United States Environmental Protection Agency
Office of Water
Washington, DC
EPA 841-R-14-008
National Coastal Condition
Assessment
2015
Laboratory Operations Manual
Version 2.0 July 2015
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NOTICE
The goal of the National Coastal Condition Assessment (NCCA) is to provide a comprehensive
assessment of the Nation's freshwater, marine shoreline and estuarine waters. The complete
documentation of overall project management, design, methods, and standards is contained in
four companion documents, including:
National Coastal Condition Assessment: Quality Assurance Project Plan EPA 841-R-14-005
National Coastal Condition Assessment: Site Evaluation Guidelines EPA 841-R-l4-006
National Coastal Condition Assessment: Field Operations Manual EPA 841-R-l4-007
National Coastal Condition Assessment: Laboratory Methods Manual EPA 841-R-l4-008
This document (Laboratory Operations Manual) contains information on laboratory methods for
analyses of the samples collected during the National Coastal Condition Assessment (NCCA). It
also provides quality assurance objectives, sample handling procedures, and data reporting
requirements. Methods described in this document are to be used specifically in work relating to
the NCCA 2015. All NCCA Cooperator laboratories must follow the guidelines presented in the
document.
With the exception of the requirements in Chapter 4 for evaluating algal toxics, mention of trade
names or commercial products in this document does not constitute endorsement or
recommendation for use. Chapter 4 requires use of a specific kit and supplemental materials
manufactured by a single firm.
More details on specific methods for site evaluation, sampling, and sample processing can be
found in the appropriate companion document.
The suggested citation for this document is:
USEPA. National Coastal Condition Assessment 2015: Laboratory Operations Manual. EPA-
841-R-14-008. U.S. Environmental Protection Agency, Office of Water, Washington, DC. 2014.
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NOTICE II
1.0 INTRODUCTION 9
2.0 GENERAL LABORATORY GUIDELINES 11
2.1 RESPONSIBILITY AND PERSONNEL QUALIFICATIONS 11
2.2 ROLES AND CONTACT INFORMATION 11
2.3 SAMPLE TRACKING 11
2.4 REPORTING 12
3.0 ALGAL TOXIN (MICROCYSTIN) IMMUNOASSAY PROCEDURE 14
3.1 SUMMARY OF THE PROCEDURE 14
3.2 HEALTH AND SAFETY WARNINGS 15
3.3 DEFINITIONS AND REQUIRED RESOURCES (PERSONNEL, LABORATORIES, AND EQUIPMENT) 15
3.3.1 Definitions 15
3.3.2 General Requirements for Laboratories 17
3.3.3 Personnel 17
3.3.4 Equipment/Materials 18
3.4 SAMPLE RECEIPT 19
3.5 PROCEDURE 20
3.5.1 Sample Preparation: Freeze-Thaw Steps 20
3.5.2 Additional Sample Preparation for Samples with Salinity>3.5 parts per thousand 21
3.5.3 Kit Preparation 22
3.5.4 Insertion of Contents into Wells 23
3.5.5 Dilutions (if needed) 28
3.6 QUALITY MEASURES 29
3.6.1 Assistance Visits 29
3.6.2 QC Samples 29
3.6.3 Summary of QA/QC Requirements 30
3.7 SAMPLE AND RECORD RETENTION 31
3.8 REFERENCES 32
4.0 BENTHIC MACROINVERTEBRATES 33
4.1 SUMMARY OF METHOD 33
4.2 HEALTH AND SAFETY WARNINGS 33
4.3 DEFINITIONS AND REQUIRED RESOURCES (LABORATORY, PERSONNEL, AND EQUIPMENT) 34
4.3.1 Definitions 34
4.3.2 Laboratory 37
4.3.3 Personnel 37
4.3.4 Equipment/Materials 38
4.4 SAMPLE RECEIPT 39
4.5 SAMPLE PREPARATION AND PICKING ORGANISMS 40
4.6 TAXONOMIC IDENTIFICATION 42
4.7 DATAENTRY 48
4.8 SAMPLE AND RECORD RETENTION 48
4.9 EXTERNAL TAXONOMIC QUALITY CONTROL 48
4.10 QUALITY ASSURANCE/QUALITY CONTROL (QA/QC) 52
4.11 REFERENCES 54
ATTACHMENT 4.1: BENTHIC MACROINVERTEBRATES: TAXONOMY BENCH SHEET (EXAMPLE) 55
5.0 WHOLE BODY FISH PROCESSING AND CONTAMINANT ANALYSIS 56
5.1 SUMMARY OF THE PROCEDURE 56
5.2 HEALTH AND SAFETY WARNINGS 56
5.3 DEFINITIONS AND REQUIRED RESOURCES (PERSONNEL, LABORATORIES, AND EQUIPMENT) 57 pq
5.3.1 Definitions 57 O
5.3.2 General Requirements for Laboratories 58 H
5.3.3 Personnel 59
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5.3.4 Equipment/Materials 59
5.4 SAMPLE RECEIPT 60
5.5 WHOLE FISH PREPARATION AND HOMOGENIZATION PROCEDURES 61
5.5.1 Sample Classification: Routine or Non-Routine 61
5.5.2 Fish Examination and Preparation 62
5.5.3 Equipment Cleaning and Rinsate Collection 64
5.5.4 Compositing and Homogenization Procedure 65
5.6 CONTAMINANT ANALYSIS: REQUIREMENTS 67
5.7 DATA ENTRY 70
5.8 QUALITY MEASURES 72
5.8.1 Assistance Visits 72
5.8.2 QC Samples 72
5.8.3 Summary of QA/QC Requirements 73
5.9 SAMPLE AND RECORD RETENTION 76
5.10 REFERENCES 76
6.0 SEDIMENT CONTAMINANT, GRAIN SIZE, AND TOC ANALYSES 78
6.1 SUMMARY OF THE PROCEDURE 78
6.2 HEALTH AND SAFETY WARNINGS 78
6.3 DEFINITIONS AND REQUIRED RESOURCES (PERSONNEL, LABORATORIES, AND EQUIPMENT) 78
6.3.1 Definitions 79
6.3.2 General Requirements for Laboratories 80
6.3.3 Personnel 80
6.3.4 Equipment/Materials 81
6.4 SAMPLE RECEIPT 81
6.5 LABORATORY ANALYSIS: REQUIREMENTS 82
6.6 DATAENTRY 85
6.7 QUALITY MEASURES 86
6.7.1 Assistance Visits 86
6.7.2 QC Samples 87
6.7.3 Summary of QA/QC Requirements 87
6.8 SAMPLE AND RECORD RETENTION 90
6.9 REFERENCES 91
7.0 WATER CHEMISTRY AND CHLOROPHYLL A 92
7.1 SUMMARY OF THE PROCEDURE 92
7.2 HEALTH AND SAFETY WARNINGS 92
7.3 DEFINITIONS AND REQUIRED RESOURCES (PERSONNEL, LABORATORIES, AND EQUIPMENT) 93
7.3.1 Definitions 93
7.3.2 General Requirements for Laboratories 94
7.3.3 Personnel 95
7.3.4 Equipment/Materials 95
7.4 SAMPLE RECEIPT 95
7.5 PREPARATION OF WATER CHEMISTRY ALIQUOTS 96
7.6 WATER CHEMISTRY AND CHLOROPHYLLS ANALYSIS: REQUIREMENTS 98
7.7 DATAENTRY 102
7.8 QUALITY MEASURES 103
7.9 SAMPLE AND RECORD RETENTION 108
7.10 REFERENCES 108
8.0 SEDIMENT TOXICITY TESTING 110
8.1 SUMMARY OF THE PROCEDURE 110
8.2 HEALTH AND SAFETY WARNINGS 110
8.3 DEFINITIONS AND REQUIRED RESOURCES (PERSONNEL, LABORATORIES, AND EQUIPMENT) 110 ^
8.3.1 Definitions Ill (J
8.3.2 General Requirements for Laboratories Ill H
8.3.3 Personnel 112 O
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8.3.4 Equipment/Materials 113
8.4 SAMPLE RECEIPT 113
8.5 TOXICITY TESTING: REQUIREMENTS 114
8.6 DATAENTRY 116
8.7 QUALITY MEASURES 117
8.7.1 Assistance Visits 118
8.7.2 QC Samples 118
8.7.3 Summary of QA/QC Requirements 118
8.8 SAMPLE AND RECORD RETENTION 120
8.9 REFERENCES 120
9.0 FISH TISSUE FILLET (GREAT LAKES) 121
10.0 MERCURY IN FISH TISSUE PLUGS 122
10.1 SUMMARY OF THE PROCEDURE 122
10.2 GENERAL REQUIREMENTS FOR LABORATORIES 122
10.2.1 Personnel 122
10.2.2 Equipment/Materials 122
10.3 SAMPLE RECEIPT 123
10.4 QUALITY MEASURES 124
10.4.1 Assistance Visits 124
10.4.2 QC Samples 124
11.0 FECAL INDICATOR: ENTEROCOCCI 126
APPENDIX A: LABORATORY REMOTE EVALUATION FORMS 127
APPENDIX B: TARGET FISH SPECIES FOR WHOLE FISH ANALYSES 134
APPENDIX C: EXAMPLE SOPS FOR MERCURY IN FISH TISSUE PLUG ANALYSES 138
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LIST OF ACRONYMS
ADT analysis decision tree
AFDM ash-free dry mass
ANC acid neutralizing capacity
ANS Academy of Natural Sciences
AQM absolute quantitation method
ASTM American Society for Testing and Materials
Avg Average
BHI brain heart infusion
BV biovolume
Ca Calcium
CAS Chemical Abstracts Service assigns unique identifiers to chemicals
CCE calibrator cell equivalents
CEQ cell equivalent
Chl-a chlorophyll-a
Cl Chloride
CO2 carbon dioxide
Ct threshold cycle
CPR cardiopulmonary resuscitation
cv curriculum vitae
DCF dilution/concentration factor
DDT dichloro-diphenyl-trichloroethane
Dl de-ionized
DIG differential interference contrast
DL detection limit
DMA Deoxyribo-nucleic Acid
DO dissolved oxygen
DOC dissolved organic carbon
DTH depositional targeted habitat
DW distilled water
ELISA enzyme-linked Immunosorbent assay
EMAP Environmental Monitoring and Assessment Program
ENT enterococci
EPA Environmental Protection Agency
ETON ethyl alcohol
FOM Field Operations Manual
g grams
GEQ genomic equivalent
CIS geographic information system
GPS global positioning device
HCI hydrogen chloride
HOPE high density polyethylene
HMOs nitric acid
HRP antibody-Horseradish Peroxidase
H2S hydrogen sulfide
H2SO4 sulphuric acid
IBD ionic balance difference
ID Identification
IM information management
IPC internal positive control
ISBN International Standard Book Number
ISO International Organization for Standardization
IT IS Integrated Taxonomic Information System (IT IS)
K potassium
kg kilograms
L Liters
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LCR
LCS
LFB
LFM
LIMS
LOM
LRL
mg
mg/kg
Mg
ml
MDL
Mn
MPCA
MSDS
N
Na
NABS
NALMS
NARS
NAWQA
ND
NELAC
NELAP
ng
NH4
NIST
NO2
NO3
NRSA
NIL
NTU
OD
ORD
OSHA
OW
PAH
PAR
PBS
PCB
PctDIFF
PDE
PCR
PE
PES
PHab
P-M
PDE
ppb
ppm
ppt
PSE
PT
PTD
QA
QAPP
Labeled Compound Recovery
Laboratory Control Sample
Laboratory Fortified Blanks
Laboratory Fortified Matrices
Laboratory Information Management System
Laboratory Operations Manual
Laboratory Reporting Limit
milligrams
milligrams per kilogram
magnesium
milliliters
method detection limit
manganese
Minnesota Pollution Control Agency
Materials Safety Data Sheet
nitrogen
sodium
North American Benthological Society
North American Lakes Management Society
National Aquatic Resource Surveys
National Water Quality Assessment Program
non-detect
National Environmental Laboratory Accreditation Conference
National Environmental Laboratory Accreditation Program
nanograms
ammonium
National Institute of Standards
nitrite
nitrate
National Rivers and Streams Assessment
no template control
Nephelometric Turbidity Units
optical density
EPA's Office of Research and Development
Occupational Safety and Health Administration
EPA's Office of Water
Polycyclic Aromatic hydrocarbons
Photosynthetically Active Radiation
phosphate buffered saline
polychlorinated biphenyl
percent difference
percent disagreement in enumeration
polymerase chain reaction
performance evaluation
performance evaluation samples
physical habitat
Palmer-Maloney (P-M) count
percent difference in enumeration
parts per billion
parts per million
parts per trillion
percent sorting efficiency
performance testing
percent taxonomic disagreement
quality assurance
Quality Assurance Project Plan
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QA/QC
QC
QCCS
QMP
qPCR
QRG
RL
RMSE
RO
RPD
ROM
RSD
RTH
Sb
SEG
SFS
Si02
S04
SOPs
SPC
S-R
SRM
SS
1MB
TN
TOC
TP
TRANS
TSN
TSS
TVS
M9
M9/9
M9/L
UNK
USGS
WSA
WQX
quality assurance/quality control
quality control
Quality Control Check Sample
Quality Management Plan
quantitative polymerase chain reaction
Quick Reference Guide
reporting limit
root mean square error
reverse-osmosis
Relative Percent Difference
relative quantitation method
Relative Standard Deviation
richest targeted habitat
antimony
Site Evaluation Guidelines
Society of Freshwater Science
silica
sulphate
Standard Operating Procedures
sample processing control
Sedgewick-Rafter count
standard reference material
salmon sperm
tetramethylbenzidine
total nitrogen
total organic carbon
total phosphorus
transect
taxonomic serial number
total suspended solids
total volatile solids
micrograms
micrograms per gram
micrograms per liter
unknown
United States Geological Survey
Wadeable Streams Assessment
Water Quality Exchange
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1.0 INTRODUCTION
This manual describes methods for laboratory analyses of the samples to be collected during the
National Coastal Condition Assessment (NCCA). The manual includes quality assurance
objectives, sample handling specifications, and data reporting requirements.
The NCCA is one of a series of water assessments conducted by States, Tribes, the U.S.
Environmental Protection Agency (EPA), and other partners. In addition to coastal waters, the
National Aquatic Resource Surveys (NARS) also focuses on rivers and streams, 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 NCCA is to address two key questions about the quality of the Nation's coastal
waters:
What percent of the Nation's coastal waters are in good, fair, and poor condition for key
indicators of water quality, ecological health, and recreation?
What is the relative importance of key stressors such as nutrients and contaminated
sediments?
The NCCA is a probability-based survey of our Nation's coastal and estuarine waters, and
designed to:
Assess the condition of the Nation's coastal and estuarine waters at national and regional
scales, including the Great Lakes;
Identify the relative importance of selected stressors to coastal and estuarine water
quality;
Evaluate changes in condition from previous National Coastal Assessments (NCA)
starting in 2000; and
Help build State and Tribal capacity for monitoring and assessment and promote
collaboration across jurisdictional boundaries.
EPA selected the sampling locations using a probability based survey design. Sample surveys
have been used in a variety of fields (e.g., 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.
The NCCA field sampling season will be during the index period of June through the end of ฃ
September. Field crews will collect a variety of measurements and samples from the statistically 2
selected sampling locations identified by geographical coordinates. The samples are shipped to Q
laboratories to evaluate the indicators identified in Table 1.1. The indicators are similar to those ^
evaluated in previous NCA. O
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Table 1.1 NCCA: Indicators
Measure/Indicator
Water
Quality
Sediment
Quality
Biological
Quality
Dissolved oxygen
PH
Temperature
Depth
Conductivity (freshwater)
Salinity (marine)
Secchi/light measurements
PAR
Nutrients:
Dissolved inorganic NO2 , NOs
NH4 ,PO4;
Total N and P
Chlorophyll a
Grain size (Silt/Clay content)
Total Organic Carbon (TOC)
Sediment chemistry
15 metals
25 PAHs
20 PCBs
14 pesticides
6 DDT metabolites
Sediment toxicity (10-day static
bioassay with Leptocheirus or Hyalella)
Whole body fish contaminants
13 metals (no Sb or Mn)
20 PCBs
14 pesticides
6 DDT metabolites
Optional: PAHs (national lab
only)
Benthic community structure
Assessment outcome
Hypoxia/anoxia
Water column characterization
Societal value and ecosystem
production
Nutrient enrichment
Influencing factor for extent and
severity for contamination
Influencing factor for extent and
severity for contamination
Risk of biological response to
sediment contamination
Biological response to sediment
exposure
Environmentally available contaminant
exposure
Biological response to site conditions
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2.0 GENERAL LABORATORY GUIDELINES
Laboratory Operations Manual
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This chapter describes the general laboratory guidelines with an overview to the quality
assurance / quality control (QA/QC) requirements. Each of the following chapters describes a
different procedure and the relevant QA/QC requirements for that particular procedure. In
addition, the Quality Assurance Project Plan (QAPP) provides a comprehensive consolidation of
the QA/QC requirements for NCCA 2015.
2.1 Responsibility and Personnel Qualifications
Each laboratory shall train its laboratory personnel in advance in the use of equipment and
procedures used for the standard operating procedure (SOP) in which they are responsible. All
personnel are responsible for complying with all of the QA/QC requirements that pertain to the
samples to be analyzed. Each laboratory follows its institutional or organizational requirements
for instrument maintenance. Appendix A identifies the specific documentation that each
laboratory must submit to demonstrate its qualifications for performing the analyses.
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 Coordinator. The Team is responsible
for overseeing all aspects of the project and ensuring that the laboratories properly adhere to the
technical and quality assurance requirements. The Team is the final authority on all decisions
regarding laboratory analysis.
The NARS Information Management (IM) Coordinator tracks the location of each NCCA
sample that involves post-processing. The coordinator will be the labs main point of contact in
regards to sample tracking and data submission.
