg)

mg/L

0-5000

0.10

0.5

± 0.05 or
±10%

±0.05 or
±10%

Sodium (Na)

mg/L

0-22,500

0.10

0.5

± 0.05 or
±10%

±0.05 or
±10%

Potassium (K)

mgL

0-170

0.10

0.5

± 0.05 or
±10%

±0.05 or
±10%

Silica (SiOz)

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

±1 or ±10%

± 1 or ±10%

True Color

PCU

0 to 1000

±5 or ±10%

Chlorophyll a

mg/L (in extract)

0.7 to
11,000

0.5

± 1.5 or ±10%

± 1.5 or
±10%

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Table 12.5 Water chemistry: quality control - laboratory samples

QC Sample

Type and

Acceptance

Description Analytes

Description

Frequency

Criteria

Corrective Action

Laboratory/

All except TSS

Once per

Control limits

Prepare and analyze new

Reagent

(ForTSS, the lab

analytical

< LRL

blank. Determine and

Blank

will filter a

baatch prior

correct problem (e.g.,

known volume

to sample

reagent contamination,

of reagent water

analysis

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

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QC Sample

Type and

Acceptance

Description Analytes

Description

Frequency

Criteria

Corrective Action

Analytical
Duplicate
Sample

All analyses

One per 10
samples

Within

precision

objective

If results are below LRL:
Prepare and analyze
duplicate from different
sample (volume
permitting). Review
precision of batch. Check
preparation of duplicate
sample.

Standard
Reference
Material
(SRM)

When available
for a particular
analyte

One analysis
in a

minimum of
five separate
batches

Manufacturers
certified range

Analyze standard in next
batch to confirm
suspected imprecision or
bias. Evaluate calibration
and QCCS solutions and
standards for
contamination and
preparation error. Correct
before any further
analyses of routine
samples are conducted.
Reestablish control by
three successive
reference standard
measurements that are
acceptable. Qualify all
sample batches analyzed
since the last acceptable
reference standard
measurement for possible
reanalysis.

Matrix

Spike

Samples

Only prepared
when samples
with potential
for matrix
interferences are
encountered

One per
batch

Control limits
for recovery
cannot exceed
100±20%

Select two additional
samples and prepare
fortified subsamples.
Reanalyze all suspected
samples in batch by the
method of standard
additions. Prepare three
subsamples (unfortified,
fortified with solution
approximately equal to
the endogenous
concentration, and
fortified with solution
approximately twice the
endogenous
concentration).

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12.4.3 Data Reporting, Review, and Management

Checks made of the data in the process of review and verification are summarized in Table 12.6. Data
reporting units and significant figures are given in Table 12.7. The NRSA 2023 - 2024 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 12.6 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:

1. If total ionic strength <100 |aeq/L
a. %IBD < ±25%.

2. If total ionic strength > 100 |aeq/L
a. %IBD <±10%.

Corrective actions:

1. Determine which analytes, if any, are the largest contributors to
the ion imbalance. Review suspect analytes for analytical error
and reanalyze.

a. Flag = unacceptable %IBD

2. If analytical error is not indicated, qualify sample to attribute
imbalance to unmeasured ions. Reanalysis is not required.

a. 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:

1. If measured conductivity < 25 |^S/cm,

a. ([measured - calculated] -r- measured) < ±25%.

2. If measured conductivity > 25 |^S/cm,

a. ([measured - calculated] -r- measured) < ±15%.

Corrective Actions:

1. Determine which analytes, if any, are the largest contributors to
the difference between calculated and measured conductivity.

2. Review suspect analytes for analytical error and reanalyze.

3. 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 12.7 Water chemistry: data reporting criteria

No. Significant

Minimum No.

Measurement

Units

Figures

Decimal Places

mg/L

Temperature

°C

pH units

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No. Significant

Minimum No.

Measurement

Units

Figures

Decimal Places

Carbon, total & dissolved organic

mg/L

ANC

M-eq/L

Conductivity

|j.S/cm at 25 °C

Calcium, magnesium, sodium, potassium,
chloride, nitrate, and sulfate

M-eq/L

Silica

mg/L

Total phosphorus

M-g/L

Total nitrogen

mg/L

Nitrate-Nitrite

mg/L

Ammonia-N

mg/L

Turbidity

NTU

True color

PCU

TSS

mg/L

Chlorophyli-a

ug/l

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) - A N("
ANC + anions + ^ cations + 2\h j

%IBD =

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 12.8. For the conductivity check, equivalent conductivities for major ions are
presented in Table 12.9.

Table 12.8 Water chemistry: constants for converting major ion concentration from mg/L to |ieq/L

Analyte

Conversion from mg/L to |J.eq/L0

Calcium

49.9

Magnesium

82.3

Potassium

25.6

Sodium

43.5

Ammonia-N

71.39

° 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.

>
=n

O
cc
O

~o
c

>
cc

q
r\i

124

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Ammonium

55.4

Chloride

28.2

Nitrate-N

71.39

Nitrate

16.1

Sulfate

20.8

Table 12.9 Water chemistry: factors to calculate equivalent conductivities of major ions.p

Ion

Equivalent Conductance per mg/L
(|4,S/cm at 25 °C)

Ion

Equivalent Conductance per
mg/L (|iS/cm at 25 °C)

Calcium

2.60

Nitrate

1.15

Magnesium

3.82

Sulfate

1.54

Potassium

1.84

Hydrogen

3.5 x 105 "

Sodium

2.13

Hydroxide

1.92 x 105

Ammonium

4.13

Bicarbonate

0.715

Chloride

2.14

Carbonate

2.82

12.5 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

p From Hillman et al. (1987).

q Specific conductance per mole/L, rather than per mg/L.

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APPENDIX A: LABORATORY REMOTE EVALUATION FORMS

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NRSA 2023/24 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 2023/24, 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 2023/24.

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 2023/24:

~ 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 2023/24.

~ 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 2023/24 LOM).

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~ Documentation attesting to experience running all analytes for the NRSA 2023/24, 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).

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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) 2023/24.

1. Utilize procedures identified in the NRSA 2023/24 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 2023/24 Quality Assurance Project Plan (QAPP) and
related Standard Operating Procedures (SOPs).

3. Have an organized IT tracking system in place for recording sample tracking and
analysis data.

4. Provide Quality Control (QC) data for internal QC check, on a quarterly basis.

5. Provide data using the template provided on the NARS Sharepoint.

6. Provide data results in a timely manner. This will vary with the type of analysis
and the number of samples to be processed. Sample data must be received no
later than March 1, 2024 (for samples collected in 2023) and March 1, 2025 (for
samples collected in 2024) 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

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NRSA 2023/24 Document Request Form - Biology Labs

As part of the NRSA 2023/24, 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 2023/24.

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 2023/24:

~ 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 2023/24.

~ 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:

~ Documentation showing participation in previous NARS for this particular indicator.

Additionally, we request that all labs provide the following information in support of your capabilities,
(these materials are required if neither of the two items above are provided):

~ A copy of your Laboratory's accreditations and certifications if applicable (i.e. NELAC, ISO, state
certifications, NABS, etc.).

~ Documentation of NABS (or other) certification for the taxonomists performing analyses (if
applicable).

~ 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 2023/24 LOM).

~ Resume of independent fish taxonomist who will verify fish vouchers (if applicable)

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This documentation may be submitted electronically via e-mail to forde.kendraffiepa.gov with a cc:
tomitchell.richardffiepa.gov and lehmann.sarah@epa.gov. Questions concerning this request can be
submitted forde.kendraffiepa.gov (202-566-0417) or mitchell.richardffiepa.gov (202-566-0644).

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Lab Signature Form - Biology Labs

certify that the

lab

located in

j will abide by the following standards in

performing biology data analysis and reporting for the National Rivers and Streams
Assessment (NRSA) 2023/24.

9. Utilize procedures identified in the NRSA 2023/24 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 2023/24 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 2023/24 Laboratory Operations
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 Sharepoint.

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 March 1, 2024 (for samples collected in 2023) and March 1, 2025 (for
samples collected in 2024) 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
2023/24.

This applies to the

biological indicator.

Signature

Date

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APPENDIX B: SAMPLE LABORATORY FORMS

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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#)

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

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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
Uniqueldentifier
)

Taxon

(see target taxon
in Table 3)

Distinct
(Y/N)

Counts of Organisms in the
Taxon:

Cumulative
Number of
Organisms
in Sample

Data
Qualifier
(Codes
in Table
4)

Is cumulative number of organisms > 500? If not, unless the sample was completed sorted, obtain
more sorted sample.

Comments:

135

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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

136

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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

137

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APPENDIX C: OTHER PERTINENT ATTACHMENTS

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Table D.0.1 Fish identification: standard common and scientific names

LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME

Perciformes

Cichlidae

Hemichromis letourneuxi

african jewelfish

Percopsiformes

Amblyopsidae

Speoplatyrhinus poulsoni

alabama cavefish

Perciformes

Percidae

Etheostoma ramseyi

alabama darter

Cypriniformes

Catostomidae

Hypentelium etowanum

alabama hog sucker

Clupeiformes

Clupeidae

Alosa alabamae

alabama shad

Cypriniformes

Cyprinidae

Cyprinella callistia

alabama shiner

Acipenseriformes

Acipenseridae

Scaphirhynchus suttkusi

alabama sturgeon

Esociformes

Umbridae

Dallia pectoralis

alaska blackfish

Clupeiformes

Clupeidae

Alosa pseudoharengus

alewife

Lepisosteiformes

Lepisosteidae

Atractosteus spatula

alligatorgar

Cypriniformes

Cyprinidae

Cyprinella xaenura

altamaha shiner

Cypriniformes

Cyprinidae

Gila alvordensis

alvord chub

Cyprinodontiformes

Cyprinodontidae

Cyprinodon nevadensis

amargosa pupfish

Cyprinodontiformes

Poeciliidae

Poecilia formosa

amazon molly

Siluriformes

Loricariidae

Pterygoplichthys pardalis

amazon sailfin catfish

Perciformes

Percidae

Percina antesella

amber darter

Petromyzontiformes

Petromyzontidae

Lampetra appendix

american brook lamprey

Anguilliformes

Angu illidae

Anguilla rostrata

american eel

Clupeiformes

Clupeidae

Alosa sapidissima

american shad

Cyprinodontiformes

Poeciliidae

Gambusia amistadensis

amistad gambusia

Cypriniformes

Cyprinidae

Pteronotropis grandipinnis

apalachee shiner

Perciformes

Percidae

Percina gymnocephala

appalachia darter

Salmoniformes

Salmonidae

Salvelinus alpinus

arctic char

Salmoniformes

Salmonidae

Coregonus autumnalis

arctic cisco

Salmoniformes

Salmonidae

Thymallus arcticus

arctic grayling

Petromyzontiformes

Petromyzontidae

Lampetra camtschatica

arctic lamprey

Perciformes

Percidae

Etheostoma cragini

arkansas darter

Cypriniformes

Cyprinidae

Notropis girardi

arkansas river shiner

Perciformes

Percidae

Etheostoma euzonum

arkansas saddled darter

Perciformes

Percidae

Etheostoma sagitta

arrow darter

Perciformes

Gobiidae

Clevelandia ios

arrow goby

Cypriniformes

Cyprinidae

Gila orcuttii

arroyo chub

Cyprinodontiformes

Goodeidae

Empetrichthys merriami

ash meadows poolfish

Perciformes

Percidae

Etheostoma cinereum

ashy darter

Syn branch iformes

Synbranchidae

Monopterus albus

asian swamp eel

Perciformes

Sciaenidae

Micropogonias undulatus

atlantic croaker

Belon iformes

Belonidae

Strongylura marina

atlantic needlefish

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LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME

Salmoniformes

Salmonidae

Salmo salar

atlantic salmon

Myliobatiformes

Dasyatidae

Dasyatis sabina

atlantic stingray

Acipenseriformes

Acipenseridae

Acipenser oxyrinchus

atlantic sturgeon

Clupeiformes

Clupeidae

Opisthonema oglinum

atlantic thread herring

Gadiformes

Gadidae

Microgadus tomcod

atlantic tomcod

Perciformes

Percidae

Etheostoma zonifer

backwater darter

Perciformes

Sciaenidae

Bairdiella icistia

bairdiella

Perciformes

Cichlidae

Herosseverus

banded cichlid

Perciformes

Percidae

Etheostoma zonale

banded darter

Cyprinodontiformes

Fundulidae

Fundulus diaphanus

banded killifish

Perciformes

Elassomatidae

Elassoma zonatum

banded pygmy sunfish

Scorpaeniformes

Cottidae

Cottus carolinae

banded sculpin

Perciformes

Centrarchidae

Enneacanthus obesus

banded sunfish

Cyprinodontiformes

Fundulidae

Fundulus cingulatus

banded topminnow

Perciformes

Percidae

Etheostoma zonistium

bandfin darter

Cypriniformes

Cyprinidae

Luxilus zonistius

bandfin shiner

Cypriniformes

Cyprinidae

Cyprinella leedsi

bannerfin shiner

Perciformes

Centrarchidae

Lepomis symmetricus

bantam sunfish

Perciformes

Percidae

Etheostoma obeyense

barcheek darter

Perciformes

Percidae

Etheostoma forbesi

barrens darter

Cyprinodontiformes

Fundulidae

Fundulus julisia

barrens topminnow

Clupeiformes

Engraulidae

Anchoa mitchilli

bay anchovy

Pleuronectiformes

Paralichthyidae

Citharichthys spilopterus

bay whiff

Perciformes

Percidae

Etheostoma rubrum

bayou darter

Cyprinodontiformes

Fundulidae

Fundulus pulvereus

bayou killifish

Cyprinodontiformes

Fundulidae

Fundulus nottii

bayou topminnow

Scorpaeniformes

Cottidae

Cottus extensus

bear lake sculpin

Salmoniformes

Salmonidae

Prosopium abyssicola

bear lake whitefish

Cypriniformes

Cyprinidae

Cyprinella formosa

beautiful shiner

Cypriniformes

Cyprinidae

Notropis rupestris

bedrock shiner

Salmoniformes

Salmonidae

Coregonus laurettae

bering cisco

Cyprinodontiformes

Poeciliidae

Gambusia gaigei

big bend gambusia

Cypriniformes

Cyprinidae

Hybopsis amblops

bigeye chub

Cypriniformes

Catostomidae

Moxostoma ariommum

bigeye jumprock

Cypriniformes

Cyprinidae

Notropis boops

bigeye shiner

Cypriniformes

Cyprinidae

Hypophthalmichthys nobilis

bighead carp

Cypriniformes

Catostomidae

Ictiobus cyprinellus

bigmouth buffalo

Cypriniformes

Cyprinidae

Nocomis platyrhynchus

bigmouth chub

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LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME

Cypriniformes

Cyprinidae

Notropis dorsalis

bigmouth shiner

Perciformes

Eleotridae

Gobiomorus dormitor

bigmouth sleeper

Perciformes

Percidae

Percina macrolepida

bigscale logperch

Cypriniformes

Cyprinidae

Rhodeus sericeus

bitterling

Perciformes

Cichlidae

Cichlasoma bimaculatum

black acara

Cypriniformes

Catostomidae

Ictiobus niger

black buffalo

Siluriformes

Ictaluridae

Ameiurus melas

black bullhead

Perciformes

Centrarchidae

Pomoxis nigromaculatus

black crappie

Siluriformes

Ictaluridae

Noturus funebris

black madtom

Cypriniformes

Catostomidae

Moxostoma duquesnei

black redhorse

Scorpaeniformes

Cottidae

Cottus baileyi

black sculpin

Perciformes

Percidae

Percina nigrofasciata

blackbanded darter

Perciformes

Centrarchidae

Enneacanthus chaetodon

blackbanded sunfish

Cypriniformes

Cyprinidae

Notropis heterodon

blackchin shiner

Perciformes

Cichlidae

Sarotherodon melanotheron

blackchin tilapia

Salmoniformes

Salmonidae

Coregonus nigripinnis

blackfin cisco

Perciformes

Percidae

Etheostoma nigripinne

blackfin darter

Cypriniformes

Catostomidae

Thoburnia atripinnis

blackfin sucker

Cypriniformes

Cyprinidae

Notropis melanostomus

blackmouth shiner

Cypriniformes

Cyprinidae

Notropis heterolepis

blacknose shiner

Cypriniformes

Cyprinidae

Phoxinus cumberlandensis

blackside dace

Perciformes

Percidae

Percina maculata

blackside darter

Perciformes

Percidae

Etheostoma duryi

blackside snubnose darter

Cypriniformes

Cyprinidae

Notropis atrocaudalis

blackspot shiner

100

Cyprinodontiformes

Fundulidae

Fundulus olivaceus

blackspotted topminnow

101

Cyprinodontiformes

Fundulidae

Fundulus notatus

blackstripe topminnow

102

Cypriniformes

Catostomidae

Moxostoma poecilurum

blacktail redhorse

103

Cypriniformes

Cyprinidae

Cyprineiia venusta

blacktail shiner

104

Cypriniformes

Catostomidae

Moxostoma cervinum

blacktip jumprock

105

Cypriniformes

Cyprinidae

Lythrurus atrapiculus

blacktip shiner

106

Cypriniformes

Cyprinidae

Luxilus zonatus

bleeding shiner

107

Perciformes

Percidae

Etheostoma blennius

blenny darter

108

Salmoniformes

Salmonidae

Coregonus hoyi

bloater

109

Perciformes

Percidae

Etheostoma sanguifluum

bloodfin darter

110

Perciformes

Gobiidae

Ctenogobius fasciatus

blotchcheekgoby

111

Cypriniformes

Cyprinidae

Erimystax insignis

blotched chub

112

Cyprinodontiformes

Poeciliidae

Gambusia senilis

blotched gambusia

113

Perciformes

Percidae

Percina burtoni

blotchside logperch

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114

Siluriformes

Ictaluridae

Ictalurus furcatus

blue catfish

115

Cypriniformes

Cyprinidae

Gila coerulea

blue chub

116

Scorpaeniformes

Cottidae

Cottus caeruleomentum

blue ridge sculpin

117

Cypriniformes

Cyprinidae

Cyprinella caerulea

blue shiner

118

Cypriniformes

Catostomidae

Cycleptus elongatus

blue sucker

119

Perciformes

Cichlidae

Oreochromis aureus

bluetilapia

120

Clupeiformes

Clupeidae

Alosa 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 hankinsoni

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

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152

Perciformes

Percidae

Percina palmaris

bronze darter

153

Perciformes

Percidae

Etheostoma burri

brook darter

154

Atheriniformes

Atherinopsidae

Labidesthes sicculus

brook silverside

155

Gasterosteiformes

Gasterosteidae

Culaea inconstans

brook stickleback

156

Salmoniformes

Salmonidae

Salvelinus fontinalis

brook trout

157

Siluriformes

Ictaluridae

Ameiurus nebulosus

brown bullhead

158

Perciformes

Percidae

Etheostoma edwini

brown darter

159

Siluriformes

Callichthyidae

Hoplosternum littorale

brown hoplo

160

Siluriformes

Ictaluridae

Noturus phaeus

brown madtom

161

Salmoniformes

Salmonidae

Salmo trutta

brown trout

162

Perciformes

Percidae

Etheostoma bison

buffalo darter

163

Cypriniformes

Cyprinidae

Nocomis raneyi

bull chub

164

Carcharhiniformes

Carcharhinidae

Carcharhinus leucas

bull shark

165

Salmoniformes

Salmonidae

Salvelinus confluentus

bull trout

166

Cypriniformes

Cyprinidae

Pimephales vigilax

bullhead minnow

167

Perciformes

Channidae

Channa marulius

bullseye snakehead

168

Gadiformes

Gadidae

Lota lota

burbot

169

Cypriniformes

Cyprinidae

Macrhybopsis marconis

burrhead chub

170

Cypriniformes

Cyprinidae

Notropis asperifrons

burrhead shiner

171

Perciformes

Cichlidae

Cichla ocellaris

butterfly peacock bass

172

Siluriformes

Ictaluridae

Noturus taylori

caddo madtom

173

Cypriniformes

Cyprinidae

Notropis cahabae

cahaba shiner

174

Cypriniformes

Cyprinidae

Hesperoleucus symmetricus

California roach

175

Perciformes

Percidae

Etheostoma osburni

candy darter

176

Cypriniformes

Cyprinidae

Notropis mekistocholas

cape fear shiner

177

Cypriniformes

Cyprinidae

Luxilus cardinalis

cardinal shiner

178

Cypriniformes

Cyprinidae

Notropis percobromus

carmine shiner

179

Perciformes

Percidae

Etheostoma collis

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

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190

Perciformes

Percidae

Etheostoma scotti

Cherokee darter

191

Perciformes

Percidae

Etheostoma etnieri

cherry darter

192

Cypriniformes

Cyprinidae

Lythrurus roseipinnis

cherryfin shiner

193

Petromyzontiformes

Petromyzontidae

Ichthyomyzon castaneus

chestnut lamprey

194

Perciformes

Percidae

Etheostoma cervus

chickasaw darter

195

Cypriniformes

Cyprinidae

Gila nigrescens

chihuahua chub

196

Cypriniformes

Cyprinidae

Notropis chihuahua

chihuahua shiner

197

Salmoniformes

Salmonidae

Oncorhynchus tshawytscha

chinooksalmon

198

Cypriniformes

Cyprinidae

Acrocheilus alutaceus

chiselmouth

199

Perciformes

Percidae

Etheostoma davisoni

choctawhatchee darter

200

Perciformes

Percidae

Etheostoma hopkinsi

Christmas darter

201

Cypriniformes

Cyprinidae

Notropis potteri

chub shiner

202

Salmoniformes

Salmonidae

Oncorhynchus keta

chum salmon

203

Salmoniformes

Salmonidae

Coregonus artedi

cisco

204

Cypriniformes

Cyprinidae

Hybopsis winchelli

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

Chitala ornata

clown knifefish

209

Perciformes

Percidae

Percina brevicauda

coal darter

210

Perciformes

Percidae

Etheostoma colorosum

coastal darter

211

Cypriniformes

Cyprinidae

Notropis petersoni

coastal shiner

212

Scorpaeniformes

Cottidae

Cottus aleuticus

coastrange sculpin

213

Salmoniformes

Salmonidae

Oncorhynchus kisutch

coho salmon

214

Perciformes

Percidae

Etheostoma ditrema

coldwater darter

215

Cypriniformes

Cyprinidae

Ptychocheilus lucius

Colorado pikeminnow

216

Scorpaeniformes

Cottidae

Cottus hubbsi

Columbia sculpin

217

Cyprinodontiformes

Cyprinodontidae

Cyprinodon elegans

comanche springs pupfish

218

Cypriniformes

Cyprinidae

Notropis amoenus

comely shiner

219

Cypriniformes

Cyprinidae

Cyprinus carpio

common carp

220

Cypriniformes

Cyprinidae

Luxilus cornutus

common shiner

221

Perciformes

Centropomidae

Centropomus undecimalis

common snook

222

Perciformes

Percidae

Percina jenkinsi

conasauga logperch

223

Cyprinodontiformes

Cyprinodontidae

Cyprinodon eximius

conchos pupfish

224

Perciformes

Cichlidae

Cichlasoma nigrofasciatum

convict cichlid

225

Perciformes

Percidae

Etheostoma coosae

coosa darter

226

Cypriniformes

Cyprinidae

Notropis xaenocephalus

coosa shiner

227

Perciformes

Percidae

Etheostoma aquali

coppercheek darter

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Perciformes

Percidae

Etheostoma basilare

corrugated darter

229

Cypriniformes

Cyprinidae

Semotilus atromaculatus

creek chub

230

Cypriniformes

Catostomidae

Erimyzon oblongus

creek chubsucker

231

Perciformes

Percidae

Etheostoma collettei

Creole darter

232

Cypriniformes

Cyprinidae

Luxilus cerasinus

crescent shiner

233

Perciformes

Gobiidae

Lophogobius cyprinoides

crested goby

234

Perciformes

Belontiidae

Trichopsis vittata

croaking gourami

235

Perciformes

Percidae

Etheostoma corona

crown darter

236

Perciformes

Percidae

Crystallaria asprella

crystal darter

237

Cypriniformes

Catostomidae

Chasmistes cujus

cui-ui

238

Perciformes

Percidae

Etheostoma susanae

Cumberland darter

239

Perciformes

Percidae

Etheostoma uniporum

current darter

240

Cypriniformes

Cyprinidae

Exoglossum maxillingua

cutlip minnow

241

Salmoniformes

Salmonidae

Oncorhynchus clarkii

cutthroat trout

242

Perciformes

Percidae

Etheostoma proeliare

cypress darter

243

Cypriniformes

Cyprinidae

Hybognathus hayi

cypress minnow

244

Perciformes

Gobiidae

Ctenogobius boleosoma

darter goby

245

Salmoniformes

Salmonidae

Coregonus johannae

deepwater cisco

246

Scorpaeniformes

Cottidae

Myoxocephalus thompsonii

deepwatersculpin

247

Salmoniformes

Osmeridae

Hypomesus transpacificus

delta smelt

248

Cypriniformes

Cyprinidae

Eremichthys acros

desert dace

249

Cyprinodontiformes

Cyprinodontidae

Cyprinodon macularius

desert pupfish

250

Cypriniformes

Catostomidae

Catostomus clarkii

desert sucker

251

Cyprinodontiformes

Cyprinodontidae

Cyprinodon diabolis

devils hole pupfish

252

Cypriniformes

Cyprinidae

Dionda diaboli

devils river minnow

253

Cypriniformes

Cyprinidae

Semotilus thoreauianus

dixie chub

254

Perciformes

Centrarchidae

Lepomis marginatus

dollar sunfish

255

Salmoniformes

Salmonidae

Salvelinus malma

dolly varden

256

Perciformes

Percidae

Percina sciera

dusky darter

257

Cypriniformes

Cyprinidae

Notropis cummingsae

dusky shiner

258

Cypriniformes

Cyprinidae

Luxilus pilsbryi

duskystripe shiner

259

Perciformes

Percidae

Etheostoma percnurum

duskytail darter

260

Cypriniformes

Cyprinidae

Rhinichthys atratulus

eastern blacknose dace

261

Cyprinodontiformes

Poeciliidae

Gambusia holbrooki

eastern mosquitofish

262

Esociformes

Umbridae

Umbra pygmaea

eastern mudminnow

263

Perciformes

Percidae

Ammocrypta pellucida

eastern sand darter

264

Cypriniformes

Cyprinidae

Hybognathus regius

eastern silvery minnow

265

Perciformes

Percidae

Etheostoma pseudovulatum

egg-mimic darter

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Siluriformes

Ictaluridae

Noturus elegans

elegant madtom

267

Perciformes

Percidae

Etheostoma baileyi

emerald darter

268

Cypriniformes

Cyprinidae

Notropis atherinoides

emerald shiner

269

Gasterosteiformes

Gasterosteidae

Gasterosteus aculeatus

espinocho

270

Perciformes

Percidae

Etheostoma etowahae

etowah darter

271

Salmoniformes

Osmeridae

Thaleichthys pacificus

eulachon

272

Perciformes

Elassomatidae

Elassoma evergladei

everglades pygmy sunfish

273

Cypriniformes

Cyprinidae

Semotilus corporalis

fallfish

274

Perciformes

Percidae

Etheostoma flabellare

fantail darter

275

Perciformes

Eleotridae

Dormitator maculatus

fat sleeper

276

Cypriniformes

Cyprinidae

Pimephales promelas

fathead minnow

277

Cypriniformes

Cyprinidae

Phenacobius crassilabrum

fatlips minnow

278

Cypriniformes

Cyprinidae

Cyprinella pyrrhomelas

fieryblack shiner

279

Cypriniformes

Cyprinidae

Phoxinus neogaeus

finescale dace

280

Perciformes

Percidae

Etheostoma pyrrhogaster

firebelly darter

281

Perciformes

Cichlidae

Cichlasoma meeki

firemouth cichlid

282

Cypriniformes

Cyprinidae

Pteronotropis signipinnis

flagfin shiner

283

Cyprinodontiformes

Cyprinodontidae

Jordanella floridae

flagfish

284

Cypriniformes

Cyprinidae

Hemitremia flammea

flame chub

285

Cypriniformes

Catostomidae

Catostomus latipinnis

flannelmouth sucker

286

Siluriformes

Ictaluridae

Ameiurus platycephalus

flat bullhead

287

Siluriformes

Ictaluridae

Pylodictis olivaris

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

Etheostoma fonticola

fountain darter

294

Scorpaeniformes

Cottidae

Myoxocephalus quadricornis

fourhorn sculpin

295

Gasterosteiformes

Gasterosteidae

Apeltes quadracus

fourspine stickleback

296

Perciformes

Percidae

Percina stictogaster

frecklebelly darter

297

Siluriformes

Ictaluridae

Noturus munitus

frecklebelly madtom

298

Perciformes

Percidae

Percina lenticula

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

Etheostoma crossopterum

fringed darter

303

Cypriniformes

Cyprinidae

Notropis buchanani

ghost shiner

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Cyprinodontiformes

Aplocheilidae

Rivulus hartii

giant rivulus

305

Cypriniformes

Cyprinidae

Gila intermedia

gila chub

306

Cyprinodontiformes

Poeciliidae

Poeciliopsis occiden talis

gila topminnow

307

Salmoniformes

Salmonidae

Oncorhynchus gilae

gila trout

308

Perciformes

Percidae

Percina evides

gilt darter

309

Clupeiformes

Clupeidae

Dorosoma cepedianum

gizzard shad

310

Perciformes

Percidae

Etheostoma vitreum

glassy darter

311

Perciformes

Percidae

Etheostoma denoncourti

golden darter

312

Cypriniformes

Catostomidae

Moxostoma erythrurum

golden redhorse

313

Cypriniformes

Cyprinidae

Notemigonus crysoleucas

golden shiner

314

Cyprinodontiformes

Fundulidae

Fundulus chrysotus

golden topminnow

315

Hiodontiformes

Hiodontidae

Hiodon alosoides

goldeye

316

Cypriniformes

Cyprinidae

Carassius auratus

goldfish

317

Perciformes

Percidae

Percina aurolineata

goldline darter

318

Perciformes

Percidae

Etheostoma parvipinne

goldstripe darter

319

Cypriniformes

Cyprinidae

Ctenopharyngodon idella

grass carp

320

Cypriniformes

Cyprinidae

Erimystax x-punctatus

gravel chub

321

Cypriniformes

Catostomidae

Moxostoma congestum

gray redhorse

322

Perciformes

Lutjanidae

Lutjanus griseus

gray snapper

323

Cypriniformes

Catostomidae

Moxostoma lachneri

greater jumprock

324

Cypriniformes

Catostomidae

Moxostoma valenciennesi

greater redhorse

325

Acipenseriformes

Acipenseridae

Acipenser medirostris

green sturgeon

326

Perciformes

Centrarchidae

Lepomis cyanellus

green sunfish

327

Cyprinodontiformes

Poeciliidae

Xiphophorus hellerii

green swordtail

328

Perciformes

Percidae

Etheostoma jordani

green breast darter

329

Perciformes

Percidae

Etheostoma chlorobranchium

greenfin darter

330

Cypriniformes

Cyprinidae

Cyprinella chloristia

greenfin shiner

331

Cypriniformes

Cyprinidae

Notropis chlorocephalus

greenhead shiner

332

Perciformes

Percidae

Etheostoma blennioides

greenside darter

333

Perciformes

Percidae

Etheostoma lepidum

greenthroat darter

334

Perciformes

Centrarchidae

Micropterus treculii

guadalupe bass

335

Cyprinodontiformes

Fundulidae

Fundulus parvipinnis

guadalupe cardinalfish

336

Cypriniformes

Cyprinidae

Dionda nigrotaeniata

guadalupe roundnose minnow

337

Perciformes

Percidae

Etheostoma oophylax

guardian darter

338

Perciformes

Eleotridae

Guavina guavina

guavina

339

Perciformes

Percidae

Etheostoma swaini

gulf darter

340

Cyprinodontiformes

Fundulidae

Fundulus grandis

gulf killifish

341

Perciformes

Percidae

Percina suttkusi

gulf logperch

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Gasterosteiformes

Syngnathidae

Syngnathus 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 lacerum

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 lawrencei

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 idus

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

jaguarguapote

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

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Scorpaeniformes

Cottidae

Cottus princeps

klamath lake sculpin

381

Petromyzontiformes

Petromyzontidae

Lampetra similis

klamath lamprey

382

Cypriniformes

Catostomidae

Catostomus snyderi

klamath largescale sucker

383

Cypriniformes

Catostomidae

Catostomus rimiculus

klamath smallscale sucker

384

Elopiformes

Elopidae

Elops saurus

ladyfish

385

Cypriniformes

Cyprinidae

Richardsonius egregius

lahontan redside

386

Cypriniformes

Cyprinidae

Couesius plumbeus

lake chub

387

Cypriniformes

Catostomidae

Erimyzon sucetta

lake chubsucker

388

Acipenseriformes

Acipenseridae

Acipenser fulvescens

lake sturgeon

389

Salmoniformes

Salmonidae

Salvelinus namaycush

lake trout

390

Salmoniformes

Salmonidae

Coregonus clupeaformis

lake whitefish

391

Perciformes

Centrarchidae

Micropterus salmoides

largemouth bass

392

Cypriniformes

Cyprinidae

Campostoma oligolepis

largescale stoneroller

393

Cypriniformes

Catostomidae

Catostomus macrocheilus

largescale sucker

394

Perciformes

Eleotridae

Eleotris amblyopsis

largescaled spinycheek

395

Cyprinodontiformes

Poeciliidae

Gambusia geiseri

largespring gambusia

396

Cypriniformes

Cyprinidae

Rhinichthys deaconi

las vegas dace

397

Cypriniformes

Cyprinidae

Phoxinus saylori

laurel dace

398

Petromyzontiformes

Petromyzontidae

Lampetra 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

Heterandria 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

Rhinichthys falcatus

leopard dace

407

Perciformes

Percidae

Percina pantherina

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

longearsunfish

416

Cypriniformes

Cyprinidae

Agosia chrysogaster

longfin dace

417

Perciformes

Percidae

Etheostoma longimanum

longfin darter

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Salmoniformes

Osmeridae

Spirinchus thaleichthys

longfin smelt

419

Perciformes

Percidae

Percina macrocephala

longhead darter

420

Perciformes

Gobiidae

Gillichthys mirabilis

longjaw mudsucker

421

Cypriniformes

Cyprinidae

Rhinichthys cataractae

longnose dace

422

Perciformes

Percidae

Percina nasuta

longnose darter

423

Lepisosteiformes

Lepisosteidae

Lepisosteus osseus

longnose gar

424

Cypriniformes

Cyprinidae

Notropis longirostris

longnose shiner

425

Cypriniformes

Catostomidae

Catostomus catostomus

longnose sucker

426

Cypriniformes

Catostomidae

Deltistes luxatus

lost river sucker

427

Elopiformes

Elopidae

Elops affinis

machete

428

Scorpaeniformes

Cottidae

Cottus bendirei

malheursculpin

429

Cypriniformes

Cyprinidae

Dionda argentosa

manantial roundnose minnow

430

Cyprinodontiformes

Poeciliidae

Gambusia rhizophorae

mangrove gambusia

431

Cyprinodontiformes

Aplocheilidae

Rivulus marmoratus

mangrove rivulus

432

Scorpaeniformes

Cottidae

Cottus klamathensis

marbled sculpin

433

Siluriformes

Ictaluridae

Noturus insignis

margined madtom

434

Scorpaeniformes

Cottidae

Cottus marginatus

margined sculpin

435

Cyprinodontiformes

Fundulidae

Fundulus confluentus

marsh killifish

436

Perciformes

Percidae

Etheostoma sellare

maryland darter

437

Perciformes

Cichlidae

Cichlasoma urophthalmus

mayan cichlid

438

Perciformes

Gobiidae

Ctenogobius claytonii

mexican goby

439

Cyprinodontiformes

Poeciliidae

Poecilia sphenops

mexican molly

440

Cypriniformes

Catostomidae

Moxostoma austrinum

mexican redhorse

441

Cypriniformes

Cyprinidae

Campostoma ornatum

mexican stoneroller

442

Characiformes

Characidae

Astyanax mexicanus

mexican tetra

443

Perciformes

Cichlidae

Cichlasoma citrinellum

midas cichlid

444

Petromyzontiformes

Petromyzontidae

Lampetra minima

miller lake lamprey

445

Cypriniformes

Cyprinidae

Notropis volucellus

mimic shiner

446

Cypriniformes

Cyprinidae

Notropis spectrunculus

mirror shiner

447

Atheriniformes

Atherinopsidae

Menidia audens

mississippi silverside

448

Cypriniformes

Cyprinidae

Hybognathus nuchalis

mississippi silvery minnow

449

Perciformes

Percidae

Etheostoma tetrazonum

missouri saddled darter

450

Cypriniformes

Cyprinidae

Moapa coriacea

moapa dace

451

Perciformes

Percidae

Percina kathae

mobile logperch

452

Cypriniformes

Catostomidae

Catostomus microps

modoc sucker

453

Hiodontiformes

Hiodontidae

Hiodon tergisus

mooneye

454

Scorpaeniformes

Cottidae

Cottus bairdii

mottled sculpin

455

Petromyzontiformes

Petromyzontidae

ichthyomyzon greeleyi

mountain brook lamprey

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Siluriformes

Ictaluridae

Noturus eleutherus

mountain madtom

457

Mugiliformes

Mugilidae

Agonostomus monticola

mountain mullet

458

Cypriniformes

Cyprinidae

Phoxinus oreas

mountain redbellydace

459

Cypriniformes

Cyprinidae

Lythrurus lirus

mountain shiner

460

Cypriniformes

Catostomidae

Catostomus platyrhynchus

mountain sucker

461

Salmoniformes

Salmonidae

Prosopium williamsoni

mountain whitefish

462

Perciformes

Cichlidae

Oreochromis mossambicus

mozambiquetilapia

463

Perciformes

Percidae

Etheostoma asprigene

mud darter

464

Perciformes

Centrarchidae

Acantharchus pomotis

mud sunfish

465

Cyprinodontiformes

Fundulidae

Fundulus heteroclitus

mummichog

466

Esociformes

Esocidae

Esox masquinongy

muskellunge

467

Perciformes

Gobiidae

Gobiosoma bosc

naked goby

468

Perciformes

Percidae

Ammocrypta beanii

naked sand darter

469

Siluriformes

Ictaluridae

Noturus placidus

neosho madtom

470

Cypriniformes

Cyprinidae

Notropis scabriceps

new river shiner

471

Perciformes

Percidae

Etheostoma nianguae

niangua darter

472

Perciformes

Cichlidae

Oreochromis niloticus

niletilapia

473

Gasterosteiformes

Gasterosteidae

Pungitius pungitius

ninespine stickleback

474

Petromyzontiformes

Petromyzontidae

Ichthyomyzon fossor

northern brook lamprey

475

Percopsiformes

Amblyopsidae

Amblyopsis spelaea

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 kansae

northern plains killifish

481

Cypriniformes

Cyprinidae

Phoxinus eos

northern redbelly dace

482

Cyprinodontiformes

Fundulidae

Fundulus catenatus

northern studfish

483

Cypriniformes

Catostomidae

Moxostoma collapsum

notchlip redhorse

484

Cypriniformes

Cyprinidae

Dionda serena

nueces roundnose minnow

485

Cypriniformes

Cyprinidae

Cyprinella callisema

ocmulgee shiner

486

Petromyzontiformes

Petromyzontidae

Ichthyomyzon bdellium

ohio lamprey

487

Perciformes

Percidae

Etheostoma okaloosae

okaloosa darter

488

Perciformes

Elassomatidae

Elassoma okefenokee

okefenokee pygmy sunfish

489

Perciformes

Percidae

Percina squamata

olive darter

490

Esociformes

Umbridae

Novumbra hubbsi

Olympic mudminnow

491

Gasterosteiformes

Syngnathidae

Microphis brachyurus

opossum pipefish

492

Perciformes

Percidae

Etheostoma radiosum

orangebelly darter

493

Perciformes

Percidae

Etheostoma bellum

orangefin darter

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Siluriformes

Ictaluridae

Noturus gilberti

orangefin madtom

495

Cypriniformes

Cyprinidae

Notropis ammophilus

orangefin shiner

496

Perciformes

Sciaenidae

Cynoscion xanthulus

orangemouth corvina

497

Perciformes

Centrarchidae

Lepomis humilis

orangespotted sunfish

498

Cypriniformes

Cyprinidae

Pteronotropis merlini

orangetail shiner

499

Perciformes

Percidae

Etheostoma spectabile

orangethroat darter

500

Cypriniformes

Cyprinidae

Oregonichthys crameri

Oregon chub

501

Cypriniformes

Cobitidae

Misgurnus anguillicaudatus

oriental weatherfish

502

Siluriformes

Loricariidae

Pterygoplichthys multiradiatus