Table 2.1 NCCA: Contact Information
Title*
EPA HQ NCCA Project
Lead
EPA HQ NCCA Project
QA Lead
EPA HQ NCCA
Laboratory Review
Coordinator
EPA HQ NARS Team
Leader
Information
Management Center
Coordinator
Name
Treda Grayson, OW
Hugh Sullivan, OW
Kendra Forde, OW
Sarah Lehmann, OW
Marlys Cappaert,
SRA International
Inc.
Contact Information
grayson.treda@epa.gov
202-566-0916
sullivan.hugh@epa.gov
202-564-1763
forde.kendra@epa.gov
202-564-0417
lehmann.sarah@epa.gov
202-566-1379
cappaert.marlys@epa.gov
541-754-4467
541 -754-4799 (fax)
*For any technical direction, laboratories under contract to EPA must contact the Task Order's
Contracting Officer's Representative (TOCOR) instead of the contacts provided in this table. For any
technical information or sample tracking, the laboratories are permitted to contact these persons.
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 sites 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
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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. Each laboratory will make arrangements with the NARS IM Coordinator, listed
above, to ensure access is granted.
When the samples arrive from the field crews, the shipments will include tracking forms (refer to
the NCCA FOM). These forms will list the samples included in the shipment. Laboratory
personnel must cross check the forms with the samples received to verify that there are not any
inconsistencies. If any sample is missing or damaged, contact the NARS IM Coordinator
immediately.
2.4 Reporting
All labs must provide data analysis information to the HQ Project Management Team and the
NARS IM Center by March 30, 2016 or as stipulated in contractual agreements. These reports
must include the data elements specified for each analytical method in this manual. The
submitted filename must use the following naming convention:
Indicator name (ex: microcystins)
Date of files submission to NARS IM Center by year, month, and day (ex: 2015_11_01)
Laboratory name (ex: MyLab)
Combined, the file name would look as follows: Microcystins_2015_l l_01_MyLab.xlsx
Before the laboratory submits the batch data to EPA, the analyst who generated the data and an
experienced data reviewer independently check and review the data, as follows:
The analyst shall review the data to ensure that:
Sample preparation information is correct and complete;
Analysis information is correct and complete;
The appropriate method and standard operating procedures were followed;
Analytical results are correct and complete; ^
Quality control samples were within established control limits; S
Blanks (where appropriate) were within the appropriate QC limits; and j
Documentation is complete. Q
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The data reviewer shall review the data package to verify that: ^
Calibration data (where appropriate) are scientifically sound and appropriate; p^
QC samples were within established control limits; ^
Qualitative and quantitative results are correct; and 55
Documentation is complete. O
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Accompanying its data submission for each batch, the laboratory shall provide a short narrative ^
that includes the following information:
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Discussion of any protocol deviations that may have occurred during sample testing;
Discussion of QC questions or issues that were encountered and the corrective measures
taken;
Definitions of any laboratory QC codes used in the data;
Summary and discussion of samples that are diluted by the presence of an interference,
non-target analyte, or target analyte; and
QC samples exceeding established control limits or parameters required by laboratory
internal analytical SOPs and an explanation of why, if known.
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 NCCA 2015 Project Lead. Unless
otherwise authorized by the Project Lead, the laboratory shall retain:
The sample materials, including vials, for a minimum of three (3) years from the date the
EPA publishes the 2015 NCCA report. During this time, the laboratory shall maintain the
materials at the temperature specified in its laboratory method. The laboratory shall
periodically check the sample materials for degradation. Unless the Project Lead arranges
for transfer of sample materials to EPA, at the end of the retention period, the laboratory
shall follow its internal protocols for disposal.
Original records, including laboratory notebooks and raw data files (including logbooks,
bench sheets, and instrument tracings), for a minimum often (10) years from the date that
EPA publishes the final report.
The Project Lead is responsible for maintaining the following:
Deliverables from contractors and cooperators, including raw data, which are permanent
as per EPA Record Schedule 258.
EPA's project records which under Schedule 501 are permanent.
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3.0
ALGAL TOXIN (MICROCYSTIN) IMMUNOASSAY
PROCEDURE
This chapter describes an immunoassay procedure that measures concentrations of total
microcystins in water samples. In applying the procedure, the laboratory uses Abraxis'
Microcystins-ADDA Test Kits (Figure 3.1; "kits"). Each kit is an enzyme-linked immunosorbent
assay (ELISA) for the determination of microcystins and nodularins in water samples.
Microcystins refers to the entire group of toxins, all of the different congeners, rather than just
one congener. Algae can produce one or many different congeners at any one time, including
Microcystin-LR (used in the kit's calibration standards), Microcystin-LA, and Microcystin-RR.
The different letters on the end signify the chemical structure (each one is slightly different),
which makes each congener different.
MICROCYS1INS NODULAHINS
ELISA KIT
mm
Figure 3.1 Microcystins: Abraxis Test Kit
(Converted from color to grayscale from James, page 3, 2010)
3.1 Summary of the Procedure
The procedure is an adaptation of the instructions provided by Abraxis for determining total
microcystins concentrations using its ELISA-ADDA kits.1 For samples samples with
salinity<3.5 parts per thousand (ppt), the procedure's reporting range is 0.15 |ig/L to 5.0 |ig/L,
although, theoretically, the procedure can detect, not quantify, microcystins concentrations as
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1 Abraxis, "Microcystins-ADDA ELISA (Microtiter Plate): User's Guide R021412." Retrieved on January 14, 2014
from
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df.
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low as 0.10 ng/L. For samples with higher concentrations of microcystins, the procedure
includes the necessary dilution steps. The procedure also provides additional sample preparation
steps for samples with salinities>3.5 ppt. The results then are adjusted by a factor of 1.75 for a
reporting range of 0.263 |ig/L to 8.75 |ig/L.
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.
2. Laboratory personnel must wear proper personal protection clothing and equipment (e.g.
lab coat, protective eyewear, gloves).
3. When working with potential hazardous chemicals (e.g., weak acid), laboratory personnel
must avoid inhalation, skin contact, eye contact, or ingestion. Laboratory personnel must
avoid contacting skin and mucous membranes with the TMB and stopping solution. If
skin contact occurs, remove clothing immediately. Wash and rinse the affected skin areas pq
thoroughly with large amounts of water. 3
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This section provides definitions and required resources for using the procedure. ^
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3.3.1 Definitions ^
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The procedure uses the following terms: g
Absorbance (A) is a measure of the amount of light absorbed by a sample at a specific 5
wavelength. A standard statistical curve is used to convert the absorbance value to the ^
concentration value of microcystins. ง
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Brackish and Seawater Samples, for the purposes of the ABRAXIS microcystins test Q
procedure, are samples with salinity greater than or equal to 3.5 parts per thousand (ppt). Q
(EPA is using different definitions for the water chemistry samples.) EPA recognizes that O
brackish water is usually defined as 0.5 ppt, and seawater as 35 ppt, but for this immunoassay ^
procedure, it is important to use additional steps described in Section 3.5.2 for any sample g
with salinity greater than or equal to 3.5 ppt. The sample labels provide the salinity levels. X
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Calibration Range is the assay range for which analysis results can be reported with j
confidence. For example, assays of undiluted samples with salinities<3.5 ppt range from the ^
reporting limit of 0.15 |ig/L to a maximum value of 5.0 |ig/L. H
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Coefficient of Variation (CV): The precision for a sample is reported in terms of the percent
CV of its absorbance values. To calculate the %CV, first calculate the standard deviation, S,
as follows:
5 =
1/2
where n is the number of replicate samples, At, is the absorbance measured for the /th
replicate. Per Section 3.5.4, 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:
5
%CV =
x 100
A
Dark or Dimly Lit: Away from sunlight, but under incandescent lighting is acceptable.
Detection Limit is the minimum concentration at which the analyte can be detected with
confidence. In other words, the outcome can be reported with confidence that it is greater
than zero (i.e., present in the sample). The detection limit is less than the reporting limit at
which the measured value of the analyte can be reported with confidence. Also see "Sample-
Specific Detection Limit."
Duplicates are defined as two aliquots of the same sample which are analyzed separately Q
using identical procedures. The results are used to evaluate the precision of the laboratory W
analyses. Per Section 3.5.4, controls are evaluated in duplicate or triplicate (i.e., three O
aliquots). ^
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NARS: National Aquatic Resource Surveys. The National Coastal Condition Assessment &o
(NCC A) is part of the NARS program. ^
O
NARS Information Management System (NARS IM): The EVI system established to 5
support all surveys, including NCCA, in the NARS program. The EVI system is used to track <5
the samples from field collection to the laboratory. ซ
O
NCCA: National Coastal Condition Assessment. Freshwater and coastal samples will be
collected during the field stage of NCCA.
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 g
procedure presents the quality control requirement in terms of %CV instead of RSD. ^
K>
Reporting Limit: A reporting limit is the point at which the measured value of the analyte O
L^
can be reported with confidence. -,
3
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Sample-Specific Detection Limit: Most samples will have a sample-specific detection equal
to the method's detection limi. For diluted samples, the sample-specific detection limit will
be the product of the method's detection limit and the dilution factor. Typical values for the
dilution factor will be 10 or 100.
Seawater Sample: See definition for brackish and seawater samples.
3.3.2 General Requirements for Laboratories
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.
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). 3
To demonstrate its ongoing commitment, the person in charge of quality issues for the Q
organization shall sign the NCCA QAPP Certification Page. W
O
3.3.3 Personnel ^
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The procedure refers to the following personnel: &o
<
Laboratory Technician: This procedure may be used by any laboratory technician who 2
is familiar with the NCCA Quality Assurance Project Plan, and this procedure in the 5
NCCA Laboratory Operations Manual (which differs from the Abraxis instructions). The Q
laboratory technician also must be familiar with the use of a multichannel pipette and ซ
plate readers. JZ
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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 o
QC contractor must be dedicated to QA/QC functions, and thus, must not be a primary ^
laboratory or a field sampling contractor for NCCA. The QC contractor is responsible for S
complying with instructions from the External QC Coordinator; coordinating and paying ^
for shipments of the performance samples to participating laboratories; comparing H
immunoassay results from the laboratories; and preparing brief summary reports. O
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3.3.4 Equipment/Materials
The procedures require the following equipment and information:
Abraxis ADDA Test Kit, Product #520011 (see items in Section 3.5.2)
Adhesive Sealing Film (Parafilm) for Micro Plates (such as Rainin, non-sterile, Cat.
No. 96-SP-100): Used to cover plates during incubation.
Data Template - See Figure 3.2
Distilled or Deionized Water: For diluting samples when necessary.
ELISA evaluation software
Glass scintillation, LC, vials (two vials of 20 mL each)
Glass vials with Teflon-lined caps of size:
o 20mL
o 4 mL (for dilutions)
Multichannel Pipette & Plastic 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; ChroMateฎ; or equivalent
readers with software to read the microtiter plates and measure absorbances). 9
Reagent Reservoirs (e.g., Costar Cat Number 4870): Plain plastic reservoir for g
reagents that accommodate the use of a multi-channel pipette. W
Test tubes (glass): For dilutions, if needed. O
Timer: For measuring incubation times. ^
Vortex Genie: For mixing dilutions. ^
^f
Whatman Glass fiber syringe filter (25mm, GF 0.45 |im filter) &o
<
Analysis of samples with salinity>3.5 ppt require additional equipment and supplies, as follows: S
o Microcystins-ADDA Seawater Sample Clean-Up Kit (Product #529912) which 5
includes the following supplies: a
Disposable 5 %" glass Pasteur pipettes ^
Disposable 9" glass Pasteur pipettes ง
Glass wool ^
Pasteur pipette bulb ^
Microcystins-ADDA Seawater Sample Treatment Solution O
Microcystins-ADDA Seawater Sample Clean-up Resin Q
o 12x75 mm test tubes ง
o Scoopula ^
o Micropipettes with disposable plastic tips H
o Vortex mixer O
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3.4 Sample Receipt
Field crews hold the microcystins samples on ice while in the field and then pack the samples in
ice for delivery to a central facility ("batching laboratory") or the State's laboratory. The
batching and State laboratories freeze the samples upon receipt. Periodically, the batching
laboratory ships samples to the microcystins laboratory. The batching and microcystins
laboratory may retain the frozen samples for several months before analysis.
Because EPA initiates tracking procedures designed to recover any missing shipment, the
laboratory personnel responsible for tracking samples must start the following login steps within
24 clock hours of receiving a delivery.
1. Report receipt of samples in the NARS IM sample tracking system (within 24 clock
hours). Alternatively, for shipments with a large number of samples, the laboratory may
email a spreadsheet with the sample login and sample condition information to NARS-IM
(see Chapter 2 for contact information).
2. Inspect each sample THE SAME DAY THEY ARE RECEIVED:
a. Verify that the sample IDs in the shipment match those recorded on the:
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. 2
b. Record the information in Table 3.1 into NARS IM, including the Condition Code for Q
each sample: ฃj
i. OK: Sample is in good condition O
ii. C: Sample container was cracked PH
iii. L: Sample container is leaking ฃl
iv. ML: Sample label is missing GO
v. W: Sample is warm (>8ฐ), record the temperature in the comment field, and
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FIELD
SAMPLE ID
DATE COLLECTED
CONDITION CODE
CONDITION
COMMENT
FORMAT
numeric
MMDDYY
text
text
DESCRIPTION
Sample id as used on field sheet (on sample
label)
Date sample was collected
Condition codes describing the condition of the
sample upon arrival at the laboratory.
Flag
OK
C
L
ML
W
Q
Definition
Sample is in good condition
Sample container is cracked
Sample or container is leaking
Sample label is missing
Sample is warm (>8ฐ)
Other quality concerns, not identified
above
Comments about the condition of the sample. If
the condition code=W then provide the
temperature
3.5 Procedure
The following sections describe the sample and kit preparation and analysis.
3.5.1 Sample Preparation: Freeze-Thaw Steps
For each frozen sample (500 mL per sample), the laboratory technician runs it through a freeze-
thaw cycle three times to lyse the cells as follows:
1. All cycles: Keep the samples in dark or dimly lit areas (i.e., away from sunlight, but
under incandescent lighting is acceptable).
2. First freeze-thaw cycle:
a. Start with a frozen 500 ml sample.
b. Thaw the sample to room temperature (approximately 25ฐ C). Swirl the sample to
check for ice crystals. At this temperature, no ice crystals should be present in the
sample.
c. Shake well to homogenize the sample, then transfer 10 mL to an appropriately
labeled clean 20 mL glass vial.
O
O
GO
GO
3. Second freeze-thaw cycle:
a. Freeze the vial.
b. Keep the large sample bottle (from the 500 mL initial sample) frozen for future
use.
c. Thaw the sample vial contents to room temperature.
4. Third freeze-thaw cycle:
a. Freeze the vial.
b. Thaw the vial contents to room temperature.
c. Filter the vial contents through a new, syringe filter (0.45 jim) into a new, labeled
20 mL glass scintillation vial. Norm-ject syringes and Whatman Glass fiber
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syringe filters (25mm, GF 0.45 jam filter) or other similar alternative are
acceptable. Use one new syringe and filter per sample.
3.5.2 Additional Sample Preparation for Samples with Salinity>3.5 parts
per thousand
For any sample with salinity of 3.5 parts per thousand (ppt) or greater (the salinity will be
marked on sample vials), the laboratory technician needs to perform the following additional
steps provided by Abraxis. 2 For all other samples (i.e. with salinity less than 3.5 ppt), the
technician skips this section (i.e., Section 3.5.2) and goes directly to kit preparation as described
in Section 3.5.3. For samples with salinity 3.5 ppt the technician:
1 . Prepares the column as follows:
a. Place a small amount of glass wool into the top of a 5 3/4" glass Pasteur pipette.
Using a 9" glass Pasteur pipette, push the glass wool into to the bottom of the 5
%" pipette to form the base of the column. The depth of the glass wool should be
approximately 5 mm. Place the column into a 12x75 mm test tube.
b. Each column will require approximately 1.5 g of Seawater Sample Clean-Up
Resin. Calculate and add the appropriate amount of Microcystins-ADDA
Seawater Sample Clean-Up Resin to a 20 mL glass vial.
c. Add distilled or deionized water at an approximately 2: 1 ratio to the Microcystins-
ADDA Seawater Sample Clean-Up Resin (for example, 10 mL of deionized or
distilled water per 5 g of Resin). Shake or vortex. m
d. Pipette the Resin in water solution into the column using the 9" Pasteur pipette. n
Avoid the formation of air bubbles in the column bed by keeping the tip of the Q
pipette at the surface of the bed being created. Fill the column to the indentation o
approximately 2 cm from the top of the pipette. This will create an approximately ง
8 cm column.
e. Allow the deionized or distilled water to drain from the column. 3. Lift the tip of
the column at least 1 cm above the surface of the water in the tube. Place the %
pipette bulb against the top of the column (do not attach the bulb to the column) ^
and push the remaining water out of the column. Avoid allowing the tip of the g
column to come into contact with the water in the tube to prevent aspiration of G
water back into the column. 5
f Place the column into an appropriately labeled 4 mL glass vial. ^
ฃ_i
2. Cleans up the sample as follows: vi
a. Add 1 mL of the sample to a clean, appropriately labeled 4 mL glass vial. Add 50 Q
uL of Microcystins-ADDA Seawater Sample Treatment Solution. Vortex. Q
O
3
2 Reformatted from Abraxis, "Microcystins in Brackish Water or Seawater Sample Preparation" Retrieved on R
January 14, 2014 from http://abraxiskits.com/uploads/products/docfiles/385 MCT- Q
ADDA%20in%20Seawater%20Sample%20Prep%20%20Bulletin%20R041112.pdf. Reproduced with permission. H
Except for Abraxis' solutions labeled as seawater, EPA has removed references to "brackish" and "seawater" which H-]
typically are defined as having different cutpoints than 3.5 ppt for salinity. %.
d
3 Additional correspondence between EPA and Abraxis notes that this step leaves the resin in the column. "^
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b. Add 375 uL of the treated sample to the top of the column. Allow the sample to
drain through the column and collect in the vial.
c. Add a second 375 uL aliquot of the treated sample to the column. Allow to drain
through the column.
d. Lift the tip of the column at least 1 cm above the surface of the sample in the vial.