Orinoco sailfin catfish

503

Perciformes

Cichlidae

Astronotus ocellatus

oscar

504

Siluriformes

Ictaluridae

Noturus lachneri

ouachita madtom

505

Cypriniformes

Cyprinidae

Lythrurus snelsoni

ouachita shiner

506

Cyprinodontiformes

Cyprinodontidae

Cyprinodon radios us

owens pupfish

507

Cypriniformes

Catostomidae

Catostomus fumeiventris

owens sucker

508

Perciformes

Centrarchidae

Ambloplites constellatus

ozark bass

509

Percopsiformes

Amblyopsidae

Amblyopsis rosae

ozark cavefish

510

Cypriniformes

Cyprinidae

Erimystax harryi

ozark chub

511

Perciformes

Percidae

Percina fulvitaenia

ozark logperch

512

Siluriformes

Ictaluridae

Noturus albater

ozark madtom

513

Cypriniformes

Cyprinidae

Notropis nubilus

ozark minnow

514

Scorpaeniformes

Cottidae

Cottus hypselurus

ozark sculpin

515

Cypriniformes

Cyprinidae

Notropis ozarcanus

ozarkshiner

516

Petromyzontiformes

Petromyzontidae

Lampetra tridentata

pacific lamprey

517

Scorpaeniformes

Cottidae

Leptocottus armatus

pacific staghorn sculpin

518

Acipenseriformes

Polyodontidae

Polyodon spathula

paddlefish

519

Cypriniformes

Cyprinidae

Lepidomeda altivelis

pahranagat spinedace

520

Cyprinodontiformes

Goodeidae

Empetrichthys latos

pahrump poolfish

521

Scorpaeniformes

Cottidae

Cottus beldingii

paiute sculpin

522

Perciformes

Percidae

Etheostoma pallididorsum

paleback darter

523

Cypriniformes

Cyprinidae

Notropis albizonatus

palezone shiner

524

Cypriniformes

Cyprinidae

Hybopsis amnis

pallid shiner

525

Acipenseriformes

Acipenseridae

Scaphirhynchus albus

pallid sturgeon

526

Cypriniformes

Catostomidae

Moxostoma pisolabrum

pealip redhorse

527

Cypriniformes

Cyprinidae

Mylocheilus caurinus

peamouth

528

Cypriniformes

Cyprinidae

Margariscus margarita

pearl dace

529

Perciformes

Percidae

Percina aurora

pearl darter

530

Cyprinodontiformes

Poeciliidae

Gambusia nobilis

pecos gambusia

531

Cyprinodontiformes

Cyprinodontidae

Cyprinodon pecosensis

pecos pupfish

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Cypriniformes

Cyprinidae

Macrhybopsis tetranema

peppered chub

533

Cypriniformes

Cyprinidae

Notropis perpallidus

peppered shiner

534

Cypriniformes

Cyprinidae

Notropis orca

phantom shiner

535

Perciformes

Percidae

Percina crassa

piedmont darter

536

Perciformes

Haemulidae

Orthopristis chrysoptera

pigfish

537

Cyprinodontiformes

Poeciliidae

Belonesox belizanus

pike killifish

538

Perciformes

Percidae

Etheostoma mariae

pinewoods darter

539

Cypriniformes

Cyprinidae

Lythrurus matutinus

pinewoods shiner

540

Perciformes

Sparidae

Lagodon rhomboides

pinfish

541

Salmoniformes

Salmonidae

Oncorhynchus gorbuscha

pinksalmon

542

Percopsiformes

Aphredoderidae

Aphredoderus sayanus

pirate perch

543

Scorpaeniformes

Cottidae

Cottus pitensis

pit sculpin

544

Petromyzontiformes

Petromyzontidae

Lampetra lethophaga

pit-klamath brook lamprey

545

Cyprinodontiformes

Fundulidae

Fundulus zebrinus

plains killifish

546

Cypriniformes

Cyprinidae

Hybognathus placitus

plains minnow

547

Cyprinodontiformes

Fundulidae

Fundulus sciadicus

plains topminnow

548

Cypriniformes

Cyprinidae

Cyprinella lepida

plateau shiner

549

Salmoniformes

Osmeridae

Hypomesus olidus

pond smelt

550

Cypriniformes

Cyprinidae

Notropis ariommus

popeye shiner

551

Cyprinodontiformes

Poeciliidae

Poeciliopsis gracilis

porthole livebearer

552

Scorpaeniformes

Cottidae

Cottus girardi

potomac sculpin

553

Cypriniformes

Cyprinidae

Macrhybopsis australis

prairie chub

554

Cypriniformes

Cyprinidae

Lythrurus bellus

pretty shiner

555

Scorpaeniformes

Cottidae

Cottus asper

prickly sculpin

556

Cypriniformes

Cyprinidae

Cyprinella proserpina

proserpine shiner

557

Cypriniformes

Cyprinidae

Opsopoeodus emiliae

pugnose minnow

558

Cypriniformes

Cyprinidae

Notropis anogenus

pugnose shiner

559

Perciformes

Centrarchidae

Lepomis gibbosus

pumpkinseed

560

Cyprinodontiformes

Fundulidae

Leptolucania ommata

pygmy killifish

561

Siluriformes

Ictaluridae

Noturus stanauli

pygmy madtom

562

Scorpaeniformes

Cottidae

Cottus paulus

pygmy sculpin

563

Salmoniformes

Salmonidae

Prosopium coulterii

pygmy whitefish

564

Cypriniformes

Catostomidae

Carpiodes cyprinus

quillback

565

Cyprinodontiformes

Goodeidae

Crenichthys nevadae

railroad valley springfish

566

Perciformes

Percidae

Etheostoma caeruleum

rainbow darter

567

Cypriniformes

Cyprinidae

Notropis chrosomus

rainbow shiner

568

Salmoniformes

Osmeridae

Osmerus mordax

rainbow smelt

569

Salmoniformes

Salmonidae

Oncorhynchus mykiss

rainbow trout

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Cyprinodontiformes

Fundulidae

Lucania parva

rainwater killifish

571

Cypriniformes

Catostomidae

Xyrauchen texanus

razorbacksucker

572

Perciformes

Sciaenidae

Sciaenops ocellatus

red drum

573

Cyprinodontiformes

Cyprinodontidae

Cyprinodon rubrofluviatilis

red river pupfish

574

Cypriniformes

Cyprinidae

Notropis bairdi

red river shiner

575

Cypriniformes

Cyprinidae

Cyprinella lutrensis

red shiner

576

Perciformes

Percidae

Etheostoma luteovinctum

redband darter

577

Perciformes

Cichlidae

Tilapia zillii

red belly tilapia

578

Perciformes

Centrarchidae

Lepomis auritus

redbreast sunfish

579

Perciformes

Centrarchidae

Lepomis microlophus

redearsunfish

580

Perciformes

Centrarchidae

Micropterus coosae

redeye bass

581

Cypriniformes

Cyprinidae

Notropis harperi

redeye chub

582

Cyprinodontiformes

Fundulidae

Fundulus rubrifrons

redface topminnow

583

Perciformes

Percidae

Etheostoma whipplei

redfin darter

584

Esociformes

Esocidae

Esox americanus

redfin pickerel

585

Cypriniformes

Cyprinidae

Lythrurus umbratilis

redfin shiner

586

Perciformes

Percidae

Etheostoma rufilineatum

redline darter

587

Cypriniformes

Cyprinidae

Notropis chiliticus

redlip shiner

588

Cypriniformes

Cyprinidae

Clinostomus elongatus

redside dace

589

Cypriniformes

Cyprinidae

Richardsonius balteatus

redside shiner

590

Cypriniformes

Cyprinidae

Nocomis asper

redspot chub

591

Perciformes

Percidae

Etheostoma artesiae

redspot darter

592

Perciformes

Centrarchidae

Lepomis miniatus

redspotted sunfish

593

Perciformes

Cichlidae

Geophagus surinamensis

redstriped eartheater

594

Cypriniformes

Cyprinidae

Nocomis effusus

redtail chub

595

Cypriniformes

Cyprinidae

Relictus solitarius

relict dace

596

Perciformes

Percidae

Etheostoma chienense

relict darter

597

Scorpaeniformes

Cottidae

Cottus perplexus

reticulate sculpin

598

Cypriniformes

Cyprinidae

Lythrurus fumeus

ribbon shiner

599

Cypriniformes

Cyprinidae

Phenacobius catostomus

riffle minnow

600

Scorpaeniformes

Cottidae

Cottus gulosus

riffle sculpin

601

Cypriniformes

Cyprinidae

Gila pandora

rio grande chub

602

Perciformes

Cichlidae

Cichlasoma cyanoguttatum

rio grande cichlid

603

Perciformes

Percidae

Etheostoma grahami

rio grande darter

604

Cypriniformes

Cyprinidae

Notropis jemezanus

rio grande shiner

605

Cypriniformes

Cyprinidae

Hybognathus amarus

rio grande silvery minnow

606

Cypriniformes

Catostomidae

Catostomus plebeius

rio grande sucker

607

Cypriniformes

Catostomidae

Carpiodes carpio

river carpsucker

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Cypriniformes

Cyprinidae

Nocomis micropogon

river chub

609

Perciformes

Percidae

Percina shumardi

river darter

610

Perciformes

Gobiidae

Awaous banana

river goby

611

Petromyzontiformes

Petromyzontidae

Lampetra ayresii

river lamprey

612

Cypriniformes

Catostomidae

Moxostoma carinatum

river redhorse

613

Cypriniformes

Cyprinidae

Notropis blennius

river shiner

614

Perciformes

Percidae

Etheostoma podostemone

riverweed darter

615

Perciformes

Centrarchidae

Ambloplites cavifrons

roanoke bass

616

Perciformes

Percidae

Percina roanoka

roanoke darter

617

Cypriniformes

Catostomidae

Hypentelium roanokense

roanoke hog sucker

618

Perciformes

Percidae

Percina rex

roanoke logperch

619

Cypriniformes

Catostomidae

Moxostoma robustum

robust redhorse

620

Perciformes

Centrarchidae

Ambloplites rupestris

rock bass

621

Perciformes

Percidae

Etheostoma rupestre

rock darter

622

Cypriniformes

Cyprinidae

Notropis suttkusi

rocky shiner

623

Cypriniformes

Cyprinidae

Lythrurus ardens

rosefin shiner

624

Cypriniformes

Cyprinidae

Hybopsis rubrifrons

rosyface chub

625

Cypriniformes

Cyprinidae

Notropis rubeIIus

rosyface shiner

626

Cypriniformes

Cyprinidae

Clinostomus funduloides

rosyside dace

627

Scorpaeniformes

Cottidae

Cottus asperrimus

rough sculpin

628

Cypriniformes

Cyprinidae

Notropis baileyi

rough shiner

629

Atheriniformes

Atherinopsidae

Membras martinica

rough silverside

630

Cypriniformes

Cyprinidae

Notropis semperasper

roughhead shiner

631

Perciformes

Gobiidae

Neogobius melanostomus

round goby

632

Salmoniformes

Salmonidae

Prosopium cylindraceum

round whitefish

633

Cypriniformes

Cyprinidae

Dionda episcopa

roundnose minnow

634

Cypriniformes

Cyprinidae

Gila robusta

roundtail chub

635

Cypriniformes

Cyprinidae

Scardinius erythrophthalmus

rudd

636

Perciformes

Percidae

Gymnocephalus cernuus

ruffe

637

Perciformes

Percidae

Etheostoma phytophilum

rush darter

638

Cyprinodontiformes

Fundulidae

Fundulus escambiae

russetfin topminnow

639

Cypriniformes

Catostomidae

Thoburnia hamiltoni

rustyside sucker

640

Cypriniformes

Cyprinidae

Notropis sabinae

sabine shiner

641

Cypriniformes

Cyprinidae

Orthodon microlepidotus

Sacramento blackfish

642

Perciformes

Centrarchidae

Archoplites interruptus

Sacramento perch

643

Cypriniformes

Cyprinidae

Ptychocheilus grandis

Sacramento pikeminnow

644

Cypriniformes

Catostomidae

Catostomus occidentalis

Sacramento sucker

645

Perciformes

Percidae

Percina vigil

saddleback darter

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Perciformes

Percidae

Etheostoma flavum

saffron darter

647

Cypriniformes

Cyprinidae

Notropis rubricroceus

saffron shiner

648

Cyprinodontiformes

Poeciliidae

Poecilia latipinna

sailfin molly

649

Cypriniformes

Cyprinidae

Pteronotropis hypselopterus

sailfin shiner

650

Cyprinodontiformes

Cyprinodontidae

Cyprinodon salinus

salt creek pupfish

651

Cyprinodontiformes

Fundulidae

Fundulus jenkinsi

saltmarsh topminnow

652

Cyprinodontiformes

Poeciliidae

Gambusia georgei

san marcos gambusia

653

Percopsiformes

Percopsidae

Percopsis transmontana

sand roller

654

Cypriniformes

Cyprinidae

Notropis stramineus

sand shiner

655

Cypriniformes

Cyprinidae

Notropis scepticus

sandbarshiner

656

Cypriniformes

Cyprinidae

Semotilus lumbee

sandhills chub

657

Cypriniformes

Catostomidae

Catostomus santaanae

santa ana sucker

658

Cyprinodontiformes

Cyprinodontidae

Cyprinodon arcuatus

santa cruz pupfish

659

Cypriniformes

Cyprinidae

Cyprinella zanema

santee chub

660

Cypriniformes

Cyprinidae

Cyprinella analostana

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 marinus

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

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684

Cypriniformes

Cyprinidae

Macrhybopsis hyostoma

shoal chub

685

Perciformes

Gobiidae

Tridentiger barbatus

shokihaze goby

686

Cyprinodontiformes

Poeciliidae

Poecilia mexicana

shortfin molly

687

Cypriniformes

Catostomidae

Moxostoma macrolepidotum