Place the pipette bulb against the top of the column (do not attach the bulb to the
column) and push the remaining sample out of the column. Avoid allowing the tip
of the column to come into contact with the sample in the vial to prevent
aspiration of the sample back into the column.
e. Lower the column back into the vial. Add 500 uL of distilled or deionized water
to the top of the column. Allow the rinse to drain through the column and collect
with the sample.
f. Lift the tip of the column at least 1 cm above the surface of the sample/rinse in the
vial. Place the pipette bulb against the top of the column (do not attach the bulb to
the column) and push the remaining rinse out of the column. Avoid allowing the
tip of the column to come into contact with the sample in the vial to prevent
aspiration of the sample back into the column.
g. Remove the column and discard (columns are single use only). Cap vial and
vortex. The sample can then be analyzed using the Abraxis Microcystins-ADDA
ELISA Kit beginning with the next section (3.5.3).
3.5.3 Kit Preparation
The technician prepares the kits using the following instructions: 2
1. Check the expiration date on the kit box and verify that it has not expired. If the kit has g
expired, discard and select a kit that is still within its marked shelf life. (Instead of W
discarding the kit, consider clearly labelling it as expired and keeping it for training o
activities.) g
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2. Verify that each kit contains all of the required contents: &o
Microtiter plate
o S5: 5.0 |ig/L O
Kit Control (KC): 0.75 |ig/L g
Antibody solution ^
Anti-Sheep-HRP Conjugate ^
Wash Solution 5X Concentrate n
Color Solution O
L^
Stop Solution j
Diluent
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3. If any bottles are missing or damaged, discard the kit. This step is important because
Abraxis has calibrated the standards and reagents separately for each kit.
4. Adjust the microtiter plate, samples, standards, and the reagents to room temperature.
5. Remove 12 microtiter plate strips (each for 8 wells) from the foil bag for each kit. The
plates contain 12 strips of 8 wells. If running less than a whole plate, remove unneeded
strips from the strip holder and place in the foil bag, ziplocked closed, and store in the
refrigerator (4-8ฐ C).
6. Prepare a negative control (NC) using distilled water.
7. The standards, controls, antibody solution, enzyme conjugate, color solution, and stop
solutions are ready to use and do not require any further dilutions.
8. Dilute the wash solution with 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.
9. Handle the stop solution containing diluted FhSCu with care. Q
3.5.4 Insertion of Contents into Wells Q
O
This section describes the steps for placing the different solutions into the 96 wells. Because of p4
the potential for cross contamination using a shaker table, the following steps specify manual ^
shaking of the kits instead mechanized shaking.
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A
B
C
D
E
F
G
H
1
SO
SO
S1
S1
S2
S2
S3
S3
2
S4
S4
S5
S5
KG
KG
KG
NC
3
NC
NC
U1
U1
U2
U2
U3
U3
4
U4
U4
U5
U5
U6
U6
U7
U7
5
U8
U8
U9
U9
U10
U10
U11
U11
6
U12
U12
U13
U13
U14
U14
U15
U15
7
U16
U16
U17
U17
U18
U18
U19
U19
8
U20
U20
U21
U21
U22
U22
U23
U23
9
U24
U24
U25
U25
U26
U26
U27
U27
10
U28
U28
U29
U29
U30
U30
U31
U31
11
U32
U32
U33
U33
U34
U34
U35
U35
12
U36
U36
U37
U37
U38
U38
U39
U39
Key:
SO-S5 = Standards;
KG = Control supplied with Kit (i.e., Kit Control);
NC = Negative Control;
U = Unknown (sample collected by the field crew);
Figure 3.2 Microcystins: Template for samples
5. Add 50 uL of the pink antibody solution to each well using the multi-channel pipettor
and a reagent reservoir. Use dedicated reagent reservoirs for each reagent to avoid
contamination from one reagent to another.
6. Place the sealing Parafilm over the wells.
7. Manually mix the contents by moving the strip holder in a rapid circular motion on the
benchtop for 30 seconds. Be careful not to spill the contents.
8. Place the plate in a dimly lit area (as defined in Section 3.3.1) for 90 minutes.
9. After 90 minutes, carefully remove the Parafilm.
10. Empty the contents of the plate into the sink, pat inverted plate dry on a stack of paper
towels, and then wash the wells of the plate three times with 250 uL of washing solution
using the multi-channel pipette. After adding the washing solution each time, empty the
solution into the sink and use the paper towels as before.
11. Add 100 jiL of enzyme conjugate solution to all wells using the multi-channel pipettor.
12. Cover the wells with Parafilm.
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13. Manually mix the contents by moving the strip holder in a rapid circular motion on the
benchtop for 30 seconds. Be careful not to spill the contents.
14. Place the strip holder in a dimly lit area for 30 minutes.
15. After 30 minutes, remove the Parafilm, decant, and rinse the wells three times again with
250 jiL of washing solution as described in step 10.
16. Add 100 jiL of color solution to the wells using the multi-channel pipette and reagent
reservoir. This color solution will make the contents have a blue hue.
17. Cover the wells with Parafilm.
18. Manually mix the contents by moving the strip holder in a rapid circular motion on the
benchtop for 30 seconds. Be careful not to spill the contents.
19. Place the plate in a dimly lit area for 20 minutes.
20. After 20 minutes, remove the Parafilm and add 50 jiL of stopping solution to the wells in
the same sequence as for the color solution. This will turn the contents a bright yellow
color. After adding the stopping solution, read the plate within 15 minutes.
21. Within 15 minutes of adding the stopping solution, use the microplate ELISA photometer 2
(plate reader) to determine the absorbance at 450 nm. The software (i.e., commercial g
ELISA evaluation program) calculates the absorbance and concentration values of the W
samples from the calibration curve and the average values for each pair. Use a 4- o
parameter standard curve fit to determine the concentrations. g
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22. Dispose of solution in plates in a lab sink. Rinse plates and sink with water to dilute the J/3
weak acid present. ^
O
23. Perform QC evaluations of the data as follows: 5
a. If the following failures occur, then the laboratory must reanalyze all samples in <
the analytical run: Q
i. Standard curve with a correlation coefficient, R, of less than 0.99 g"
ii. Standards SO-S5 must have decreasing absorbance values. First, calculate H
the average values for each standard. That is, if Ai is the absorbance >H
average for Si, then the absorbance averages must be: Q
Ao> Ai _
iii. The average absorbance of the standard SO less than 0.8 (i.e., Ao < 0.8). S
iv. Two or more negative control sample results report detectable ^
concentrations of microcystins (i.e., values > 0.1 |ig/L). If this occurs, then R
evaluate possible causes (e.g., cross-contamination between samples), and o
if appropriate, modify laboratory processes before the next analytical run. ^
v. Results for control samples of outside the acceptable range of 0.75 +/-
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b. If either, or both, of the following situations occur, then the sample must be
reanalyzed (maximum of two analyses,4 consisting of the original analysis and, if
necessary, one reanalysis):
i. The concentration value registers as HIGH (exceeds the calibration
range).5 Dilute the sample for the reanalysis per Section 3.5.5.
ii. The %CV > 15% between the duplicate absorbance values for a sample.
24. If the sample has a salinity of 3.5 ppt or greater, then convert the results by multiplying
by 1.75. If the assay was non-detected, then the detection limit is 0.175 |ag/L. The
reporting limit is 0.263 |ag/L. The calibration range is 0.263 |ag/L to 8.75 |ag/L.
25. Record the results, even if the data failed the quality control requirements in #23b, for
each well in EPA's data template (see Table 3.2 for required elements). The required
entries are for the following columns:
a. TYPE indicates the sample type using one of the following codes: SO-S5 for
standards; KC or NC for controls; and U for unknown sample.
b. CONC contains the numeric concentration value. Two special cases:
i. Non-detected concentrations: If the sample is non-detected, then provide
the sample-specific detection limit which is 0.1 |ig/L if the sample is
undiluted with a salinity<3.5 ppt in the sample. See step 24 for reporting
values for samples with salinity >3.5 ppt. See Section 3.5.5 for calculating
the sample-specific detection limit for a diluted sample. pj
ii. If the result shows that it is "HI," this indicates that the sample value is ง
outside of the calibration range and must be diluted and re-run using Q
another analytical run. Leave the CONC column blank and record 'HF in Q
the DATA FLAG column. O
p4
c. DATA FLAGS have codes for the following special cases: PH
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 m
|ig/L (for undiluted samples with salinity <3.5 ppt; see step 24 for samples 3.5 ppt); ง
iii. HI if the concentration value registers as HIGH (exceeds the calibration P
range). a
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 ง
used for all failures to facilitate data analysis. ^
ii. Q for any other quality issue (describe in COMMENTS) ^
e. DILUTION FACTOR is only required if the sample was diluted. O
PH
O
4 In its data analyses, EPA compares the microcystins data values to 10 ug/L, which is the World Health R
Organization threshold for moderate risk. If a sample is diluted once following the procedures in Section 3.5.5 and Q
the concentration still registers as HIGH, the concentration is recorded as >50 ug/L which is greater than the WHO H
threshold. EPA does not require additional dilution to obtain a more precise value, but a laboratory may choose to i]
increase the dilution of the sample and report the associated concentration value. ^
5 A value of HIGH is not a QA/QC failure, but rather indicates a necessity to find the correct dilution to get it within j
calibration. "^
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f. DUP AVG and DUP CV are required for duplicate samples and control samples
(use all three values if the controls are used in triplicate).
Table 3.2 Microcystins: Required Data Elements
STAGE
LOGIN
ANALYSI
S
FIELD
LAB ID
DATE RECEIVED
SITE ID
VISIT NUMBER
SAMPLE ID
DATE
COLLECTED
CONDITION CODE
CONDITION
COMMENT
TECHNICIAN
ANALYSIS DATE
ANALYSIS TIME
KIT EXPIRE DATE
KIT ID
R2
TYPE
LOCATION
FORMAT
Character
MMDDYY
Character
Numeric
Numeric
MMDDYY
Character
Character
Character
MMDDYY
24-hour time
MMDDYY
Character
Numeric
Character
Character
DESCRIPTION
Name or abbreviation
for QC laboratory
Date sample was received by lab
NCCAsite ID code as
tracking form (blank if
recorded on sample label or
standard or control)
sequential visits to site (1 or 2) (blank if standard or
control)
6-digit Sample ID number as recorded on sample
jar or tracking form (blank if standard or control)
Date sample was collected (blank if standard or
control)
Sample condition upon arrival at the laboratory
(blank if standard or control)
Flag
Blank or N
OK
C
L
ML
W
Definition
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 iswarm (>8ฐ)
Comments about the condition of the sample. If the
condition code='W then provide the temperature
Name or initials of technician performing the
procedure
Date when samples are inserted into the wells per
Section 3.5.4
Time when 1st sample
Section 3.5.4
is inserted into the wells per
Expiration date on kit box
Kit identification code.
a unique code to each
If one does not exist, assign
kit.
R2 from curve fit to the average absorbance values
for the standards. Value is between 0 and 1 .
Type of solution being
tested in the well
Code Definition
KG Kit Control
NC Negative Control
SO,S1,S2,S3, Standard
S4, S5
U Sample of unknown
concentration
Location of well in the kit (e.g., B5 would be the
fifth well from the left in the second row B)
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STAGE
FIELD
SALINITY
CONG
ABSORBANCE
DILUTION
FACTOR
CV_ABSORB
AVG_ABSORB
AVG_CONC
DATA FLAG (if
appropriate)
QUAL FLAG
COMMENTS
FORMAT
Numeric
Numeric
Numeric
Numeric
Numeric
Numeric
Numeric
Character
QCF/Q
Character
DESCRIPTION
If the sample vial has the salinity marked on the
vial, record the value in units of parts per thousand.
Otherwise, leave blank.
Concentration or sample-specific detection limit of
contents of well in ug/L. Sample-specific detection
limit should be 0.1 ug/L for a sample with salinity
<3.5 ppt which hasn't been diluted. (Detection limit
is 0.1 75 ug/L for samples with salinity >3.5 ppt)
Absorbance value
1 0, 1 00, etc for number of times the sample was
diluted. If not diluted, leave blank or record 1
Calculated %CV of duplicate values of absorbance
fora sample. Only calculated forTYPE=U, KG, or
NC. Enter %CV. Value is between 0 and 100%.
Calculated average of absorbance values for a
sample. Only provided forTYPE=U, KG, NC, or
SC. Average value of the original sample and its
duplicate (or replicates for KG and NC).
Calculated average of
sample. Substitute for
reporting limit.
concentration values for a
any value below the
Data qualifier codes associated with specific
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
ND
HI
J
QCF
Q
Definition
Concentration below
detection.
Result indicated a high
concentration (i.e., outside
calibration range)
Concentration above
detection but below
reporting limit.
QC failure
Other quality concerns, not
identified above
Explanation for data flag(s) (if needed) or other
comments.
3.5.5 Dilutions (if needed)
Dilutions if needed are prepared as follows (using clean glass tubes):
1. 1:10 dilution
a. Add 900 jiL of distilled water to a clean vial. (Note: Dilutions may also be made
using the kit's diluent rather than distilled water.)
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b. Pipette 100 jiL from the sample into the vial. (To provide more accurate dilutions
and less chance of contaminating the diluent, add the diluent to the vial before the
sample.)
c. Mix by vortexing.
d. Multiply final concentration and Abraxis' detection limit by 10 to obtain the
sample-specific detection limit.. For example, for a sample with salinity<3.5 ppt,
Abraxis' detection limit is 0.1 |ig/L and the sample-specific detection would be
1.0|ig/L for a 1:10 dilution.
2. 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 jiL 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 by 100 to obtain the
sample-specific detection limit. For example, for a sample with salinity<3.5 ppt,
Abraxis' detection limit is 0.1 jig/L and the sample-specific detection would be 10
|ig/L for a 1 : 100 dilution.
3 . Other dilutions can be calculated in the same manner as #1 and #2 if needed.
3.6 Quality Measures
This section describes the quality assurance and quality control measures used to ensure that the g
data will meet NCCA's requirements. W
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3.6.1 Assistance Visits g
Assistance visits are intended to familiarize EPA with actual procedures being implemented by >H
different laboratories; and to ensure a clear and consistent understanding of procedures and ^
activities by both EPA and the laboratories. If EPA decides to conduct an assistance visit, a ^
qualified EPA scientist or contractor will administer a checklist based upon the steps described in O
this chapter. EPA will develop, review and approve the checklist prior to conducting an R
assistance visit. ง
3.6.2 QC Samples B
The External QC Coordinator will instruct the QC contractor to provide one or two identical sets H
of freshwater and/or seawater performance test samples to all participating laboratories. If the
laboratory will assay both freshwater and seawater samples, then it will receive both sets (i.e.,
freshwater and seawater). Each set will contain five samples to test the expected range of
concentrations in the NCCA samples. S
For the contract laboratory, the QC contractor will provide the first set to be run with the first set R
of samples and a second set to be run at the midpoint of the assigned samples. If available, a o
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
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Each laboratory will run the QC samples following the same procedures used for the other
samples. The External QC Coordinator will compare the results and assess patterns in the data
(e.g., one laboratory being consistently higher or lower than all others). Based upon the
evaluation, the External QC Coordinator may request additional information from one or more
laboratories about any deviations from the Method or unique laboratory practices that might
account for differences between the laboratory and others. With this additional information, the
External QC Coordinator will determine an appropriate course of action, including no action,
flagging the data, or excluding some or all of the laboratory's data.
3.6.3 Summary of QA/QC Requirements
Table 3.3 provides a summary of the quality control requirements described in Sections 3.5 and
3.6.
Table 3.3 Microcystins: Sample analysis quality control activities and objectives
Quality
Control
Activity
Description and Requirements
Corrective Action
Kit - Shelf Life
Is within its expiration date listed on kit box.
If kit has expired, then discard or
clearly label as expired and set
aside for training activities.
Kit - Contents
All required contents must be present and in
acceptable condition. This is important
because Abraxis has calibrated the standards
and reagents separately for each kit.
If any bottles are missing or
damaged, discard the kit.
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Calibration
All of the following must be met:
Standard curve must have a correlation
coefficient of >0.99;
Average absorbance value, Ao, for SO must be
>0.80; and
Standards SO-S5 must have decreasing
average absorbance values. That is, if Ai is the
average of the absorbance values for Si, then
the absorbance average values must be: Ao>
Ai > A2 > As > A4 >As
If any requirement fails:
Results from the analytical run are
not reported.
All samples in the analytical run
are reanalyzed until calibration
provides acceptable results. At its
discretion, the lab may consult with
EPA for guidance on persistent
difficulties with calibration.
Kit Control
The average concentration value of the
duplicates (or triplicate) must be within the
range of 0.75 +/- 0.185 ug/L. That is, the
average must be between 0.565 ug/L and
0.935 ug/L.
Negative Control
The values for the negative control replicates
must meet the following requirements:
All concentration values must be < 0.15 ug/L
(i.e., the reporting limit; and
one or more concentration results must be
nondetectable (i.e., <0.10 ug/L)
If either requirement fails:
Results from the analytical run are
not reported
The lab evaluates its processes,
and if appropriate, modifies its
processes to correct possible
contamination or other problems.
The lab reanalyzes all samples in
the analytical run until the controls
meet the requirements.
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Sample
Evaluations
All samples are run in duplicate. Each
duplicate pair must have %CV<15% between
its absorbance values.
If %CV of the absorbances for the
sample>15%, then:
Record the results for both
duplicates using different start
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Quality
Control
Activity
Description and Requirements
Corrective Action
dates and/or start times to
distinguish between the runs..
Report the data for both duplicate
results using Quality Control
Failure flag "QCF"; and
re-analyze the sample in a new
analytical run. No samples are to
be run more than twice.
If the second run passes, then the
data analyst will exclude the data
from the first run (which will have
been flagged with "QCF"). If both
runs fail, the data analyst will
determine if either value should be
used in the analysis (e.g., it might
be acceptable to use data if the CV
is just slightly over 15%).
Results Within
Calibration Range
All samples are run in duplicate. If both of the
values are less than the upper calibration
range (i.e., < 5.0 ug/L for undiluted samples
with salinity<3.5 ppt; < 8.75 ug/L for undiluted
samples with salinity >3.5 ppt), then the
requirement is met.
If a result registers as "HIGH", then
record the result with a data flag of
"HI." If one or both duplicates
register as 'HIGH,' then the sample
must be diluted and re-run. No
samples are to be run more than
twice. The lab reports both the
original and diluted sample results.
External Quality
Control Sample
External QC Coordinator, supported by QC
contractor, provides 1-2 sets of identical
samples to all laboratories and compares
results.
Based upon the evaluation, the
External QC Coordinator may
request additional information from
one or more laboratories about any
deviations from the Method or
unique laboratory practices that
might account for differences
between the laboratory and others.
With this additional information, the
External QC Coordinator will
determine an appropriate course of
action, including no action, flagging
the data, or excluding some or all
of the laboratory's data.
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3.7 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.
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After the stated time periods, the laboratory shall follow its internal protocols for disposal.
3.8 References
Abraxis, "Microcystins-ADDA ELISA (Microtiter Plate)," Product 520011, R021412, Undated.
Retrieved January 2014 from
http://www.abraxiskits.com/uploads/products/docfiles/278 Microcystin%20PL%20ADDA%20u
sers%20R120214.pdf.
Abraxis, "Microcystin-ADDA ELISA Kit, Detailed Procedure," Undated. Retrieved January
2014 from http://www.abraxiskits.com/uploads/products/docfiles/253 PN520011FLOW.pdf.
Abraxis, "Microcystins in Brackish Water or Seawater Sample Preparation" Undated. Retrieved
on January 2014 from http://abraxiskits.eom/uploads/products/docfiles/3 85JVICT-
ADDA%20in%20Seawater%20Sample%20Prep%20%20Bulletin%20R041112.pdf.
Loftin, K.A., et al., "Comparison of Two Cell Lysis Procedures for Recovery of Microcystins in
Water Samples from Silver Lake in Dover, Delaware, with Microcystin Producing
Cyanobacterial Accumulations," in USGS Open-File Report 2008 -1341. 2008. Retrieved April
2013 from http://pubs.usgs.gov/of/2008/1341/pdf/of2008 1341.pdf.
James, R., et al., "Environmental Technology Verification Report: Abraxis Microcystin Test
Kits: ADDA ELISA Test Kit; DM ELISA Test Kit; Strip Test Kit," in Environmental ง
Technology Verification System Center 2010. Retrieved March 2013 from 3
http://nepis.epa. gov/Adobe/PDF/P 1OOEL6B .pdf g
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Kamp, L. (Abraxis) "Re: question about instructions for brackish water or seawater"; Email to g
M. Smith (EPA). June 23, 2015. >H
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4.0 BENTHIC MACROINVERTEBRATES
This chapter describes the steps for identifying benthic macroinvertebrate organisms in samples
collected in coastal waters and the Great Lakes during the 2015 National Coastal Condition
Assessment (NCCA). Field crews preserve samples in the field with formalin and ship them to a
central holding facility or directly to the laboratory. Because NCCA samples generally have
fewer than 400 organisms, this procedure requires the laboratory to fully sort and identify all
organisms in the sample. If, upon initial inspection, a sample appears likely to have more than
400 organisms, contact the EPA HQ Laboratory Review Coordinator (see contact information in
Chapter 2) for processing instructions. (EPA may require use of the subsampling procedures
such as those described in the Laboratory Operations Manual for the 2013-2014 National Rivers
and Streams Assessment (NRSA)).6
In the following discussion, Sections 4.1, 4.2, and 4.3 summarize the procedure; health and
safely concerns; and definitions and required resources. Section 4.4 provides the steps for
acknowledging sample receipt. Section 4.5 provides the steps for preparing and picking
organisms from the sample. Sections 4.6 - 4.8 provide the steps for the taxonomy identification;
data entry; and sample and record retention. Sections 4.9 and 4.10 describe EPA's external
review of laboratory operations and quality measures. Section 4.1 1 identifies references used in
developing the procedure. Attachment 4.1 provides an example of a taxonomic bench sheet.
4.1 Summary of Method
The procedure describes the steps for picking and identifying organisms from sediment samples.
This section provides a summary of the procedure and quality control measures.
The sorter evenly distributes each sample across a tray(s) and then picks all organisms from the
sample. During the identification step, a taxonomist identifies all organisms to the target
taxonomic levels for the survey and discards materials that do not meet the identification criteria.
For each species or lowest identifiable taxonomic level, the taxonomist includes at least one
representative organism in the laboratory ' s reference collection for NCCA 20 1 5 . ^
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As part of the quality control measures, a second taxonomist will re-identify a subset (usually p4
10%) of the samples to quantify enumeration and taxonomic precision, or consistency, as percent pq
difference in enumeration (PDE) and percent taxonomic disagreement (PTD), to help target ^
corrective actions, and ultimately to help minimize problems during data analysis. P
4.2 Health and Safety Warnings O
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In addition to the laboratory's requirements, persons using this procedure must abide by the S
following health and safety procedures: o
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2;
6 USEPA, 2013, National Pavers and Streams Assessment 2013-14: Laboratory Operations Manual EPA 841-B-12- pq
010. PQ
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1. Wear proper personal protection clothing and equipment (e.g. lab coat, protective
eyewear / goggles).
2. When working with potential hazardous chemicals (e.g. Rose Bengal) or biological
agents (benthic organisms and sediments), avoid inhalation, skin contact, eye contact, or
ingestion. If skin contact occurs, remove clothing immediately and wash / rinse
thoroughly. Wash the affected skin areas thoroughly with large amounts of soap and
water.
4.3 Definitions and Required Resources (Laboratory, Personnel, and
Equipment)
This section provides definitions and required resources for using this procedure. Section 4.3.1
defines the terms used throughout the procedure. Section 4.3.2 describes the expertise required
for each laboratory using the procedure. Section 4.3.3 describes the roles and responsibilities of
the personnel involved in the procedure. Section 4.3.4 identifies the equipment necessary to
apply the procedure in preparing, sorting, and identifying benthic macroinvertebrate organisms
in samples.
4.3.1 Definitions
The procedure uses the following throughout the document:
Dissecting microscope: Microscope 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 4.6 provides the steps used to ^
identify which taxa are flagged. p
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Good quality digital photograph: Good quality means that other taxonomists can PQ
readily identify the taxon from one or multiple photographs and the library can readily H
locate the photographs. To ensure that the photographs meet these objectives, the image 3
must be:
Taken through the microscope at a high enough resolution so that the key g
diagnostic features are distinguishable and clear. Include all features that would
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o Only one taxon in the photo. If necessary, the laboratory may edit (e.g.,
crop) the digital photograph and save the file with a new filename as
specified below. Both the original and edited files must be included in the
digital library.
o A scale bar or measurements in an appropriate location to indicate the size
of the specimen.
o One specimen that lies flat on the surface instead of tilted (to the extent
practicable).
Saved using a format that preserves the image in the highest resolution possible.
Saved with a filename that is consistent within the digital library and shall include
the following elements in the order listed below:
o NCCA2015
o Laboratory name (or abbreviation)
o Sample number
o Taxa name
o Magnification (if applicable, otherwise indicate no magnification as "Ix")
o Date (format YYYYMMDD) that the photograph was taken.
o Appendage of "e" if the photograph was edited (e.g., cropped).
For example, on September 8, 2015, laboratory ABC identified the specimen in
sample 1234 to be a Capitella capitata and took a digital photograph at a
resolution of 40x and then cropped the photograph to eliminate extraneous
material. The filenames of the original and edited photographs would be:
NCCA2_ABC_1234_ capitella capitata_40x_20150908.gif and
NCCA2_ABC_1234_ capitella capitata_40x_20150908e.gif.
Elutriate: Circulate water over the sample in order to wash away the lighter or finer
particles of the detritus.
Inorganic material: Material that is not capable of further decay (e.g., gravel, sand, silt)
Integrated Taxonomic Information System (ITIS): Database with standardized, vi
reliable information on species nomenclature and their hierarchical taxonomic H
classification. ^
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NARS: National Aquatic Resource Surveys. The National Coastal Condition Assessment H
(NCC A) is part of the NARS program. 3
NARS Information Management (IM) System: The IM system established to support Q
all surveys, including NCCA, in the NARS program. The IM system is used to track the g
samples from field collection to the laboratory. <
^
NCCA: National Coastal Condition Assessment. The samples are collected during the u
field stage of NCCA. H
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Organic material: Material derived from living organisms that is capable of further
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 independent
sorter (B) for a sample (sorter B sorts through pickate and counts only organisms missed
by Sorter A).
PSE = A x 100
A + B (i)
Percent disagreement in enumeration (PDE): measure of taxonomic precision
comparing the number of organisms, ni, counted in a sample by the primary taxonomist
with the number of organisms, ซ2, counted by the internal or external QC taxonomist.
n, -n7
PDE = -xlOO
ni
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, TV is the total number of
organisms in the larger of the two counts.
PTD =
1-
comPpos
xlOO
(3)
Pickate: This is the remaining material left from the tray after the sorter has removed all
benthic macroinvertebrates. This could include small stones, sticks or leaves, etc.
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 &o
original identifications for the sample. H
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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.
a)
4.3.2 Laboratory
The procedure may be used by any laboratory that demonstrates competency in analytical work
and quality procedures as documented by any one or more of the following::
1. Analytical work: To demonstrate its expertise, the laboratory shall provide EPA with one
or more of the following:
a. Memorandum that identifies the relevant services that the laboratory provided for
the National Aquatic Resource Surveys in the past five years.
b. Memorandum describing experience with analyses that are the same or similar to
the requirements of this method.
c. 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.
d. Memorandum that describes the laboratory's participation in round robin studies
and/or performance studies.
e. 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 NCCA 2015 QAPP Certification Page.
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3. Reporting standardized data. To demonstrate its expertise, the laboratory shall provide p
EPA with a memorandum that confirms that the laboratory has a computerized 53
Laboratory Information Management System (LIMS) routinely used to track samples and PQ
record laboratory results. The memorandum also shall confirm that the laboratory will H
use LIMS to record and report results from the procedure. S
4.3.3 Personnel
The procedure may be used by any person who has received training in processing and
identification of benthic macroinvertebrates. For purposes of this procedure, EPA assumes that
the following personnel are responsible for performing specific duties:
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Internal Taxonomy QC Officer provides oversight of daily operations, sample
processing, monitors QC activities at the laboratory to determine conformance, and
conducts performance and systems audits of the procedures. The laboratory must retain
documentation for the qualifications for the Internal Taxonomy QC Officer meeting the
following requirements. The laboratory must provide, or otherwise make available, this
documentation to EPA upon request. The Internal Taxonomy QC Officer is an
experienced taxonomist who:
1. Demonstrated an initial enumeration and identification proficiency (as measured
by PDE<5% and PTD<15%.
2. 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 administrative in nature, the External QC Coordinator is not required to have
laboratory experience, although such experience would be preferable.
External QC Taxonomists, are selected by the External QC Coordinator (after
consultation with EPA experts), and have demonstrated expertise and experience to be
used as a quasi "gold standard" for taxonomic evaluations.
Taxonomists are trained, and have considerable experience, in identifying benthic
macroinvertebrates, i.e., taxonomy. It is also important that the taxonomist maintains
contact with other taxonomists through professional societies and other interactions, and
keeps up with the pertinent literature, since systematics and species identifications change
over time. EPA prefers, but does not require, that the freshwater 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
NCCA samples to the EPA Project QC Officer.
Sorters are laboratory technicians who have basic training in laboratory procedures. An
"experienced" sorter is one that has achieved >90% sorting efficiency in 5 consecutive
samples.
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4.3.4 Equipment/Materials H
The procedure requires the following equipment and materials for sample preparation 53
(subsampling), sorting, and taxonomic identifications. PQ
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4.3.4.1 Sample Preparation (Subsampling) and Sorting S
Equipment/Materials ฃ
U.S. 35 sieve (500 urn) g
Round buckets g
Standardized, possibly, gridded screen (40 Mesh (380-um openings, T304 stainless steel
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Permanent ink pen (e.g Pigma Micronฎ pen)
Dropper
Fine-tipped forceps (watchmaker type, straight and curved)
Vials with caps or stoppers
Sample labels for vials
70-80% ethanol
Stereo zoom microscope (6-1 OX magnification)
4.3.4.2 Taxonomy Identification Equipment/Materials
Stereo dissecting microscope with fiber optics light source (50-60X magnification)
Compound microscope (10, 40, and 100X objectives, with phase-contrast capability)
Digital camera with high resolution capability mounted on a microscope
Petri dishes
Microscope slides (1" x 3" flat, precleaned)
Cover slips (appropriately sized)
CMCP-10 (or other appropriate mounting medium)
Permanent ink pen (e.g Pigma Micronฎ pen)
Dropper
Fine-tipped forceps (watchmaker type, straight and curved)
Vials with caps or stoppers
Sample labels for vials
70 - 80% non-denatured ethanol in plastic wash bottle
Taxonomic Bench Sheet (Attachment 4.1 provides an example)
Hand tally counter
4.4 Sample Receipt
Because EPA initiates tracking procedures designed to recover any missing shipment, the
laboratory personnel should start the following login steps within 24 clock hours of receiving a
delivery.
1. Record receipt of samples in the NARS IM system (within 24 clock hours) and the ^
laboratory's Information Management System (LIMS). Assign the appropriate ^
chronological bench number to each sample. Alternatively, for shipments with a large p4
number of samples, the laboratory may email a spreadsheet with the sample login and pq
sample condition information to NARS-IM (see Chapter 2 for contact information). b
s
2. Inspect each jar THE SAME DAY THEY ARE RECEIVED: g
a. Add 70-80% formalin to the jar, if necessary (i.e., to cover the contents completely). O
b. Verify that the site identification and sample number on the label also appear on the o
chain of custody form in the shipment. ^
c. Notify the EPA HQ Laboratory Review Coordinator (see contact information in
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3. Store the sample containers at room temperature until sorting begins. If the sample will
be stored for a long time before sorting, replace the formalin with ethanol for better
preservation of the organisms.
4. Maintain the chain-of-custody form with the samples; it will be needed if the samples are
transported to any other location (e.g., for taxonomic identification, external QC
evaluation).
5. Verify that the login information includes the required data elements in Table 4.1. After
completing all required elements, provide the information to the data entry personnel.
Table 4.4.1 Benthics Macroinvertebrates Login: Required Data Elements
FIELD
LAB NAME
LAB ID (optional)
DATE RECEIVED
SITE ID
VISIT NUMBER
SAMPLE ID
DATE COLLECTED
SALINITY
CONDITION_CODE
COND COMMENTS
FORMAT
Character
Character
MMDDYY
Character
Numeric
Numeric
Date
Numeric
Character
Character
DESCRIPTION
Name of lab
Lab sample id
Date sample was received by lab
NCCA site identification code as used on sample label
Sequential visits to site (1 or 2, if specified on label)
Sample number as used on field sheet (on sample label)
Date sample was taken
Salinity: Value is provided on the sample label
Condition codes describing the condition of the sample upon
arrival at the laboratory.
Flag
OK
C
L
ML
NP
Q
Definition
Sample is in good condition
Sample container is cracked
Sample or container is leaking
Sample label is missing
Not enough preservative used
Other quality concerns, not identified above
(explain in COND_COMMENTS)
Explanation for Q FLAG (if needed)
4.5 Sample Preparation and Picking Organisms
This section describes the steps for the sorter in preparing the sample and picking organisms.
1. Remove the lid from the sample container and remove the internal sample label.
2. Carefully decant the formalin from the sample container by pouring the fluid through a sieve
(U.S. 35) into a separate container. Inspect the mesh of the sieve for any organisms and
return any organisms found to the sample container so they can be included in the sample
sort process.
3. Remove sieved organisms from the sample container and place into a sorting tray.
4. Sort all samples under a minimum of 6x (maximum of lOx) dissecting microscope. Remove
the macroinvertebrates from the detritus with forceps. In general, do not remove:
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o Empty snail or bivalve shells
o Organisms of water surface-dwelling or strict water column2 arthropod
taxa, and meiofauna.
o Incidentally-collected terrestrial taxa.
o Fragments such as legs, antennae, gills, wings, or tails.
For Oligochaeta, attempt to remove only whole organisms or fragments that include the head.
In other words, do not remove fragments without the head.
o In case of uncertainties, place the organism in the sort vial for the
taxonomist to make the final determination.
5. Place picked organisms of the same type into a single set of jars and vials containing 70-80%
ethanol.
6. This QC step is performed if: 1) the sorter (sorter A) has not reached 90% proficiency in 5
consecutive samples (referred to as the "proficiency QC check" below); or 2) this sample is
the 1 in 10 sample QC check for experienced sorters (referred to as the "periodic QC check"
below). For this step, a second sorter (sorter B):
o Performs QC checks using the same power microscope as the sorter;
o Extracts any missed organisms found in the pickate from Sorter A and
places them into the sample vial, or other suitable sample vial;
o Notes the number of organisms missed; and
o Adds that number to the final count of the sample.
o Calculates the PSE for the sample (see Section 4.3.1 for definition;
equation 1). If the PSE is:
<90% and the sample is the:
Proficiency QC check, a second sorter must check the next
5 samples until the original sorter has PSE>90% for 5
consecutive samples.