shorthead redhorse

688

Scorpaeniformes

Cottidae

Cottus confusus

shorthead sculpin

689

Salmoniformes

Salmonidae

Coregonus zenithicus

shortjaw cisco

690

Salmoniformes

Salmonidae

Coregonus reighardi

shortnose cisco

691

Lepisosteiformes

Lepisosteidae

Lepisosteus platostomus

shortnose gar

692

Acipenseriformes

Acipenseridae

Acipenser brevirostrum

shortnose sturgeon

693

Cypriniformes

Catostomidae

Chasmistes brevirostris

shortnose sucker

694

Scorpaeniformes

Cottidae

Cottus greenei

shoshone sculpin

695

Acipenseriformes

Acipenseridae

Scaphirhynchus platorynchus

shovelnose sturgeon

696

Cypriniformes

Cyprinidae

Macrhybopsis meeki

sicklefin chub

697

Cypriniformes

Cyprinidae

Hypophthalmichthys molitrix

silver carp

698

Cypriniformes

Cyprinidae

Macrhybopsis storeriana

silver chub

699

Petromyzontiformes

Petromyzontidae

Ichthyomyzon unicuspis

silver lamprey

700

Perciformes

Sciaenidae

Bairdiella chrysoura

silver perch

701

Cypriniformes

Catostomidae

Moxostoma anisurum

silver redhorse

702

Cypriniformes

Cyprinidae

Notropis photogenis

silver shiner

703

Cypriniformes

Cyprinidae

Notropis shumardi

silverband shiner

704

Cypriniformes

Cyprinidae

Notropis buccatus

silverjaw minnow

705

Cypriniformes

Cyprinidae

Notropis candidus

silverside shiner

706

Cypriniformes

Cyprinidae

Notropis stilbius

silverstripe shiner

707

Clupeiformes

Clupeidae

Alosa chrysochloris

skipjack herring

708

Cypriniformes

Cyprinidae

Notropis uranoscopus

skygazershiner

709

Perciformes

Percidae

Etheostoma smithi

slabrock darter

710

Perciformes

Percidae

Etheostoma boschungi

slackwater darter

711

Perciformes

Gobiidae

Ctenogobius pseudofasciatus

slashcheekgoby

712

Cypriniformes

Cyprinidae

Erimystax cahni

slender chub

713

Siluriformes

Ictaluridae

Noturus exilis

slender madtom

714

Scorpaeniformes

Cottidae

Cottus tenuis

slender sculpin

715

Perciformes

Percidae

Percina phoxocephala

slenderhead darter

716

Cypriniformes

Cyprinidae

Pimephales tenellus

slim minnow

717

Scorpaeniformes

Cottidae

Cottus cognatus

slimy sculpin

718

Perciformes

Percidae

Etheostoma gracile

slough darter

719

Cypriniformes

Cyprinidae

Notropis buccula

smalleye shiner

720

Perciformes

Centrarchidae

Micropterus dolomieu

smallmouth bass

721

Cypriniformes

Catostomidae

ictiobus bubalus

smallmouth buffalo

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722

Cypriniformes

Catostomidae

Moxostoma breviceps

smallmouth redhorse

723

Perciformes

Percidae

Etheostoma microlepidum

smallscale darter

724

Perciformes

Centropomidae

Centropomus parallelus

smallscalefat snook

725

Perciformes

Eleotridae

Eleotris perniger

smallscaled spinycheek

726

Prist iformes

Pristidae

Pristis pectinata

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 simoterum

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 eremus

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

Percops iformes

Amblyopsidae

Typhlichthys subterraneus

southern cavefish

740

Pleuronect iformes

Paralichthyidae

Paralichthys lethostigma

southern flounder

741

Perciformes

Percidae

Percina austroperca

southern logperch

742

Cyprinodontiformes

Poeciliidae

Xiphophorus maculatus

southern platyfish

743

Cypriniformes

Cyprinidae

Phoxinus erythrogaster

southern redbellydace

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 stellifer

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 rathbuni

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

Leiostomus xanthurus

spot

758

Cypriniformes

Cyprinidae

Erimonax monachus

spotfin chub

759

Cyprinodontiformes

Fundulidae

Fundulus luciae

spotfin killifish

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Cypriniformes

Cyprinidae

Cyprinella spiloptera

spotfin shiner

761

Perciformes

Percidae

Etheostoma squamiceps

spottail darter

762

Cypriniformes

Cyprinidae

Notropis hudsonius

spottail shiner

763

Perciformes

Centrarchidae

Micropterus punctulatus

spotted bass

764

Siluriformes

Ictaluridae

Ameiurus serracanthus

spotted bullhead

765

Perciformes

Percidae

Etheostoma maculatum

spotted darter

766

Lepisosteiformes

Lepisosteidae

Lepisosteus oculatus

spotted gar

767

Perciformes

Sciaenidae

Cynoscion nebulosus

spotted seatrout

768

Perciformes

Eleotridae

Eleotris picta

spotted sleeper

769

Cypriniformes

Catostomidae

Minytrema melanops

spotted sucker

770

Perciformes

Centrarchidae

Lepomis punctatus

spotted sunfish

771

Perciformes

Cichlidae

Tilapia mariae

spotted tilapia

772

Percopsiformes

Amblyopsidae

Forbesichthys agassizii

spring cavefish

773

Perciformes

Elassomatidae

Elassoma alabamae

spring pygmy sunfish

774

Perciformes

Percidae

Percina uranidea

stargazing darter

775

Cypriniformes

Cyprinidae

Phenacobius uranops

stargazing minnow

776

Cyprinodontiformes

Fundulidae

Fundulus dispar

starhead topminnow

777

Pleuronectiformes

Pleuronectidae

Platichthys stellatus

starry flounder

778

Cypriniformes

Cyprinidae

Cyprinella whipplei

st eelcolor shiner

779

Perciformes

Percidae

Etheostoma punctulatum

stippled darter

780

Cyprinodontiformes

Fundulidae

Fundulus bifax

stippled studfish

781

Perciformes

Percidae

Etheostoma derivativum

stone darter

782

Siluriformes

Ictaluridae

Noturus flavus

stonecat

783

Perciformes

Percidae

Etheostoma fragi

strawberry darter

784

Cypriniformes

Cyprinidae

Erimystax dissimilis

streamline chub

785

Perciformes

Percidae

Etheostoma striatulum

striated darter

786

Perciformes

Percidae

Percina notogramma

stripeback darter

787

Perciformes

Moronidae

Morone saxatilis

striped bass

788

Perciformes

Percidae

Etheostoma virgatum

striped darter

789

Cypriniformes

Catostomidae

Moxostoma rupiscartes

striped jumprock

790

Perciformes

Gerreidae

Eugerres plumieri

striped mojarra

791

Mugiliformes

Mugilidae

Mugil cephalus

striped mullet

792

Cypriniformes

Cyprinidae

Luxilus chrysocephalus

striped shiner

793

Perciformes

Percidae

Etheostoma kennicotti

stripetail darter

794

Cypriniformes

Cyprinidae

Macrhybopsis gelida

sturgeon chub

795

Siluriformes

Loricariidae

Hypostomus plecostomus

suckermouth catfish

796

Cypriniformes

Cyprinidae

Phenacobius mirabilis

suckermouth minnow

797

Salmoniformes

Osmeridae

Hypomesus pretiosus

surf smelt

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798

Perciformes

Centrarchidae

Micropterus notius

suwannee bass

799

Cypriniformes

Cyprinidae

Notropis procne

swallowtail shiner

800

Perciformes

Percidae

Etheostoma fusiforme

swamp darter

801

Percopsiformes

Amblyopsidae

Chologaster cornuta

swampfish

802

Perciformes

Percidae

Etheostoma swannanoa

swannanoa darter

803

Perciformes

Centropomidae

Centropomus ensiferus

swordspine snook

804

Siluriformes

Ictaluridae

Noturus gyrinus

tadpole madtom

805

Cypriniformes

Catostomidae

Catostomus tahoensis

tahoe sucker

806

Cypriniformes

Cyprinidae

Notropis maculatus

taillight shiner

807

Perciformes

Percidae

Etheostoma tallapoosae

tallapoosa darter

808

Cypriniformes

Cyprinidae

Cyprinella gibbsi

tallapoosa shiner

809

Cypriniformes

Cyprinidae

Notropis braytoni

tamaulipas shiner

810

Perciformes

Percidae

Percina aurantiaca

tangerine darter

811

Elopiformes

Megalopidae

Megalops atlanticus

tarpon

812

Perciformes

Centropomidae

Centropomus pectinatus

tarpon snook

813

Perciformes

Percidae

Etheostoma barbouri

teardrop darter

814

Cypriniformes

Cyprinidae

Notropis telescopus

telescope shiner

815

Cypriniformes

Cyprinidae

Tinea tinea

tench

816

Cypriniformes

Cyprinidae

Phoxinus tennesseensis

tennessee dace

817

Cypriniformes

Cyprinidae

Notropis leueiodus

tennessee shiner

818

Perciformes

Percidae

Etheostoma olmstedi

tessellated darter

819

Perciformes

Percidae

Percina carbonaria

texas logperch

820

Cypriniformes

Cyprinidae

Notropis amabilis

texas shiner

821

Cyprinodontiformes

Poeciliidae

Gambusia speciosa

tex-mex gambusia

822

Cypriniformes

Cyprinidae

Cyprinella labrosa

thicklip chub

823

Cypriniformes

Cyprinidae

Gila crassicauda

thicktail chub

824

Clupeiformes

Clupeidae

Dorosoma petenense

threadfin shad

825

Perciformes

Gobiidae

Eucyclogobius newberryi

tidewater goby

826

Perciformes

Gerreidae

Eucinostomus harengulus

tidewater mojarra

827

Perciformes

Percidae

Etheostoma tippecanoe

tippecanoe darter

828

Perciformes

Percidae

Etheostoma lachneri

tombigbee darter

829

Cypriniformes

Cyprinidae

Exoglossum laurae

tonguetied minnow

830

Siluriformes

Ictaluridae

Trogloglanis 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

Etheostoma trisella

trispot darter

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836

Percopsiformes

Percopsidae

Percopsis omiscomaycus

trout-perch

837

Perciformes

Gobiidae

Proterorhinus marmoratus

tubenose goby

838

Perciformes

Percidae

Etheostoma gutselli

tuckasegee darter

839

Cypriniformes

Cyprinidae

Gila bicolor

tui chub

840

Perciformes

Embiotocidae

Hysterocarpus traskii

tule perch

841

Perciformes

Percidae

Etheostoma inscriptum

turquoise darter

842

Perciformes

Percidae

Etheostoma tuscumbia

tuscumbia darter

843

Perciformes

Percidae

Etheostoma douglasi

tuskaloosa darter

844

Cypriniformes

Cyprinidae

Rhinichthys umatilla

umatilla dace

845

Cypriniformes

Cyprinidae

Oregonichthys kalawatseti

umpqua chub

846

Cypriniformes

Cyprinidae

Rhinichthys evermanni

umpqua dace

847

Cypriniformes

Cyprinidae

Ptychocheilus umpquae

umpqua pikeminnow

848

Cypriniformes

Cyprinidae

Gila atraria

Utah chub

849

Scorpaeniformes

Cottidae

Cottus echinatus

Utah lake sculpin

850

Cypriniformes

Catostomidae

Catostomus ardens

Utah sucker

851

Cyprinodontiformes

Poeciliidae

Xiphophorus variatus

variable platyfish

852

Perciformes

Percidae

Etheostoma variatum

variegate darter

853

Siluriformes

Loricariidae

Pterygoplichthys disjunctivus

vermiculated sailfin catfish

854

Perciformes

Percidae

Etheostoma chermocki

vermilion darter

855

Perciformes

Gobiidae

Gobioides broussonetii

violet goby

856

Cypriniformes

Cyprinidae

Gila seminuda

virgin chub

857

Cypriniformes

Cyprinidae

Lepidomeda mollispinis

virgin spinedace

858

Cypriniformes

Catostomidae

Moxostoma pappillosum

v-lip redhorse

859

Perciformes

Percidae

Etheostoma perlongum

waccamaw darter

860

Cyprinodontiformes

Fundulidae

Fundulus waccamensis

waccamaw killifish

861

Atheriniformes

Atherinopsidae

Menidia extensa

waccamaw silverside

862

Salmoniformes

Osmeridae

Hypomesus riipponensis

wakasagi

863

Siluriformes

Clariidae

Clarias batrachus

walking catfish

864

Perciformes

Percidae

Sander vitreus

walleye

865

Perciformes

Cichlidae

Oreochromis urolepis

wami tilapia

866

Perciformes

Centrarchidae

Lepomis gulosus

warmouth

867

Cypriniformes

Catostomidae

Catostomus warnerensis

warner sucker

868

Cypriniformes

Cyprinidae

Luxilus coccogenis

warpaint shiner

869

Perciformes

Percidae

Etheostoma bellator

warrior darter

870

Cypriniformes

Cyprinidae

Lythrurus alegnotus

warrior shiner

871

Perciformes

Percidae

Etheostoma nuchale

watercress darter

872

Cypriniformes

Cyprinidae

Notropis greenei

wedgespot shiner

873

Cypriniformes

Cyprinidae

Notropis texanus

weed shiner

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LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME

874

Cypriniformes

Cyprinidae

Rhinichthys obtusus

western blacknose dace

875

Petromyzontiformes

Petromyzontidae

Lampetra richardsoni

western brook lamprey

876

Cyprinodontiformes

Poeciliidae

Gambusia affinis

western mosquitofish

877

Perciformes

Percidae

Ammocrypta clara

western sand darter

878

Cypriniformes

Cyprinidae

Hybognathus argyritis

western silvery minnow

879

Cyprinodontiformes

Fundulidae

Fundulus blairae

western starhead topminnow

880

Perciformes

Moronidae

Morone chrysops

white bass

881

Siluriformes

Ictaluridae

Ameiurus catus

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 flavimanus

yellowfin goby

910

Siluriformes

Ictaluridae

Noturus flavipinnis

yellowfin madtom

911

Cypriniformes

Cyprinidae

Notropis lutipinnis

yellowfin shiner

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LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME

912

Perciformes

Percidae

Etheostoma juliae

yoke darter

913

Perciformes

Percidae

Sander lucioperca

zander

914

Cypriniformes

Catostomidae

Moxostoma cf. poecilurum

apalachicola redhorse

915

Salmoniformes

Salmonidae

Salmo salar

atlantic salmon juvenile

916

Atheriniformes

Atherinopsidae

Menidia menidia

atlantic silverside

917

Cypriniformes

Cyprinidae

Hypophthalmichthys nobilis

bighead carp

918

Cypriniformes

Cyprinidae

Mylopharyngodon piceus

black carp

919

Perciformes

Pomatomidae

Pomatomus saltatrix

bluefish

920

Perciformes

Percidae

Etheostoma meadiae

bluespar darter

921

Salmoniformes

Salmonidae

Oncorhynchus clarkii Utah

bonneville cutthroat trout

922

Cypriniformes

Catostomidae

Moxostoma cf. lachneri

brassy jumprock

923

Anura

Ranidae

Rana catesbeiana

bullfrog

924

Anura

Ranidae

Rana catesbeiana

bullfrog tadpole

925

Scorpaeniformes

Cottidae

Cottus Chattahoochee

Chattahoochee sculpin

926

Salmoniformes

Salmonidae

Oncorhynchus tshawytscha

chinooksalmon (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 mykiss

cutbow

934

Perciformes

Percidae

Etheostoma planasaxatile

duck darter

935

Perciformes

Percidae

Etheostoma orientale

eastrim darter

936

Perciformes

Centropomidae

Centropomus parallelus

fat snook

937

Esociformes

Esocidae

Esox americanus vermiculatus

grass pickerel

938

Clupeiformes

Clupeidae

Brevoortia patronus

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 mykiss

rainbow trout (steelhead)

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LINE NO. ORDER FAMILY SCIENTIFIC NAME COMMON NAME

950

Anura

Ranidae

Rana aurora

red-legged frog

951

Salmoniformes

Salmonidae

Oncorhynchus mykiss gairdneri

redband rainbow trout

952

Caudata

Salamandridae

Taricha 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

Hypophthalmichthys 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

Salmo X Salvelinus trutta x fontinalis

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 mykiss

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

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LINE NO.

ORDER

FAMILY

SCIENTIFIC NAME

COMMON NAME

988

Anura

Ranidae

Rana clamitans

green frog

989

Caudata

Ambystomatidae

Dicamptodon aterrimus

idaho giant salamander

990

Petromyzontiformes

Petromyzontidae

Lampetra similis

klamath river lamprey

991

Anura

Ranidae

Lithobates pipiens

leopard frog

992

Caudata

Ambystomatidae

Ambystoma macrodactylum

longtoed salamander

993

Anura

Ranidae

Rana yavapaiensis

lowland leopard frog

994

Anura

Ranidae

Rana muscosa

mountain yellow-legged frog

995

Anura

Hylidae

Pseudacris regilla

pacific tree frog

996

Anura

Ranidae

Lithobates blairi

plains leopard frog

997

Salmoniformes

Salmonidae

Oncorhynchus mykiss x aguabonita

rainbow x golden trout

998

Anura

Bufonidae

Bufo punctatus

red-spotted toad

999

Cypriniformes

Cyprinidae

Richardsonius X Rhinichthys balteatus x osculus

redside shinerx speckled dace

1000

Anura

Ranidae

Rana pretiosa

spotted frog

1001

Anura

Ranidae

Rana sylvatica

wood frog

1002

Anura

Bufonidae

Bufo woodhousii

woodhouse's toad

1003

Anura

Ranidae

Lithobates chiricahuensis

chiricahua leopard frog

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Laboratory Operations Manual
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Templates will be provided on the NARS SharePoint.

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APPENDIX E: EXAMPLE SOP FOR ASH FREE DRY MASS ANALYSIS OF
PERIPHYTON BIOMASS

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•SOP
. *?>*>¦¦*

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SOP

FOR

The DeteiminaticMa. of Ash Free Dry Mass
WRS 73A.5

Biennial Reviews*

Date

EPA Reviewer (WAS; if

WAC OR;.

Date entered

br:

QA Manager

*Listed EPA re\iewer has personally A erified, or has been assured directly or
indirectly by those currenth using this SOP, that it is being used, essentiaUy

ns written and tfcat no revisions are necessary.

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Standard Operating Procedure for the
Determination of Ash Free Dry Mass

WRS 73A.5

Willamette Research Station
Analytical Laboratory
200 SYV 35a Street
Corvallis, Oregon

Prepared by Karen Baxter
December 2002
Revised In Diaune Mever

Api U 2012

Contract £p-D-ll~#27
Dynunac Corporation

O.
D_
<

170

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Standard Operating Procedure for the
Determination of Ash Free Dry Mass
WRS 73A.5

list of Anitas CmiMbrting
to ins SOP

Ongnndly pre§»tiei by Karen Baxter, 2002

Revision!:

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Standard Ope rating Procedure for the
Determinntioii of Ash Free Dry Mass
WRS 73A.5

TaMe of Contents

10 Scope and Appleitieo. 6

2,# Sunmmy of Method. 6

3.0 Defkniban* 6

4 0 Health and Safety Warnings 7

5.0 OmtiaiiS 7

6 0 Intofemce 7

7.0 Equipment and Supplies 1

8.0 Reagents md Standards I

9.0 Sample Handling aad Storage §

10 0 Quality Control 9

110 Calibration ad Staudanlizmtian 9

12.0 Procedure 9

13 J Data Analysis and CaEaiations 10

14.® MtftgiMoHn U

15.® 11

16.0 Waste Management 11

17.0 ftnonari QnalifiratmiB II

18.0 References 12

19.0 Tables, Diagrams. Flowcharts, mi Validation Data 14

20.0 A{|xsniix.A 15

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Standard Operating Procedure for the
Deternmintioa of Ash Free Dry Mass

WRS "3A.5

1.0 Scope and Appficatnn

1.1 His method u designed for flie bboxataty fctenninEtioa of ash free dry
xnasg(AFDM}mperiphytoii. Sanpl«weiecoIecteii)floiringllK
procedures described in FisM Operations Manual for Jlfafaabfc Streams
(PecketaL, 2005),

1.2 The differences between flis vnson (WKS 73A3} and the previous
version, (WRS 73A.4) include editorial dimges is well as the addition of
die personnel qualifications section.

2.0 Summary of Method

2.1 SinopfelllM wiriMmaf«eed-ak
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1J Gistss-fita- filter: Whatman GF/F glass-filet filter dices (=0.7 pn particle
leteifioii).

3.4 QCS: A Quality Control Simple is nm at least oik* per nm to dancmstrate
inter-urn cansisteuEy and for inpficale pmsioo. He <}C8 is an in house
reference standard made by filtering penphyfm rich water through a
Whatman GF/F glass fiber filter Treated filters are frozen until needed.

3 5 Reagent Water: Water passed through a reverse osmosis and a

deionizatkiii system to produce A5TM Type I jengent water (>18
iMCTiihnK resistivity) (3pecifica]tkraD-li93-99e], ASTM1999).

4.0 Health and Safety Warnings

41 ftusoml Protecttw Equipment (PEE) is advised for amylaitxKiifioxy

procedure*. <

4.2 Use heat protection suck as oven mils, and tongs to prevent contact with

4.3 Allow muffle iniace to cool to 2:0tTC before opening door to access

4.4 This method does not address all safety issues associated with its use. He _

laboratory staff is. responsible for safety ceBtkietmg lab work mi chemical u.

analysis in accordance with flue MHEE1L-WED Health. and Safety °

Handbook (U S EPA. 2010),, the applicable Dynamac HaziniBis ^

Activity Safety Plans, and the Material Safety DaM Sheets for the specific ^

chemicals. <

5.0 Cautions $

5.1 Fillers placed m the forced-air avm should be cavesed mil foil to prevent ^

filter from being blown, out of identifying aluminum weigfa-boat. g

f J Interferences ^

6.1 Samples must be stored in the desiccator after the drying process to ensure ^

that there is no increase in weight derived im atmospheric moisture. ^

LL
CL

l/l

<
X

X
Q

CL
CL
<

174

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7.0 Eqaipment and Supplies

Nat*. Track Htmas, ngp&ars andpart numbers are for mfbrmationalpurposes only
Mo endorsement is implied. Equivalent performance may be achieved using
appamms and materials other than those specified hem, but demonstration of
equivalent performance that meets the requirements of this method is the

mspomibjliiy &f laboratory

7.1 Analytical balance with refutation to 0.1 mg

7.2 Gas fiber ffltas {GF/F or epiualat)

7.3 Fon»i-iir oven, with over tanperataire control mil set at i temperature of
WC

7.4 AhimrTnim weigh-boats

7 5 Muffle fimma.ee wilt temperature capacity of 55tPC

7 6 Tangs

7.7 Safety glasses

7.5 Nitrite glovw
7.9 Lab coat

ft.0 Reagents and Standards

81 liesgpat Water

8.2 HST willed weight set (D.OOIO—200g} to check balance calibration.

8 3 QCS: A large volume (qiproabinately 5 gallons bulk sample) of

jmiphjfcn. rich water is collected lam a local stream. Bitch.es of QCS
smiles (fiUm) ans prepared at one time mi frozen for iiime we in the
AFDM analyses To j^epaie the QCS samples, 50-mL attqpwts are
JUhhI ihrangh GF/F filters pnerifiusly baked in a muffle ftamaee at 550°C
Ik 30 minutes, and the fitters me frozen (-20°Q ia aluminum foil. Two
previously frozen QCS samples (filter) are processed with each batch of
AFDM sample filters.