Periodic QC check, then a second sorter examines the
original sorter's samples since the last QC check for missed
organisms. The original sorter must again demonstrate
proficiency by achieving a PSE>90% in 5 consecutive
samples. vi
>90% and the sample is the: H
Proficiency QC check, the sample counts towards the 1 in 5 ฃ
consecutive samples used to establish proficiency. P5
Periodic QC check, no corrective action is required. b
o Records the results from the QC step. The laboratory must record the W
results from all QC steps, even if they exceed the frequency required by g
this step. The laboratory must provide the sorter QC results to EPA upon O
request. Q
O
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H
2Strict water column taxa are those that do not have at least one life stage that is benthic (i.e., ง
bottom-dwelling). PQ
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7. Remove the remaining material left on the sorting pan (i.e. material such as sticks, organic
debris) and place it in a separate container with preservative (70-80% ethanol). Label the
container "Pickate," on both internal and external labels.
8. Label the vials and jars of sorted organisms and material using permanent ink (e.g., using a
Pigma Micronฎ pen). Internal sample labels should be made of cotton rag paper or an
acceptable substitute.
9. Retain the vials and materials for the time period specified in Section 4.8.
10. Thoroughly clean all sample preparation and sorting equipment and make sure all equipment
is free of organisms prior to sorting the next sample.
4.6 Taxonomic Identification
The taxonomist performs the following steps in identifying the benthic macroinvertebrate
organisms:
1. Upon receipt of a set of sample vials from the sorter:
a. Compare all site identification codes and sample numbers on the form with those
entered on the labels of samples, and resolve any discrepancies with the sorter.
b. Determine if any vials are broken. For any broken vial, attempt to recover as
much of the sample as possible. Describe the damage in the LAB_COMMENTS
field in the database.
c. Maintain the chain-of-custody form with the sample vials; it will be needed to
return/store them.
2. Empty one sample vial at a time into a small Petri dish. Add 70-80% ethanol to keep the
organisms covered. Remove the internal sample label and complete the top portion of a
Taxonomic Bench Sheet (for an example, see Attachment 4.1), using the information
from the label. Depending on the type of organisms, select the appropriate step:
a. For all Chironomidae organisms, extract the organisms from the Petri dish.
i. Prepare slide mounts using CMCP-10 (or CMC-9, CMC-10, or other 02
media) and applying a coverslip. All organisms must be visible, which H
generally means a maximum of 10-20 organisms per slide. Label the slides ฃ
with the same sample identification code or log-in number as the ethanol S
organisms. H
ii. If the laboratory prefers to use another method than slide mounting, the W
EPA External QC Coordinator will grant a waiver if the following applies: g
1) The request is for a laboratory located at a single location. For o
example, EPA would not consider the combined qualifications of a Q
prime contract laboratory and its subcontract laboratories. Instead, ;<
for whichever laboratories met the requirements, EPA would ^
evaluate and grant (or deny) a waiver for the prime contract a
laboratory separate from each of its subcontractor laboratories. H
ง
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2) The request for a waiver must identify and describe a minimum of
three studies. For each study, the external QC evaluation must
demonstrate that the laboratory met or exceeded the NCCA QC
requirements (i.e., PDE<5% and PTD<15%) for its Chironomidae
organisms.
3) The laboratory agrees to mount the organisms on slides if it fails
one of the periodic (NCCA) external QC evaluations, as follows:
a. It must mount all Chironomidae organisms in samples
processed since the previous external QC evaluation (i.e.,
for which it met the PDE and PTD requirements).
b. It must continue to mount all Chironomidae organisms for
the unprocessed samples.
b. For all other organisms, remove similar organisms to other dishes (keep these
covered with 70-80% ethanol).
3. View the sample to ensure that all necessary diagnostic characters have been observed,
according to the taxonomic key or other literature using:
a. A stereo dissecting microscope for organisms in dishes.
b. A compound microscope for slides of Chironomidae and Oligochaeta organisms
4. Identify organisms to the lowest practical taxonomic level (species is the target for all
organisms with the exception of meiofauna, (due to being smaller than 0.5 mm).
Additional exceptions include Oligochaeta (Class) and Chironomidae (Family) in
samples from marine, polyhaline and mesohaline regions ONLY. If a laboratory or
individual taxonomist is having trouble reaching species for a taxonomic group (not for
an individual organism which might be damaged or otherwise difficult to identify), the
lab must contact the NCCA project lead for guidance. Add any necessary data qualifiers
(see list provided with Required Data Elements in Table 4.2).
a. Enter the Taxonomic Serial Number (TSN) as it appears in the column "Unique
Identifier" of the taxa list provided by EPA.
b. Note whether the identification of a group of organisms is distinct (Distinct=Y/N)
from other organisms in the same sample as follows:
i. If the organisms can be identified to the target level, then Distinct="Y." ^
ii. If an organism cannot be identified to the target level then assign values as p
follows: 53
1) If at least some of the organisms in the sample can be identified to ffl
the target level, then: H
a. Distinct="Y" for organisms identified at the target level; 3
and ฃ
b. Distinct="N" for organisms that were identified at a higher g
taxonomic level (e.g., family) that may contain a target P^
level taxa already identified in a given sample (e.g., genus).
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Veneroida, thenMacoma would be distinct, but Tellinidae
and/or Veneroida would not be Distinct.
2) If none of the organisms in the sample could be identified at the
target level, then:
a. Distinct="Y" for organisms identified at the lowest
taxonomic level (e.g., family); and
b. Distinct="N" for organisms identified at a higher level
(e.g., order).
c. For example, if a taxonomist can identify a number of
Veneroida (Order) families, but a number of the organisms
could not be taken past Veneroida, then the individual
families would be distinct, but the order would not be
distinct.
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Record the identifications. For example, using the taxonomic bench sheet in Attachment
4.1, record the identification in the Column labeled "taxon." Enter the number of larvae,
pupae, and adults, or total count (e.g. mollusks), if appropriate life history column does
not apply, of each taxon under the appropriate columns.
iii. If the target taxonomic level cannot be achieved due to immature or
damaged organisms this should be noted in the data file in the QA_FLAG
field (e.g., QA_FLAG=IM). Table 4.2 provides other codes for the
QA_FLAG field.
iv. If damaged organisms can be identified, they are counted ONLY if the:
1) Fragment includes the head, and, in the case of arthropods, the
thorax;
2) Oligochaetes have a sufficient number of segments in the head;
3) Mollusk shell (bivalve or gastropod) is occupied by a organism;
4) Organism is the sole representative of a taxon in the sample.
v. If a unique taxon is determined for which the appropriate taxonomic level
is not available in the literature and there are other taxa in that taxonomic
level:
1) Provide good quality digital photographs of the organism to
outside experts for identification; and
2) Include the tentative identification in the database with a data
qualifier code of QA_FLAG='UN' so that these organisms can be
distinguished from other organisms in the data analysis.
3) When the outside expert identifies the organism, update the
database with the correct identification.
5. Compare taxa names from the taxa list provided by EPA to the names used for the
identifications. Check the non-matches for the following common problems and correct
them.
a. Abbreviations
b. Extra information identifiers (e.g., sp., spp.,, nr., cf, genus 1, w/ hair chaetae)
c. Extra character (e.g., "?", "Acentrella ?turbida", blank space)
d. The word "probably" or "prob" (e.g., "Microcylloepus prob. similis")
e. Double names (e.g., Callibaetis callibaetis) &o
f Common misspellings p
g. Tribes/subfamilies/subgenus sometimes may not appear 53
h. Species with incorrect genus (Hydatopsyche betteni) PQ
i. Split level taxonomy (e.g., Cricotopus/Orthocladius) H
Invalid name (e.g., taxonomic change, synonym; Sphaeriidae vs. Pisiidae) 3
6. Complete the identification by entering the totals for each developmental stage and the Q
total number of each taxon in the cells at the bottom of the sheet. Cross-check to be sure P^
the totals were summed correctly.
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8. Make two copies of the bench sheet or computer file used to record the identifications.
They are distributed as follows: 1) the project file; and 2) EPA's External QC
Coordinator.
9. 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 citations will be kept on file with
the Internal Taxonomic QC Officer, who will periodically review the reference collection
to ensure that it is complete.
10. Verify that the reference collection contains at least one organism that represents each
genus (or lowest taxonomic level) identified from all sample. For any missing references,
choose an appropriate organism(s) from the sample to represent a taxon name in the
master taxa list:
a. Place the physical specimen in the reference library.
b. Place two labels in the sample container to identify: organisms placed in the
reference collection, and those in the non-reference organisms.
c. Obtain a good quality representative digital photographs of the specimen (see
instructions in Section 4.3.1).
11. If the Internal Taxonomy QC Officer selects the sample for a QC check, the Internal
Taxonomy QC Officer re-counts and re-identifies the organisms in the sample following
the same steps above for the original taxonomist. One in 10 of the taxonomist's samples
must be checked. The Internal Taxonomy QC Officer records the independent
verifications on a bench sheet or computer file. The Internal Taxonomy QC Officer will
also supply a list of taxa that were found to be problematic during their QC sorting check,
which can be submitted in an Excel or Word document format. (If the Internal Taxonomy
QC Officer performs the QC check more frequently, then all QC data must be submitted.)
12. Carefully return the rest of the organisms to the original sample vial, fill with 70-80%
ethanol, and cap tightly. &o
S
13. Re-package the samples and slide-mounted organisms carefully, and sign and date the 53
chain-of-custody form. Return or store the samples according to laboratory protocols and PQ
requirements in Section 4.8. H
I
14. Verify that all required data elements in Table 4.2 have been recorded by the taxonomist ^
and Internal Taxonomy QC Officer. If the results were recorded on paper, provide the Q
Taxonomic Bench Sheet to the data entry personnel. P^
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Table 4.2 Benthic Macroinvertebrates Taxonomic Identification: Required Data o
Elements H
H
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FIELD
LAB NAME
FORMAT
Character
DESCRIPTION
Name of lab
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FIELD
LAB ID (optional)
DATE RECEIVED
SITE ID
VISIT NUMBER
SAMPLE ID
DATE COLLECTED
DATE TAXON
ANALYST NAME
QC VERIFICATION
FAMILY
SUBFAMILY
TRIBE
GENUS GROUP
GENUS
SPECIES
TSN
LAB TIN (OPTIONAL)
TAXANAME
ABUNDANCE
LARVAE
ABUNDANCE PUPAE
ABUNDANCE ADULT
ABUNDANCE TOTAL
DISTINCT
CITATION
QA FLAG (if
appropriate)
QA COMMENTS
LAB COMMENTS
FORMAT
Character
Date
Character
Numeric
Numeric
Date
Date
Character
Character
Character
Character
Character
Character
Character
Character
Numeric
Character
Character
Numeric
Numeric
Numeric
Numeric
Character
Character
Character
Character
Character
DESCRIPTION
Lab sample id
Date sample was received by lab
NCCA
site identification code as used on sample label
Sequential visits to site (1 or 2, if specified on label)
Sample number as used on field sheet (on sample label)
Date sample was taken
Date that the taxonomist started identifying organisms in the
sample
Name of taxonomist or Internal Taxonomy QC Officer (if
record provides results of QC check)
Y if the record provides the results from the QC check
Taxonomic family
Taxonomic subfamily
Taxonomic tribe
Taxonomic genus group (e.g., thienemannimyia)
Taxonomic genus
Taxonomic species
Taxonomic Serial Number as defined by "Uniqueldentifier"
in taxa list provided by EPA. If taxon is not in this list,
provide citation for reference used to identify organism in
CITATION field
Lab taxa ID number
Unique taxon name in the taxa list provided by EPA
Number of individual larvae or immature bugs
Number of individual pupae
Number of individual adults
Total number of individuals
Distinct taxa in sample (y/n) (See description in Section 4.6)
Citation for reference used to identify organism, if taxon not
present in taxa list provided by EPA database
QA/QC flag (lab may use its own flags, if defined in
QA COMMENTS field or provided to NARS IM team)
Flag
DD
IM
IN
NP
NT
S
UN
Q
Definition
Damaged Organism, poor condition or fragments
Immature
Indeterminate (explain in QA_COMMENTS field)
Not enough preservative used
Not able to meet target level for identification (may
be used with other codes, or explain in
QA COMMENTS field)
Sample shipping problem (explain in
QA COMMENTS field)
Unknown. Identification is tentative. Organism has
been sent to expert taxonomist for definitive
identification.
Other quality concerns, not identified above
Explanation for QA FLAG (if needed)
General laboratory analysis comments
H
ง
PQ
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4.7 Data Entry
Tables 4.1 and 4.2 identify the required data elements that the sorting and taxonomic laboratories
must provide to EPA, preferably in EPA's data template, available separately from EPA. In
addition, the laboratory must provide the resume or curriculum vitae for each taxonomist who
identifies benthic macroinvertebrates for the NCCA samples. The resume or cv for each
taxonomist is submitted once to EPA's External QC Coordinator.
4.8 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.
4.9 External Taxonomic Quality Control
EPA requires that all NCCA laboratories ("primary laboratories") participate in the External
Taxonomic Quality Control Evaluation. Each taxonomist must participate in the QC evaluation,
even if the taxonomist is under subcontract with, or consulting for, another firm.
In contrast to the internal QC evaluation in Section 4.6 that verify adherence to the procedures
and ensures in-laboratory consistency between taxonomists, the purpose of the external QC
evaluation is to ensure consistency between laboratories and taxonomists. To achieve this
objective, EPA compares the primary laboratory results to those from a second laboratory, ^
considered a quasi "gold standard" for taxonomic evaluations. p
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The External QC Coordinator, who is an EPA staff member, is responsible for selecting and PQ
managing the "QC contractor." To eliminate the appearance of any inherent bias, the QC H
contractor must be dedicated to QA/QC functions, and thus, must not be a primary laboratory or 3
a field sampling contractor for NCCA. The QC contractor is responsible for complying with ฃ
instructions from the External QC Coordinator; obtaining and managing the secondary g
laboratory; coordinating and paying for shipments of the QC samples between locations; P^
comparing sample identifications by different laboratories; facilitating reconciliation
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The External QC Coordinator will arrange for the QC contractor to conduct a minimum of two
QC evaluations. To the extent practicable, the External QC Coordinator and QC contractor will
schedule batch evaluations evenly throughout the project period.
Each QC evaluation consists of the following steps:
1. In consultation with the QC contractor, the External QC Coordinator determines an
appropriate time to conduct the evaluation based upon the total number of samples
assigned to the laboratory, the delivery schedule, processing schedule, and the following
constraints:
a. Availability of samples from other laboratories. For example, if three state
laboratories are each processing less than 30 samples, the External QC
Coordinator might combine their samples into one batch for the QC evaluation.
b. If a primary laboratory is responsible for processing 100 samples or more for the
NCCA, the External QC Coordinator will split their samples into several batches
(e.g., each 50 to 100 samples) so that EPA can evaluate and correct performance
on an ongoing basis.
2. The External QC Coordinator provides the QC contractor with a list of laboratories and
processed samples. Sample identification includes the site identification code, sample
number, and taxonomist who performed the identifications.
3. The QC contractor randomly selects 10% of the samples from each NCCA laboratory,
subject to the following constraints:
a. If the primary laboratory received fewer than 30 samples, then the QC contractor
randomly selects three samples for the evaluation.
b. For each taxonomist identified on the list, the QC contractor ensures that the
selection includes one or more of his/her samples.
c. The External QC Coordinator may elect to provide an initial evaluation of the
national laboratory by selecting a small batch from the samples that the laboratory
completed in the first 2-3 months.
4. The QC contractor provides a list of the QC samples, and instructions, to the External QC ^
Coordinator and each primary laboratory participating in the evaluation. Although the p
External QC Coordinator and QC contractor may tailor the instructions for the 53
participating taxonomists' preferences, the instructions are likely to specify the PQ
following: H
a. Pack and ship the QC samples to the central holding facility designated by the QC 3
contractor. Instructions are likely to require that the: ฃ
i. Shipments contain chain-of-custody documentation for all slides and Q
containers. P^
ii. Containers (e.g., slides, vials) include the site identification code and
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considerations (e.g., size of animals and amount of ethanol needed for
preservation, amount of ethanol allowed in a single shipment to meet DOT
shipping requirements).
b. Track the QC samples using forms provided by the QC contractor.
c. Email a spreadsheet with the data for the QC samples to the External QC
Coordinator. (EPA requires that all labs use its spreadsheet template for recording
the taxonomic data.)
5. The QC contractor reviews the condition of the QC samples (e.g., verifies that the
containers do not identify taxon for any organism) and ships the samples to the secondary
laboratory along with instructions and the EPA template for reporting data.
6. Within 24 hours of receipt, the secondary laboratory:
a. Notifies the QC contractor that it has received the samples;
b. Faxes or emails any additional receipt records, including discrepancies, within 24
hours; and
c. Completes any other instructions from the QC contractor.
7. The secondary laboratory:
a. Re-identifies and re-counts following the procedures in the Method, except does
not:
i. Develop a reference library.
ii. Photograph organisms unless the taxa are identified for reconciliation
discussion.
iii. Perform any internal QC checks.
b. Records the required data elements in Section 4.7.
c. Enters the data using EPA's spreadsheet template for the taxonomic data.
d. Emails the completed spreadsheet to the QC contractor.
8. The QC contractor compares the original taxonomic results (i.e., data) generated by the
primary laboratory to the taxonomic results generated by the secondary laboratory for
each sample. As part of this evaluation, the QC contractor calculates PDE and PTD using
the equations in Section 4.3.1 and compares their values to the QC requirements in the ^
Section 4.10. $
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9. If any samples exceed the PDE or PTD limits in Section 4.10, the QC contractor consults PQ
with the External QC Coordinator to determine if reconciliation calls are necessary to H
resolve differences. The External QC Coordinator may decide that a reconciliation call is 3
unnecessary if there appears to be an obvious explanation for differences, few samples ฃ
are affected, or other reasons. Q
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10. The QC contractor schedules and facilitates reconciliation teleconferences with EPA and
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ii. The QC contractor provides the participants with a spreadsheet that
includes:
1. List of samples and taxon identifications for discussion;
2. Relevant data from the primary and secondary laboratories; and
3. PDE and PTD values.
iii. The primary and secondary laboratories provide participants with the
relevant reference (or citation) and photograph for each taxonomic
identification for the discussion.
iv. The QC contractor emails a meeting announcement for a convenient time
for all participants. The email identifies instructions for accessing the
External QC Coordinator's toll-free teleconference line.
b. Within a week after the teleconference, the QC contractor sends an email to the
External QC Coordinator and other teleconference participants that summarizes:
i. Agreements to use common nomenclature for discrepancies;
ii. Commitments to reevaluate identifications by reexamining samples;
iii. Application of changes that are appropriate for all samples, not just the
QC samples (e.g., common nomenclature)
iv. Items that will not be resolved for some reason (e.g., sample degraded
during shipment).
11. After completing the reconciliation calls, the participants complete the following steps:
a. Secondary laboratory:
i. Reexamines samples as deemed necessary during the reconciliation call
ii. Updates its database with changes to:
1. QC samples per reexamination and other items in the QC
contractor email; and
2. Non-QC samples as appropriate (e.g., nomenclature changes apply
to all samples, not just QC samples).
iii. Provides database to QC contractor.
b. QC contractor confirms that the secondary laboratory (i.e., its subcontractor)
completed its assignments before allowing the secondary laboratory to move to
the next step.
c. Secondary laboratory stores its original records, including laboratory notebooks ^
and the reference library, for a minimum of 10 years from the date that EPA p
publishes the final report. 53
d. Secondary laboratory and QC contractor follow steps 4 and 5 above to return the PQ
samples to the primary laboratory. H
e. After receiving the samples (and tracking per step 4), the primary laboratory: 3
i. Reexamines samples as deemed necessary during the reconciliation call; ฃ
ii. Updates its database with changes to: Q
1. QC samples per reexamination and other items in the QC P^
contractor email; and
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identified in the QC contractor's email summary of the teleconferences
(from Step lO.b).
f. QC contractor provides EPA with a report or memorandum that:
i. Identifies the participating laboratories, with the following information
about each laboratory:
1 . Laboratory name
2. Address
3. Contact person (name, telephone, and email)
ii. Quantifies the taxonomic precision (PDE and PTD) as they were prior to
the reconciliation call;
iii. Assesses data acceptability;
iv. Highlights taxonomic problem areas;
v. Identifies any discrepancies for which the External QC Coordinator
determined that a reconciliation teleconference was not necessary;
vi. Identifies primary and secondary laboratory commitments to change its
identifications or provide additional review of any organisms; and
vii. Provides recommendations for improving precision for other samples not
included in the QC evaluation.
12. After review, the External QC Coordinator:
a. Submits the report, and draft technical direction with next steps for the laboratory,
to the EPA staff managing or coordinating with the primary laboratory.
b. Determines if significant differences within the batch of QC samples warrant re-
identification of samples by the primary laboratory and a second QC evaluation
by the secondary laboratory. If deemed necessary, EPA will instruct the primary
laboratory to include the samples for review with the next batch of QC samples.
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 chapter. The
objective of the visit would be to:
Confirm the laboratory is properly implementing the steps in the method.
Assist with questions from laboratory personnel. vi
Suggest corrections if any errors are made. H
4.10 Quality Assurance/Quality Control (QA/QC)
PQ
Equation 4.1 Percent sorting efficiency (PSE) ง
Number of organisms found by the sorter (A) compared to the combined (total) number of found ง
by the sorter (A) and the number recovered by the QC Officer (B) from Sorter A's pickate for a ^
sample. PSE should be >90%. g
A O
PSE = xioo a
A+B H
w
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Equation 4.2 Percent disagreement in enumeration (PDE)
Measure of taxonomic precision comparing the number of organisms, ni, counted in a sample by
the primary taxonomist with the number of organisms, ซ2, counted by the internal or external QC
taxonomist. PDE should be <5%.
PDE =
ป!-
^xlOO
+n
Equation 4.3 Percent taxonomic disagreement (PTD)
Measure of taxonomic precision comparing the number of agreements (positive comparisons,
comppos) of the primary taxonomist and internal or external QC taxonomists. In the following
equation, TV is the total number of organisms in the larger of the two counts. PTD should be
PTD =
( comp
pos
N
xlOO
Table 4.3 Benthic Macroinvertebrates: Measurement Data Quality Objectives
Variable or
Measurement
Sort and Pick
Identification
Precision
90% a
85% b
Accuracy
90% a
95% c
NA = not applicable;a As measured by PSE; b As measured by (100%-PTD);c As measured by (100%-PDE)
Table 4.4 Benthic Macroinvertebrates: Laboratory quality control
Check or
Sample
Description
Frequency
Acceptance Criteria
Corrective Action
SAMPLE PROCESSING AND SORTING
Sample pickate
examined by
another sorter
10% of all
samples
(minimum of 1)
completed per
sorter
PSE > 90%
If < 90%, examine all residuals
of samples by that sorter and
retrain sorter
IDENTIFICATION
Duplicate
identification by
Internal Taxonomy
QC Officer
Independent
identification by
outside, expert,
taxonomist
1 in 10 samples
per taxonomist,
All uncertain taxa
PTD < 15%
Uncertain identifications
to be confirmed by expert
in particular taxa
If PTD >1 5%, reidentify all
samples completed by that
taxonomist since last meeting
the acceptance criteria, focusing
on taxa of concern
Record both tentative and
independent IDs
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Check or
Sample
Description
External QC
Use of
widely/commonly
accepted
taxonomic
references by all
NCCA labs
Prepare reference
collection
Frequency
10% of all
samples
completed per
laboratory
For all
identifications
Each new taxon
per laboratory
Acceptance Criteria
PDE < 5%
PTD<15%
All keys and references
used by each lab must be
on bibliography prepared
by one or more additional
NCCA labs or in The taxa
list provided by EPA. This
requirement demonstrates
the general acceptance of
the references by the
scientific community.
Complete reference
collection to be
maintained by each
individual laboratory
Corrective Action
If PDE > 5%, implement
recommended corrective
actions.
If PTD> 15%, implement
recommended corrective
actions.
If a lab proposes to use other
references, the lab must obtain
prior permission from External
QC Officer before submitting the
data with the identifications
based upon the references.
Internal Taxonomy QC Officer
periodically reviews data and
reference collection to ensure
reference collection is complete
and identifications are accurate
DATA VALIDATION
Taxonomic
"reasonableness"
checks
All data sheets
Taxa known to occur for
coastal waters or Great
Lakes.
Second or third identification by
expert in that taxon
4.11 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.
USEPA. 2012. National Rivers and Streams Assessment 2013-2014: Laboratory Operations
Manual. EPA-841-B-12-010. U.S. Environmental Protection Agency, Office of Water,
Washington, DC.
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Attachment 4.1: Benthic Macroin vertebrates: Taxonomy Bench Sheet (example)
Laboratory Information
Project ID
Station Name
Station Location
Station Number
Sample Information
Sample ID
Site ID
Date Collected
Field Crew ID
Taxonomist Name
Date 1st Organism Identified in Sample:
QC Check? Y/N
TSN
(Use # in
Uniqueldentifier
from taxa list
provided by
EPA)
Taxon
Distinct
(Y/N)
Counts of Organisms in the
Taxon:
Total
(any
stage)
Larvae
Pupae
Adults
Cumulative
Number of
Organisms
in Sample
Data
Qualifier
(Codes
in Table
4.2)
Comments:
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5.0 WHOLE BODY FISH PROCESSING AND CONTAMINANT
ANALYSIS
This chapter describes fish processing and analysis requirements for whole body fish samples.
The purpose is to determine concentrations of contaminants in fish samples collected in the 2015
NCCA and related studies. The laboratory shall perform analysis to determine the lipid content,
concentrations of metals, mercury, pesticides, and PCBs found in fish within coastal waters and
Great Lakes. EPA also may require the national contract laboratory to analyze the samples for
PAHs; however, EPA will not require the State laboratories to analyze for them.
At each sampling site, the Field Operations Manual (FOM) instructs the crews to collect five fish
of the same species (or 10 sea urchins of any species) and similar size for each sample. The crew,
or EPA's batch laboratory, then ships the fish specimens on dry ice to the laboratory.
In the following discussion, Sections 5.1, 5.2, and 5.3 summarize the procedure; health and
safety concerns; and definitions and required resources. Section 5.4 provides the steps for
acknowledging sample receipt. Section 5.5 provides the steps for creating whole fish composites.
Sections 5.6 - 5.7 provide the minimum requirements that the laboratory must meet in
performing the contaminant analyses and the required data elements. Section 5.8 describes &o
EPA's external review of laboratory operations and other quality measures. Section 5.9 identifies ^
references used in developing the procedure. j
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5.1 Summary of the Procedure ฃ
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This chapter describes the fish processing and contaminant determination of whole fish samples Ej
collected for EPA's 2015 National Coastal Condition Assessment (NCCA). To ensure consistent H
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2. Laboratory personnel must wear proper personal protection clothing and equipment O
(e.g. lab coat, protective eyewear, gloves).
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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 acid. If skin
contact occurs, remove clothing immediately. Wash and rinse the affected skin areas
thoroughly with large amounts of water.
4. When operating grinding equipment, the laboratory personnel must exercise caution.
5.3 Definitions and Required Resources (Personnel, Laboratories, and Equipment)
This section provides definitions and required resources for using the procedure.
5.3.1 Definitions
The procedure uses the following terms:
Detection Limit is the minimum concentration at which the analyte can be detected with
confidence. In other words, the outcome can be reported with confidence that it is greater
than zero (i.e., present in the sample). Also see "Sample-Specific Detection Limit."
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Duplicates are defined as two aliquots of the same sample which are analyzed separately ^
using identical procedures. The results are used to evaluate the precision of the laboratory <3
analyses. ^
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Fish Composite: Each composite consists of all parts of the fish including the head, skin, ^
internal organs, muscle, and bones. For sea urchins, it includes only the gonad tissue because ง
it is essentially the only tissue present. Unless otherwise specified, references to "fish" Sj
include "sea urchins." With the exception of sea urchins, NCCA does not provide support for H
analyses of any other invertebrates such as crustacean (e.g., lobster, crabs). ^
o
NARS: National Aquatic Resource Surveys. The National Coastal Condition Assessment @
(NCCA) is part of the NARS program.
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concentration measured in the spiked part; C is the concentration measured in the unspiked
part; and s is the known concentration amount for the spike. The following equation is used
to calculate the percent recovery:
Cs- C
%RS = X 100
s
Relative Standard Deviation (RSD): The precision at each concentration is reported in
terms of the RSD. To calculate the RSD, first calculate the standard deviation, S, as follows:
5 =
n
V(CS- C)
1/2
where n is the number of replicate samples, C, is the concentration measure for the kth
sample, and C is the average concentration of the replicate samples. Then, RSD is calculated
as:
5
RSD =
x 100
Reporting Limit: A reporting limit is the point at which the measured value of the analyte
can be reported with confidence.
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Routine sample: A routine composite sample consists of individual adult fish of a single ^
species that meet EPA' s length requirement (Length of smallest fish in the composite must be at <3
least 75% of the length of the longest fish),, and sufficient number offish to meet target mass of 300 %
grams. See Section 5.5.1 for more information. H
%
Sample-Specific Detection Limit: Most samples will have a sample-specific detection equal g
to the method's detection limit. For diluted samples, the sample-specific detection limit will 5
be the product of the method's detection limit and the dilution factor. Typical values for the ^
dilution factors will be 10 or 100.
Spiked Sample: See Percent Recovery definition for purpose of spiked samples.
TOCOR: Task Order Contracting Officer's Representative is EPA's contact person for
laboratories under contract to EPA.
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Competency: To demonstrate its competency, the laboratory shall provide analyte and matrix GO
specific information to EPA. EPA will accept one or more of the following as a demonstration ^
of competency: Q
Memorandum that identifies the relevant services that the laboratory provided for the O
National Aquatic Resource Surveys in the past five years. pq
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Documentation detailing the competency of the organization, including professional
certifications for fish-related analyses, membership in professional societies, and
experience with analyses that are the same or similar to the requirements of this method.
Also, the lab must provide a demonstration of competency with fish samples in achieving the
method detection limits, accuracy, and precision targets.
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 NCCA QAPP Certification Page.
5.3.3 Personnel
The procedure refers to the following personnel:
Laboratory Technician: This procedure may be used by any laboratory technician who ^
is familiar with the NCCA Quality Assurance Project Plan, and this procedure in the ^
NCCA Laboratory Operations Manual.
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5.4 Sample Receipt
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. The laboratory must inspect the samples promptly on
receipt. As samples arrive, the laboratory must:
1 . Log the samples into the National Aquatic Resource Survey Information Management
system (NARS-EVI) within 24 clock hours. Alternatively, for shipments with a large
number of samples, the laboratory may email a spreadsheet with the sample login and
sample condition information to NARS-IM (see Chapter 2 for contact information).
2. Check that each shipping container has arrived undamaged. Check the temperature of one
of the samples in the cooler using a thermometer that reads to at least -20 ฐC (i.e., the
expected temperature of frozen samples), or an infra-red (IR) temperature "gun" and
record the reading. Record the condition and temperature of the sample in the database
using the codes in Table 5.1.
3. Compare the information on the label on each individual fish specimen to the sample
tracking form for each composite and verify that each specimen was included in the
shipment and is properly wrapped and labeled. The crew labels each fish specimen using ^
the sample identification code and appends a specimen identification code. For example, ^
if the sample number is "NCCA15-1 1 11," then the crew might label specimen "A" as
Table 5.1 Whole Body Fish Login: Required Data Elements _ Q
PQ
Variable
SITE ID
SAMPLE
DATE COLLECT
Type
Character
Character
Date
Description
Site identification code
Sample number
Date that the field crew collected the
sample
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Variable
Type
ARRIVAL_TEMP
NUMBER_FISH
SAMPLE_WT
CONDITION_CODE
COND_COMMENT
Description
Numeric
Numeric
Numeric
Characte
r
Characte
r
Temperature of sample upon arrival at the
laboratory (fish should be frozen).
Number of fish in the sample
Total weight of sample (all fish)
Condition codes describing the condition of
the sample upon arrival at the laboratory;
leave blank for control
Flag
OK
C
L
ML
NF
Q
Definition
Sample is in good condition
Sample wrapping is cracked
Sample or container is
leaking
Sample label is missing
Sample is not at proper
temperature
Other quality concerns, not
identified above
Explanation for Q FLAG (if needed)
5.5 Whole Fish Preparation and Homogenization Procedures
This section describes the whole fish preparation and homogenization procedures. As described
in Section 5.5.1, if a laboratory determines that a sample is non-routine, the laboratory contacts
the EPA HQ NCCA Laboratory Review Coordinator (Chapter 2 provides contact information)
for additional instructions before continuing with the compositing and homogenization
procedures in Section 5.5.2. Section 5.5.3 describes rigorous equipment cleaning and rinsate
collection steps used before the compositing and homogenization steps in Section 5.5.4.
5.5.1 Sample Classification: Routine or Non-Routine
Each sample is either a "routine" composite sample, or a "non-routine" composite sample, based
on the following definitions:
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Routine sample - A routine composite sample consists of individual adult fish of
a single species that meet EPA's length and other requirements. For example, the
species must be one of the target species identified in Appendix B of this LOM.
The laboratory homogenizes the fish to prepare one composite sample.
Non-routine sample - A non-routine sample is any sample that does not meet the
definition of a routine sample. When field crews collect non-routine samples,
depending on the circumstances, EPA will provide instructions for processing, or
possibly destroying, the non-routine samples. These instructions also may include
discarding some of the fish in the composite sample based on size before
proceeding with homogenizing. For non-routine composites, the laboratory
homogenizes only the designated specimens, i.e., those that EPA identifies by
specimen number.
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Note: Non-routine samples do not include samples from an incorrect sampling location, an
unnecessary duplicate sample, or inappropriate fish species. EPA does not plan on using these
"invalid" samples, so it is imperative that the sample preparation laboratory not process any
sample without specific instructions from EPA. Therefore, laboratories shall retain such samples
in frozen storage until EPA determines the appropriate course of action, which may include
processing the sample. If the status of any composite sample in the instructions is not clear, the
laboratory must contact EPA and wait for clarification.
5.5.2 Fish Examination and Preparation
This section describes the steps for fish examination and preparation.
1 . Put on powder-free nitrile gloves (if not already gloved) before unpacking individual fish
specimens. For sea urchins, wear thick rubber gloves to provide protection from the
urchin spines. As samples are unpacked and unwrapped, inspect each fish carefully for
any damage (e.g., tears in the skin or punctures in the gut). Document any damage in
comments per Table 5.2.
2. The field crews measured the total length of each fish specimen in the field and recorded
those lengths on the sample tracking form. Because of the importance of length
measurements, EPA requires laboratories to perform a second series of measurements of ^
the length for each fish. Because it may be difficult to reproduce the field measurements ^
offish length when the specimens are still partially frozen, begin processing the j
specimens in the following steps: ^
a. Lay them out in order by specimen number (e.g., the portion of the sample ID H
9. Identify and record the species of each fish specimen. Confirm that the species is one of Q
the target species listed in Appendix B of this LOM. pq
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10. Determine if the sample is routine or non-routine (per classification definitions in Section O
5.5.1) and record its classification and any applicable fish code from Table 5.3. Return 5
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any non-routine sample to the freezer and contact the EPA HQ NCCA Laboratory
Review Coordinator for processing instructions (see Chapter 2 for contact information).