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t J Sample Handling and Storage

9 I Samples are neceived, logged into the laimatorj data system, and stored in.
a deep freeze at -20QC.

92 Sauries may be stored ia the freeze- fbr sevenl months pior to analysis
(APHA. 1998. Method 10300 D).,

1# J Quality Control

10.1 QCS is an aliquot from a bulk sample that is filtered feoogti a GF/F filter
then stored frozen at -20°C until needed Two filters are processed nith

each batch of AFDM filters to monitor inter-nia consistency and

pmsioB.

10.2 A filler Hank is processed each ran as a tudkgpjuni check.
11J Calibration ani Standardization

111 Analytical balances: CaHbfatiom is checked to within 2% of the

lieorefeat weigjht with certified standard weigbfe. A weight with a mass
similar to the wagHMtt-stniple mast should Ik used to check the
calitnatkm.

12 J Procedure

121 Remove tie sample from freezer and record sample ID, sample volumes,
and collection date on laboratory data sheets (see Section 19.0).

12.2 Remove filter from field packaging and place Mo am mgpwei (labeled)
afamnMHi weigh-best He wofji-feoat label should match ifp with the
comet sample ID number found on the weigh sheet If sample filter is
folded within lie oodiifiige 'tube, unfold pior to drying. Not*: son* of
the emtnfinge tubes way cmtain a small amount of wafer (tea flan 10
mL) This should be Irmsfermi to the mmgk<-bmi with the sample. Ifthe
sample contains more than 10 mL efwahi, the stmph is ti eaied
diffwmtbf, see appendix A forprocwdim.

12.3 Cover samples will foil ts prevent filters front blowing out of weigh-boat
ail dry the sample in a forced-air owed at WC fa 24 hours

12.4 Place warm, dry samples in a desiccator to cool. Weigh samples in
aluminum weigh-borts and lecaxd to the nearest 0 1 mg, Remove (he

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wagh-boat+samiletonbalance. Ensure ftetalmitxkziexoecL 'His.is
fine initial sample weight

12.5 Place tie samples in a muffle furnace ami being the tempaatuie to 550°C.

Ash the simples in a muffle .itamiee by balding Ami at 550°C for 30

12.fi ABow the iimace to cool to ajjposiinaleJj 200"C. Remove tie samples
from lie muffle furnace and place in a desiccator to finish cooing. After
ashing. the precipitate should appear gray md dumbly

12.7 Saturate fc cooled simples with reagent water to rehvdrate tte days.

12 8 Cover tangles with feal to pewit filters ftnm Mowing out and dry the

samples in a finced-air oven at 60°C fin" 24 boms.

12.9 Store the dry samples in a desiccator to cooL Calibrate balance and
leconL Weigh the samples m aluminum weigh-boats and record fine
weight mi the datasheet (see Section 19 0) to the nearest 0.1 mg Remove

the anipfc and weigh-boat from balance and ensure the balance is zeroed
This is tie final (ash fiee) sample weight

13.0 Data Analysis and Cakuhtions

13.1 He lab does not receive all of the requited field data to standardize. Ash
Free Dry Mass results to a unit area Therefore initial and final weights
(second value) in reported by electronic spreadsheet to EPA aid the raw

data is used to calculate the AEDM using the folouring eafaiiitkio:

AH*, {pa,,) (g) = Initial wt <^g)—Final wt (g)

AFDN^v ^Him fmmatmiiimmi {g/mL) = AHJMm (g) / Filtered volume (mL)

AFDMLh^Bw^rnta^i (gfcmP)=jMFDMuh * [Total volume (mL) / area
scraped (an2)]

Where:

Filtered volume = vahnme of sample filtered in the field (usually 25 mL)
Total volume = volume of sample coflecltai (usually 500 mL)

Area scraped = (# of transects)*(area delimiter (cm?)) (usually 12 cm2 x 11
transects = 132 em2!

13 .2 AH results are recorded an a datasheet (see Section 19.0). Datasheets are

filed in the AFDM binder created and maintained for thai calendar year.

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Results are entered into electronic format and entries are verified by a.

second pecan.

14.1 Method Performance

14.1 Method performance is measured through analysis of filter blanks, bulk
caUectxoiuQCS samples. audpraasinii (Quality Asamice Flan, 2012}.
Blank measurements should not pin or lose any weight after complete

ashing and drying process. Two fitter* prepared from i bulk sample are
analyzed each analysis batch ani results are monitored over tune.
Duplicate m i-v"' > '.re ji-*; "< i based on the QC'S duplicates.

14.2 2®» QCS Mils had a mean. AFDM of 7.3 mg
15 J Pollution Prevention

15.1 The chemicals used in this method pose little threat to the environment

15.2 Whenever possible waste is reduced the source. When waste cannot be
feasibly reduced the waste is collected for recycling

16.0 Waste Management

16 1 It is the laborataay's responsibility to comply with all federal, state and

local refpMnns gcweiBijig waste management, and to protect die
anrimnnEnt by winmwb«tg gad c-outroling all Biases fina 'fane hoods
aMijoijsAoperatiEHB. QMifjlaiicewifijals«wigefcchn^|ieinB'lS and
regulations is required

17 6 Contact the Dynamac Environmental, Health aud Safety Manager for

guidance <¦. waste collection and disposal.. FarfliriijariBfanBitiaiion
waste maxngemoit, consult 7>r Waste MtauQ*mant Manual fir
Laboratory Personnel, and loss is Berter: Laborawiy Chmticai
Managammijar Waste RaehaSm, both available from the American
Chemical Society's Department of Government Relations ami Science
Policy, 1155 16* Street N.W, Washington, DC 20036.

17.0 Personnel Qualifications

It is required that qualified tedmicians with appropriate taing ami background
ml conduct the procedures described in this document Ttase using this SOP

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will have demonstrated that they ire able to pedum ill aspects of the SOP

thoKMgkly and inJepenieHilj prior to ccUecting research data.

17.1 Training

17.1.1 Not ieqmnd of anyone wis prepared or significantly contributed to

the preparation of this SOP.

17.1.2 Personnel assigned to implement the tasks in this SOP are required to
read and understand the prateiures herein. The EPA Principe
iHwestigilOT or designee (or contractor Woii Plan Manager),

"trainee" will isriew the contents of this document with lie EPA (or
coBfai^c) personnel assigned to thjs SOP and observe its
implementation from shit' to finish. If hands-on training appears to I*
learned, it will "be provided.

II this SOP is for an instrument, proficiency is demonstrated Mowing
tie applicable sections of fine Danraistratioa of Analyst Proficiency
paragraph of ORD policy PPM13.4 Minimum QA/QC Procedures fox
the OKD Labontniies Coeductmg Research.

17.2 Training' Documentation

17.2.1 EPA, mm-contract staff: when the trainer has detenmned a tramee can
soccessfiiily implement this SOP m the collection. of research data,
he/she will provide to the Division QA Manger withm 30 days, tie
SOP name, tame, trainer, aid date of"the debanination. He QAM
will add this Mbimatian to Hue SOP table in WED's QA database.,

17.2.2 Contract stiff: saecfssfii training will be documented on the
Employe Training Record fam and signed by the supervisor, A copy
of the contractor Employee Acceptance Fonn for this SOP signed by
contract staff, will be forwanfai to the contract Program Quality

Assurance Manager He Work Plan Mansgo- is encouraged, to inform
the WACOR when staff u newly trained on this SOP. providing names
of trainee and trainer,, aad applicable date. The WACOR will pass this
mfixmution on to tie WED QAM.

18.0 References

APHA. Standard Methods for the Examination of Water and
Wastewater. 20^ Edition. 1998. American Public Health

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Association, 1015 Fifteealb Street, N W, WaesJungbm DC, 20005,
Method 10300 D.

Society far Testing ¦

D1193-99el ASTMr Fbiladd^riaa, PA,

CJeiowi, Di, and D.V Peck (Ed* ). Envncamaital Monitoring and
Assessment Program: Integrated Qnality Assurance Project Plan for the
SimficeWaim Resouiw Omap, 1994 Activities. EPA
Rev. 2 00. U.S. EnvinnuDeatai Ftotectian Agency, las Vegas, NV 1994.

Pftck, D V . J M. liEoedjak, and D i. Kkmm In pea, Western Pint
Study Field Operations Manual for WadeaMe Streams. U.S.

Environmental Protection Agency. Washington. DC. 2005

l/l

Quality Assurance Ban, Willamette Keseardx Station, Analytical <

laboratory. Dynamac Cotporation. Revision 4, May 2012. Corvallts,

Oregon.

U.S. EPA. Western Ecology Diwiaoii, IfcalSi anl SaMy HanAotA, U.S. O

Envnmmeiifal Protection Agency, National Health and Emnroameiital ^

Eff«isReaaniiLaliiai*toiy3Coc*allis,OE JF7333, Janmy 2010. J

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2#J Apptmiix A

Procedure for Ash Free Dry Mass Problem Samples

Samples nctnttdfivm the field that contain more' than JO mL of water osr* coKadmred to
bt problem samples due to the -nutcr noshing sample off of tit* fiber used to detwmim
ash free dr, mari. Samples are subjected to extra filtration In order to mirimm the soBit
MMtmiatjfoM tk« wafer in the tube

1. Remove the sample fan freessr ami record sample ID, sample volumes, and
colectioB date on laboratory data sheets (see Section 19.0). In Hie ennments,
recuii '(he amount of wafer eadb. problem sanpfc cxatains.

2. Allow samples with mare than 10 mL of water to fully defrost before beginning
the sample preparation.

3. Assemble cleaned filter apparatus with new. previously baked„ GF/F filter and
apply vacuum.

4. Whenever possible, remove the sample filtw fiom fc centrifuge tube and pi tee in

8H. »wph boat. Empty the witef fen tW c®ntnfog6 tot® in to the

filtration apparatus. Using reagent water, rinse the centri&ge tube to ensure all
sample nnieiiaJ is filtered.

5. Rinse 'lie filter apparatus with reagent water to maximize the recovery of sample.

6. Continue suction for stleast 30 seconds ifis i visible water pan through the
filter.

7. Carefully remove the filter from 'the apparatus and place it in tie an™ ahirmi—
weigh boat u fc aiigna! fiher

i. Cover samples with foil to prevent filtas from Mowing out and dry the sample in
finoed-air oven at60°C for 24 hours.

9 Place wann, dry samples in ¦ desiccator to cool. Weigh samples in aluminum
wagUwats axkl record to fc nearest 0. i mg). This is tbe imhal samrte weight.

10 Plaice fc samples in i mnffle fiimace md bnng the fanparatiire to 550°C. Ash
the samples in a mnffle fiimace by hnMmg at 530"C for 30 minutes.

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11. Allow tie furnace to cool to ajpmxiHHtelf 2WC. Remove tie samples from the
muffle furnace and place in a desiccator to finish cooling. After ashing, lie
pedjiMe should appear gray aui crumbly

12. Saturate the cooled simples with reifgnt waiter to rehjiisie the clays.

13. Cover samples with foi to pewent filters from Mowing out and dry the samples in
a farad-ask own at 60°C for 24 hams.

14 Store tbe dry samples m a desiccator to cool Weijpi the aunties m dumiaum
weigh-boits and record the weight oh the datasheet (see Section I S:.§) to he
neanest 01 mg His u the final (ash fiee) sample weight

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APPENDIX F: EXAMPLE SOPS FOR MERCURY IN FISH TISSUE PLUG
ANALYSIS

Please see separate attachement for Appendix G

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