11. Verify that all required data elements, per Tables 5.2 and 5.3, have been recorded. If any
elements are missing, then enter them into the database.
12. Rinse each fish with deionized water and remove any adhering slime as a precautionary
measure to treat for possible contamination from sample handling in the field. Use
HDPE wash bottles for rinsing fish and for cleaning homogenization equipment and
utensils. Do NOT use Teflonฎ wash bottles for these procedures.
13. Return to freezer for storage until ready to homogenize the sample. If the laboratory
intends to proceed directly to homogenization, then allow the sample to partially thaw
while cleaning the equipment as described in the next section.
Table 5.2 Whole Body Fish: Data Elements for Each Fish Specimen
Variable
SITE ID
SAMPLE
SPECIMEN ID
SPECIES
FISH WT
WT UNIT
FISH LEN
LEN UNIT
COMMENT
Type
Character
Character
Character
Character
Numeric
Character
Numeric
Character
Character
Description
Site identification code
Sample number
Identification code assigned to a
single fish
Species offish
Weight offish
Units offish weight (kg, Ib)
Length offish
Units offish length (cm, in)
Comment about condition offish
observations
or other
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Table 5.3 Whole Body Fish: Data Elements from Examination of Each Sample
Variable
SITE ID
SAMPLE
Type
Character
Character
SAMPLE CLASS
FISH CODE
Description
Site identification code
Sample number
Character
Character
Sample classification: Routine or Non-
routine
Codes describing any deviations from the
FOM criteria for fish collection for each
sample
Flag
SP
LE
NS
WT
Definition
Not all specimens are of the
same species
Not all specimens lengths are
within 75% of longest fish
Specimen number is fewer
than minimum of 5 or greater
than 20 maximum
Mass does not meet
minimum of 140 grams
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Variable
Type
Description
LL
LS
Q
Longest fish exceeds 400 mm
maximum length
Shortest fish below 100 mm
minimum length
Other quality concerns, not
identified above
* Field crews are required to collect a minimum of 300 grams, but the minimum required for laboratory
analyses is 140 grams.
5.5.3 Equipment Cleaning and Rinsate Collection
This section describes the rigorous cleaning required to protect against cross-contamination of
samples. To verify that the cleaning procedures are effective, EPA requires the collection of
rinsate samples as described below.
1. Before processing any sample, thoroughly clean all of the homogenization equipment.
Disassemble the homogenization equipment (i.e., blender, grinder, or other device) and
thoroughly clean all surfaces and parts that contact the sample. Similarly, clean all
knives, cutting boards, and other utensils used The cleaning steps are as follows:
a. Wash with a detergent solution (phosphate- and scent-free) and warm tap water
b. Rinse three times with warm tap water
c. Rinse three times with deionized (DI) water
d. Rinse with acetone
e. Rinse three times with DI water
f Rinse with (not soak in) 5% nitric acid
g. Rinse three times with DI water
h. Allow the components to air dry
i. Reassemble the homogenization equipment
2. Once per batch (i.e., once per maximum of 20 samples), collect rinsate samples for use in
assessing any equipment contamination. To minimize the number of project samples that
might be affected by cross contamination, collect the normal rinsate samples on the first
day that samples in a batch of 20 are processed. Ideally (not required), the laboratory will
vary the point at which the rinsates are collected on that first day over the course of the
project (e.g., between the 1st and 2nd samples for one batch, the 2nd and 3rd samples for
another batch, etc.). Prior to reassembling the homogenization equipment, use the
following steps to prepare enough rinsate samples for the relevant QA/QC activities:
a. Prepare each hexane rinsate sample by pouring a 100-mL portion of pesticide-
grade hexane over all parts of homogenization equipment, including the cutting
boards and knives, and collect it in a clean glass container. Place an additional
100-mL aliquot of clean hexane in a similar glass container for use as a solvent
blank. Allow the solvent to evaporate from the equipment. Per QA/QC
requirements, the laboratory will analyze the rinsate and solvent blank for the
Polychlorinated biphenyls (PCBs), pesticides, and Polycyclic Aromatic
hydrocarbons (PAHs) selected for NCCA analysis.
b. Once the hexane has evaporated, prepare each DI water rinsate using 250 mL of
DI water. Collect the DI water rinsate in a clean glass or HOPE container. Place a
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second aliquot of DI water in a separate similar clean container for use as a blank.
Acidify these two samples to pH < 2 with nitric acid. Per QA/QC requirements,
the laboratory will analyze the rinsate and blank samples for metals and mercury.
c. Store the rinsates and blanks at a cold, not freezing, temperature (<6 ฐC).
5.5.4 Compositing and Homogenization Procedure
This section describes the steps for a "batch" homogenization method that uses the entire
homogenized volume of all fish specimens to prepare the composite. In contrast to an
"individual" method that would combine equal weights of tissue from each specimen, the batch
homogenization method uses the complete specimens regardless of each individual specimen's
proportion to one another. The steps are as follows:
1. Change gloves between samples. The technician may use the same gloves in handling all
fish within a given sample.
2. Partially thaw samples for ease of grinding during homogenization.
3. For sea urchins, prepare the sea urchin for compositing by cracking open the shell of each
sea urchin in the sample. From all of the sea urchins in the sample, extract and composite
only the gonad tissue. (The gonad tissue is essentially the only tissue present in sea ^
urchins.) ฃo
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4. Process each sample using a size-appropriate homogenization apparatus (e.g., automatic S
grinder or high-speed blender). If difficulties arise with the samples sticking to
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self-sealing plastic freezer bag to avoid sample loss due to breakage. Freeze the tissue
aliquots at -20 ฐC, and maintain samples in the freezer until analysis.
8. For one sample in every batch (same batch as specified for the rinsate samples collected
in Section 5.5.3), the laboratory conducts triplicate analyses of the lipid content to
confirm that the grinding has resulted in an homogeneous sample. As with the collection
of rinsate samples, the laboratory performs the homogeneity testing on the first day on
which samples in a batch of 20 are processed. However, the sample chosen for
homogeneity testing must be one that yields enough tissue mass to support the added
mass needed for triplicate lipid aliquots (15 to 30 g).
a. The laboratory selects one sample processed on the first day of every batch that
will provide well over 140 g of total tissue mass.
b. From that sample, place three 5- to 10-g aliquots in clean glass or plastic
containers of suitable size and label as appropriate.
c. Calculate the mean lipid content (in percent), the standard deviation (SD), and the
relative standard deviation (RSD) as follows:
mean %lipids =
2(%lipids: -meanlipids)
RSD =
mean
If the RSD of the triplicate results is: %
Less than or equal to the QC criterion, then the homogenization effort is ^
judged to be sufficient for all samples in that QC batch. JZ
Otherwise, corrective action consists of regrinding all of the aliquots from ^
each composite sample in the affected batch until meeting the QC Q
criterion. This may entail retrieving all sample aliquots (see Table 5.4) O
from the freezer, allowing them to partially thaw, homogenizing them PH
again, determining new lipids results, and performing a new E
homogenization QC determination. New sample containers are required E
for any rehomogenized samples. Also, follow the steps in Section 5.5.3 for ^
cleaning the equipment between each composite sample in o
rehomogenizing the samples.
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e. For this sample analyzed in triplicate, record the lipid content measured in the
first analysis.
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9. Before homogenizing the next sample, clean the grinding equipment and all other
sample preparation equipment using the procedures described in Section 5.5.3.
Table 5.4. Whole Body Fish: Initial Aliquot Requirements
Analysis
Mercury
Metals other
than mercury
PCBs
Pesticides
PAHs (only by
EPA request)
Lipids
Maximum*
Target
Mass
5 -10g
5-10g
30 - 35 g
30 - 35 g
30 - 35 g
10- 15g
140 g
Sample Jar Requirements
50-mL HOPE straight-sided jar with foil-lined lid, or conical HOPE
tube with snap top
50-mL HOPE straight-sided jar with foil-lined lid, or conical HOPE
tube with snap top
1 25-mL straight-sided amber or clear glass jar with
PTFE-lined lid
1 25-mL straight-sided amber or clear glass jar with
PTFE-lined lid
1 25-mL straight-sided amber or clear glass jar with
PTFE-lined lid
Laboratory's choice, as this aliquot will be used in-house to determine
the lipid content of the sample
*ln the event that insufficient fish tissue mass exists to prepare the required number of aliquots, contact
EPA for instructions.
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5.6 Contaminant Analysis: Requirements
The laboratory shall perform analysis of the homogenized composites to determine the lipid
content, concentrations of metals, mercury, pesticides, and PCBs. EPA also may require the
national contract laboratory to analyze the samples for PAHs; however, EPA will not require the
State laboratories to analyze for them. With the exception of sea urchins, NCCA does not
provide support for analyses of any other invertebrates such as crustaceans (e.g., lobster, crabs).
After preparing the fish composites as described in Section 5.5, laboratories may choose to use
any analysis method, including those in Table 5.5, that measures contaminants to the levels of
the method detection limits identified in Table 5.6. In addition, the method must meet the target
precision of 30% and the target accuracy as follows:
Metals: 20%
Organics (PCBs, pesticides, and PAHs): 35%
The laboratory must store the fish samples frozen at a maximum of -20ฐ C and complete the
analyses within one year.7
7 NCCA allows for a 1-year holding time because of the sheer volume of sample collected in a short amount of time.
Generally, EPA recommends different holding times, see for example Appendix J "Recommended procedures for
preparing whole fish composite homogenate samples" in Guidance for Assessing Chemical Contaminant Data for
Use in Fish Advisories, Volume 1 (Fish Sampling and Analysis), 3rd Edition, 2000. EPA #823-B-00-007. Retrieved
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Table 5.5 Whole Body Fish: Analytical Methods
Analysis
Metals (except Mercury)
Mercury
PCBs, Pesticides, PAHs
Percent Lipids
Extraction
Any method using microwave
assisted digestion8
EPA Method 3540C11
Any method using hexane
Methods that Meet the QA/QC
Requirements (any method that
meets the QA/QC requirements
is acceptable)
EPA Method 6020A9
EPA Method 24510
EPA Method 827012
EPA Method 9071 B13
Table 5.6 Whole Body Fish: Lipids and Required Contaminants
Type
LIPID
METAL
UNITS
% Wet
Weight
ug/wet g
(mg/L)
Parameter
% LIPID
Aluminum
Arsenic
Cadmium
Chromium
Copper
Iron
Lead
Mercury
Nickel
Selenium
Silver
Tin
Vanadium
Zinc
CAS
Number
7429-90-5
7440-38-2
7440-43-9
7440-47-3
7440-50-8
7439-89-6
7439-92-1
7439-97-6
7440-02-0
7782-49-2
7440-22-4
7440-31-5
7440-62-2
7440-66-6
PCB
Numbe
r
(where
applicabl
e)
MDL
Targe
t
10.0
2.0
0.2
0.1
5.0
50.0
0.1
0.01
0.5
1.0
0.3
0.05
1.0
50.0
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from
http://water.epa.gov/scitech/swguidance/fishshellfish/techguidance/risk/upload/2009 04 23 fish advice volumel
vlcover.pdf.
8 For example, see Method 3150A "Microwave Assisted Acid Digestion of Sediments, Sludges, Soils, and Oils,"
retrieved from http://www.epa.gov/osw/hazard/testmethods/sw846/pdfs/3051a.pdf.
9 For example, Method 6020A "Inductively Coupled Plasma-Mass Spectrometry" retrieved from
http://www.epa.gov/epawaste/hazard/testmethods/sw846/pdfs/6020a.pdf.
10 For example, Method 245.7 "Mercury in Water by Cold Vapor Atomic Fluorescence Spectrometry, Revision 2.0"
(EPA-821-R-05-001, February 2005), retrieved from
http://water.epa.gov/scitech/methods/cwa/bioindicators/upload/2007 07 10 methods method 245 7.pdf.
11 For example, see Method 3540C "Soxhlet Extraction" retrieved June 27, 2014 from
http://www.epa.gov/osw/hazard/testmethods/sw846/pdfs/3540c.pdf.
12 For example, Method 8270D "Semivolatile Organic Compounds by Gas Chromatography/Mass Spectrometry
(GC/MS) retrieved from http://www.epa.gov/osw/hazard/testmethods/sw846/pdfs/8270d.
13 Method 9171B "n-Hexane Extractable Material (HEM) for Sludge, Sediment, And Solid Samples," retrieved from
http://www.epa.gov/osw/hazard/testmethods/sw846/pdfs/9071b.pdf.
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Type
PCB
PEST
UNITS
ng/wet g
(M9/L)
ng/wet g
(M9/L)
Parameter
2,2',3,3',4,4',5,5',6,6'-
Decachlorobiphenyl
2,4'-Dichlorobiphenyl
2,2',3,4',5,5',6-Heptachlorobiphenyl
2,2',3,3'4,4',5,6-Octachlorobiphenyl
2, 2', 3, 4 ,5,5',6-Heptachlorobiphenyl
2,2',3,3',4,4'-Hexachlorobiphenyl
2,2',3,3'4,4',5-Heptachlorobiphenyl
2,2',3,4,4',5'-Hexachlorobiphenyl
2,2',4,4',5,5'-Hexachlorobiphenyl
2,2',3,3',4,4',5,5',6-
Nonachlorobiphenyl
2,3,3',4,4'-Pentachlorobiphenyl
2,2',4,5,5'-Pentachlorobiphenyl
2,3',4,4',5-Pentachlorobiphenyl
2,3,3',4,6'-Pentachlorobiphenyl
3,3',4,4',5-Pentachlorobiphenyl
2,2',3,5'-Tetrachlorobiphenyl
3,3',4,4'-Tetrachlorobiphenyl
2,2',5,5'-Tetrachlorobiphenyl
2,3',4,4'-Tetrachlorobiphenyl
2,2',5-Trichlorobiphenyl
2,4,4'-Trichlorobiphenyl
2,4'-DDD
2,4'-DDE
2,4'-DDT
4,4'-DDD
4,4'-DDE
4,4'-DDT
Aldrin
Alpha-BHC
Beta-BHC
Delta-BHC
Alpha-Chlordane
Gamma-Chlordane
Dieldrin
Endosulfan 1
Endosulfan II
Endosulfan Sulfate
Endrin
Endrin Aldehyde
Endrin Ketone
Heptachlor
Heptachlor Epoxide
Hexachlorobenzene
Lindane
Mi rex
CAS
Number
2051-24-3
34883-43-7
35065-29-3
52663-78-2
52663-68-0
38380-07-3
35065-30-6
35065-28-2
35065-27-1
40186-72-9
32598-14-4
37680-73-2
31508-00-6
38380-03-9
57465-28-8
41464-39-5
32598-13-3
35693-99-3
32598-10-0
37680-65-2
7012-37-5
53-19-0
3424-82-6
789-02-6
72-54-8
72-55-9
50-29-3
309-00-2
319-84-6
319-85-7
319-86-8
5103-71-9
5566-34-7
60-57-1
959-98-8
33213-65-9
1031-07-8
72-20-8
7421-93-4
53494-70-5
76-44-8
1 024-57-3
118-74-1
58-89-9
2385-85-5
PCB
Numbe
r
(where
applicabl
e)
209
8
180
195
187
128
170
138
153
206
105
101
118
110
126
44
77
52
66
18
28
MDL
Targe
t
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
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Type
PAHs*
UNITS
Parameter
Cis-Nonachlor
Oxychlordane
Trans-Nonachlor
Acenaphthene
Acenaphthylene
Anthracene
Benz(a)anthracene
Benzo(b)fluoranthene
Benzo(k)fluoranthene
Benzo(g,h,i)perylene
Benzo(a)pyrene
Benzo(e)pyrene
Biphenyl
Chrysene
Dibenz(a,h)anthracene
Dibenzothiophene
2,6-Dimethylnaphthalene
Fluoranthene
Fluorene
lndeno(1 ,2,3-c,d)pyrene
1 -Methylnaphthalene
2-Methylnaphthalene
1 -Methylphenanthrene
Naphthalene
Perylene
Phenanthrene
Pyrene
2,3,5-Trimethylnaphthalene
CAS
Number
5103-73-1
26880-48-8
39765-80-5
83-32-9
208-96-8
120-12-7
200-280-6
205-99-2
207-08-9
191-24-27-2
50-32-8
192-97-2
92-54-4
218-01-9
53-70-3
132-65-0
581-42-0
205-99-2
86-73-7
1 93-39-5
90-12-0
91-57-6
832-69-9
91-20-3
198-55-0
85-01-8
129-00-0
2245-38-7
PCB
Numbe
r
(where
applicabl
e)
MDL
Targe
t
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
* EPA also may require the national contract laboratory to analyze the samples for PAHs; however, EPA
will not require the State laboratories to analyze for them.
5.7 Data Entry
Tables 5.1 (Section 5.4), 5.2 (Section 5.5), 5.3 (Section 5.5), and 5.7 (below) identify the
required data elements that laboratories must provide to EPA, preferably in EPA's data template,
available separately from EPA.
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Table 5.7 Whole Body Fish: Data Elements for Each Sample
Variable
SITEJD
SAMPLE
REPEAT
DATE COLLECT
Type
Character
Character
Numeric
Date
Description
Site identification code or type of QC sample (e.g., LAB
BLANK)
Sample number, LCS, QCCS, Blank, Matrix Spike, or
Rinsate
Duplicate or Triplicate (otherwise blank)
Date that the field crew collected the sample
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Variable
PARAMETER
CAS NO
LABNAME
METHOD
ANALYST
REVIEWER
INSTRUMENT
DATE
PREPARED
DATE ANALYSIS
Type
ARRIVAL_TEMP
NUMBER_FISH
SAMPLE_WT
SAMPLE_CLASS
CONDITION
CODE
COND COMMEN
T
FISH CODE
Character
Character
Character
Character
Character
Character
Character
Date
Date
Description
Numeric
Numeric
Numeric
Character
Character
Character
Character
Temperature of sample upon arrival at the
laboratory (fish should be frozen).
Number of fish in the sample
Total weight of sample (all fish)
Sample classification: Routine or Non-
routine
Condition codes describing the condition of
the sample upon arrival at the laboratory;
leave blank for control
Flag
OK
C
L
ML
NF
Definition
Sample is in good condition
Sample wrapping is cracked
Sample or wrapping is
leaking
Sample label is missing
Sample is not at proper
temperature
Explanation for Q FLAG (if needed)
Codes describing any deviations from the
criteria for fish collection for each sample
Flag
SP
LE
NS
WT
LL
LS
Q
Definition
Not all specimens are of the
same species
Not all specimens lengths
are within 75% of longest
fish
Specimen number is fewer
than minimum of 5 or
greater than 20 maximum
Mass does not meet
minimum of 500 grams
Longest fish exceeds 400
mm maximum length
Shortest fish below 100 mm
minimum length
Other quality concerns, not
identified above
Analyte name
CAS Registry number corresponding to the analyte
Laboratory name (abbreviation)
Laboratory method used
Last name or initials of person who performed the analysis
Last name or initials of the person who provided a
separate independent review of the data
Identification of instrument used for the analysis - provide
enough information to identify the particular instrument in
the laboratory
Date that the sample homogenization started
Date that the sample analysis started
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Variable
QC_BATCH_LOT
HOLDING TIME
MATRIX
MDL
LRL
DILUTION
RECOVERY
RESULT
REASON
RESULT QUAL
UNIT
QC_CODE
COMMENT
Type
Character
Y/N
Character
Numeric
Numeric
Numeric
Numeric
Numeric
Character
Character
Character
Character
Character
Description
Unique laboratory quality control lot numbers assigned to
the batch of samples. The lot number must associate each
batch of field samples to the appropriate rinsates,
laboratory control sample, matrix spike, laboratory
duplicate, and method blank samples.
Analysis performed within holding time
Fish
Lab method detection limit (based upon lab's historical
data)
Lab reporting limit (based upon lab's historical data)
Dilution of sample (blank or 1 if no dilution)
Only for appropriate QC samples
Concentration value
Reason for qualification in RESULT_QUAL (usually blank)
Data qualifier (usually blank)
Unit of measurement for RESULT, MDL, and RL
Apply laboratory defined QC codes and describe in the
comments field. Provide set of laboratory's code as part of
the case narrative
Explain situation that created QC code, or any unusual
aspects of the analysis
5.8 Quality Measures
This section describes the quality assurance and quality control measures used to ensure that the
data will meet NCCA's requirements.
5.8.1 Assistance Visits
Assistance visits are intended to familiarize EPA with actual procedures being implemented by
different laboratories; and to ensure a clear and consistent understanding of procedures and
activities by both EPA and the laboratories. If EPA decides to conduct an assistance visit, a
qualified EPA scientist or contractor will administer a checklist based upon the steps described in
this chapter.
5.8.2 QC Samples
Once or twice during the performance period, the External QC Coordinator will provide one or
two identical sets of QC samples to all participating laboratories. Each set will contain up to five
QC samples. As determined by the External QC Coordinator, the QC samples may be synthetic;
aliquots of additional samples collected at NCC A sites; or reference samples obtained from an
organization such as the National Institute of Standards. Each laboratory will run the QC samples
following the same procedures used for the other samples. The External QC Coordinator will
compare the results to the expected value and determine consistency between laboratories (e.g.,
determine if one laboratory is consistently higher or lower than all others). Based upon the
evaluation, the External QC Coordinator may request additional information from one or more
laboratories about any unique laboratory practices that might account for differences between the
laboratory and others. The contractor shall analyze the external QC samples using the same
procedures as those for the field samples.
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5.8.3 Summary of QA/QC Requirements
QC protocols are an integral part of all analytical procedures to ensure that the results are reliable
and the analytical stage of the measurement system is maintained in a state of statistical control.
The laboratory must conduct QC analyses for each batch of samples. Each batch shall consist of
no more than 20 samples. Unique laboratory quality control lot numbers must be assigned to
each batch of samples. The lot number must associate each batch of field samples to the
appropriate measures such as laboratory control sample, matrix spike, laboratory duplicate, and
method blank samples. Also, each laboratory QC samples (i.e., preparation and instrument
blanks, laboratory control sample (LCS), spike/duplicate, etc.) must be give a unique sample
identification. Table 5.8 provides a summary of the quality control requirements.
Table 5.8 Whole Body Fish: Quality control activities
Quality
Control
Activity
Description and Requirements
Corrective Action
Demonstrate
competency for
analyzing fish
samples with the
required methods
Demonstration of competency with fish
samples in achieving the method detection
limits, accuracy, and precision targets
EPA will not approve any
laboratory for NCCA sample
processing if the laboratory cannot
demonstrate competency. In other
words, EPA will select another
laboratory that can demonstrate
competency for its NCCA samples.
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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 in a frozen state.
Assign appropriate condition code
identified in Table 5.2.
Store sample
appropriately.
While stored at
the laboratory,
the sample must
be kept at a
maximum
temperature of
-20ฐ C.
Check the temperature of the freezer per
laboratory's standard operating procedures.
Record temperature of sample
upon arrival at the laboratory. If at
any other time, samples are
warmer than required, note
temperature and duration in
comment field.
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Determine if all
fish meet the
criteria
Evaluate if the sample contains fish of the
same species and are similar in size (within
75%), and provides enough material to run the
analysis
Contact the EPA HQ NCCA
Laboratory Review Coordinator*
for a decision on fish selection
and/or chemical analysis.
Analyze sample
within holding
time
The test must be completed within the holding
time (i.e., 28 days for mercury; 6 months for
other metals; and 1 year for all others). 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.
Perform once at
the start of each
batch to evaluate
Control limits for recovery cannot exceed
100ฑ20%.
First, prepare and analyze one
additional LCS. If the second blank
meets the requirement, then no
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Quality
Control
Activity
Description and Requirements
Corrective Action
the labeled
compound
recovery (LCR) in
a Laboratory
Control Sample
(LCS). This tests
the performance
of the equipment.
further action is required. If the
second LCS fails, then determine
and correct the problem before
proceeding with any sample
analyses.
Perform once at
the start of each
batch to evaluate
the entire
extraction and
analysis process
using a Method
Blank
Control limits cannot exceed the laboratory
reporting level (LRL).
First, prepare and analyze one
additional blank. If the second
blank meets the requirement, then
no further action is required. If the
second blank fails, then determine
and correct the problem (e.g.,
homogenization, reagent
contamination, instrument
calibration, or contamination
introduced during filtration) before
proceeding with any sample
analyses. Reestablish statistical
control by analyzing three blank
samples. Report values of all
blanks analyzed.
Check calibration
immediately
before and
immediately after
the sample batch
is run
(abbreviated as
QCCS for quality
control check
sample)
Results must be ฑ10% of each other or as
specified in method criteria
If calibration fails before analysis,
recalibrate and reanalyze QCCS
until it passes. If check fails after
all samples in the batch have been
analyzed, verify the QCCS
reading. If the QCCS reading fails
a second time, then reanalyze all
samples in the batch and report
both sets of results. For the first
run, include a data qualifier that
indicates that the QCCS reading
taken immediately following the
first run failed. For the second run,
include a data qualifier that
indicates that it is the second set
and whether the QCCS reading
immediately following that second
run passed. No sample is to be
analyzed more than twice.
Evaluate rinsates
for first sample in
each batch. This
evaluation is a
surrogate for
assessing cross-
contamination.
Results must be below the LRL.
If first rinsate is above LRL,
analyze rinsate from a second
sample. If second rinsate sample
also has results above the LRL,
then assign a data qualifier to all
samples in the batch for the
parameters with results above the
LRL in the rinsates. Also, improve
procedures for cleaning all
surfaces, knives, and
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Quality
Control
Activity
Description and Requirements
Corrective Action
homogenization equipment
between samples.
Compare lipids in
triplicate for the
first sample in
each batch. This
evaluation is a
surrogate for
assessing
homogenization.
Substitute the LRL for any value below the LRL
before calculating the RSD. If the RSD of the
triplicate results is <20%, then the
homogenization effort is judged to be sufficient
for all samples in the batch.
If the RSD could not be achieved,
then regrind all samples in the
batch one or more times as
described in Section 5.5
Compare results
of one laboratory
duplicate sample
or matrix spike
duplicate sample
for each batch
Results must be within the target precision goal
in Table 5.8.1 (30% for all analytes).
If both results are below LRL, then
conclude that the test has passed.
Otherwise, prepare and analyze a
split from different sample in the
batch. If the second result is within
the target precision goal (see
Table 5.8.1) of the original sample,
then report the data and findings
for both QC samples. However, if
the two results differ by more than
the target precision goal, review
precision of QCCS measurements
for batch; check preparation of split
sample; etc. and report evaluation
and findings in the case narrative.
Consult with the EPA HQ NCCA
Laboratory Review Coordinator* to
determine if reanalysis of the entire
batch (at the laboratory's expense)
is necessary. If no reanalysis is
necessary, report and quantify all
samples in batch. If reanalysis is
necessary, then report all QC
sample and the 2nd analysis of the
batch. If the second set also is
unacceptable, then assign a data
code to each sample in the batch.
Compare results
of one matrix
spike sample per
batch to evaluate
performance in
matrix
Evaluate performance after the first 3 batches.
Ideally, control limits for recovery will not
exceed the target accuracy goal (Table 5.8.1),
but this may not be realistic for all parameters
with this matrix.
If both results are below LRL, then
conclude that the test has passed
for the batch. Otherwise, if any
results are not within the target
accuracy goal for the 3 batches,
within 2 working days, contact the
EPA HQ NCCA Laboratory Review
Coordinator* to discuss method
performance and potential
improvements. Continue to
perform the test for every batch.
Report the results from the original
analysis, the matrix spike, matrix
spike duplicate, and %recovery.
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Quality
Control
Activity
Description and Requirements
Corrective Action
Maintain the
required MDL
identified in the
Section 5.6
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 within each indicator
type as follows:
Metals in ug/g or ppm.
PCBs, pesticides, and PAHs in ng/g or ug/L.
If dry units are reported for any
sample (QC or field), reanalyze the
sample and report only the
reanalysis results. If it is not
possible to provide the results in
wet units, then assign a QC code
and describe the reason for dry
units in the comments field of the
database.
Maintain
completeness
Completeness objective is 95% for all
parameters.
Contact EPA HQ NCCA
Laboratory Review Coordinator*
immediately if issues affect
laboratory's ability to meet
completeness objective.
*Chapter2 provides contact information for the EPA HQ NCCA Laboratory Review Coordinator.
Laboratories under contract to EPA must contact the Task Order's Contracting Officer's Representative
(TOCOR) instead of the Laboratory Review Coordinator.
5.9 Sample and Record Retention
The laboratory shall retain:
1. The sample materials, including vials, for a minimum of 3 years from the date the EPA
publishes the final report. During this time, the laboratory shall freeze the materials. The
laboratory shall 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.
5.10 References
All references are from U.S. Environmental Protection Agency:
Guidance for Assessing Chemical Contaminant Data for Use in Fish Advisories, Volume 1 (Fish
Sampling and Analysis), 3rd Edition, 2000. Appendix J "Recommended procedures for preparing
whole fish composite homogenate samples". EPA #823-8-00-007. Retrieved from
http://water.epa.gov/scitech/swguidance/fishshellfish/techguidance/risk/upload/2009 04 23 fish
advice volume 1 vlcover.pdf.
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Method 245.7 "Mercury in Water by Cold Vapor Atomic Fluorescence Spectrometry, Revision
2.0" (EPA-821-R-05-001, February 2005), retrieved from
http://water.epa.gov/scitech/methods/cwa/bioindicators/upload/2007_07_10_methods_method_2
45 7.pdf.
Method 3150A "Microwave Assisted Acid Digestion of Sediments, Sludges, Soils, and Oils,"
retrieved from http://www.epa.gov/osw/hazard/testmethods/sw846/pdfs/3051a.pdf
Method 6020A "Inductively Coupled Plasma-Mass Spectrometry" retrieved from
http://www.epa.gov/epawaste/hazard/testmethods/sw846/pdfs/6020a.pdf
Method 8270D "Semivolatile Organic Compounds by Gas Chromatography/Mass Spectrometry
(GC/MS) retrieved from Method 8270D "Semivolatile Organic Compounds by Gas
Chromatography/Mass Spectrometry.
Method 9171B "n-Hexane Extractable Material (HEM) for Sludge, Sediment, And Solid
Samples," retrieved from http://www.epa.gov/osw/hazard/testmethods/sw846/pdfs/907 Ib.pdf.
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6.0 SEDIMENT CONTAMINANT, GRAIN SIZE, AND TOC ANALYSES
This chapter describes the analysis requirements for sediment samples. The purpose is to
determine concentrations of contaminants, grain size, and total organic carbon (TOC) in
sediment samples collected in the 2015 NCCA and related studies. The laboratory shall perform
analysis to determine the moisture content, concentrations of metals, mercury, pesticides, and
PCBs found in sediments in coastal waters and Great Lakes.
At each sampling site, the Field Operations Manual (FOM) instructs the crews to collect
sediment samples. The field crew then ships the samples on wet ice to either its own state
laboratory or EPA's batching laboratory. Once the samples arrive, the laboratory will freeze the
samples for the contaminant analyses and refrigerate the grain size and TOC samples.
In the following discussion, Sections 6.1, 6.2, and 6.3 summarize the procedure; health and
safety concerns; and definitions and required resources. Section 6.4 provides the steps for
acknowledging sample receipt. Sections 6.5 - 6.6 provide the minimum requirements that the
laboratory must meet in performing the contaminant analyses and the required data elements.
Section 6.7 describes EPA's external review of laboratory operations and other quality measures.
Section 6.8 identifies references used in developing the procedure.
6.1 Summary of the Procedure
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This chapter describes the contaminant, grain size, and TOC determination of sediment samples W
collected for EPA's 2015 National Coastal Condition Assessment (NCCA). As described in >H
Section 6.5, unless otherwise contractually bound by other requirements, the laboratory may
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6.3.1 Definitions
The procedure uses the following terms:
Detection Limit is the minimum concentration at which the analyte can be detected with
confidence. In other words, the outcome can be reported with confidence that it is greater
than zero (i.e., present in the sample). Also see "Sample-Specific Detection Limit."
Duplicates are defined as two aliquots of the same sample which are analyzed separately
using identical procedures. The results are used to evaluate the precision of the laboratory
analyses.
NARS: National Aquatic Resource Surveys. The National Coastal Condition Assessment
(NCCA) is part of the NARS program.
NARS Information Management System (NARS IM): The EVI system established to
support all surveys, including NCCA, in the NARS program. The EVI system is used to track
the samples from field collection to the laboratory.
NCCA: National Coastal Condition Assessment. Freshwater and coastal samples will be
collected during the field stage of NCCA.
_!
Percent Recovery: Recovery is measured by comparing the concentrations of a sample split G
into two parts; and one part is spiked with a known concentration value. G is the g
concentration measured in the spiked part; C is the concentration measured in the unspiked
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TOC: Total Organic Carbon
TOCOR: Task Order Contracting Officer's Representative is EPA's contact person for
laboratories under contract to EPA.
6.3.2 General Requirements for Laboratories
Competency. To demonstrate its competency, the laboratory shall provide analyte and matrix
specific information to EPA. EPA will accept one or more of the following as a demonstration
of competency:
Memorandum that identifies the relevant services that the laboratory provided for the
National Aquatic Resource Surveys in the past five years.
Documentation detailing the competency of the organization, including professional
certifications for water-related analyses, membership in professional societies, and
experience with analyses that are the same or similar to the requirements of this method.
Demonstration of competency with sediment samples in achieving the method detection
limits, accuracy, and precision targets.
Quality assurance and quality control requirements.
To demonstrate its competency 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). J
^
To demonstrate its ongoing commitment, the person in charge of quality issues for the <
organization shall sign the NCCA QAPP Certification Page. O
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6.3.3 Personnel 9
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The procedure refers to the following personnel: W
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Laboratory Technician: This procedure may be used by any laboratory technician who jz
is familiar with the NCCA Quality Assurance Project Plan, and this procedure in the <
NCCA Laboratory Operations Manual. O
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External QC Coordinator is an EPA staff person who is responsible for selecting and ง
managing the "QC contractor." To eliminate the appearance of any inherent bias, the JZ
QC contractor must be dedicated to QA/QC functions, and thus, must not be a primary 5
laboratory or a field sampling contractor for NCCA. The QC contractor is responsible for ^
complying with instructions from the External QC Coordinator; coordinating and paying ง
for shipments of the performance samples to participating laboratories; comparing o
immunoassay results from the laboratories; and preparing brief summary reports. fc
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6.3.4 Equipment/Materials
The analytical methods, selected by the laboratory, specify the required equipment.
6.4 Sample Receipt
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. The laboratory must inspect the samples promptly on
receipt. As samples arrive, the laboratory must:
1 . Log the samples into the National Aquatic Resource Survey Information Management
system (NARS-EVI) within 24 clock hours. Alternatively, for shipments with a large
number of samples, the laboratory may email a spreadsheet with the sample login and
sample condition information to NARS-IM (see Chapter 2 for contact information).
2. Check that each shipping container has arrived undamaged. Check the temperature of one
of the samples in the cooler using a thermometer that reads from 21 ฐC (i.e., room
temperature) down to -20 ฐC or lower (i.e., the expected temperature of frozen samples),
or an infra-red (IR) temperature "gun" and record the reading. Field crews ship sediment
samples on wet ice; the batch laboratory freezes the sample and ships with dry ice.
Record the condition and temperature of the sample in the database using the codes in
Table 6.1.
3. Verify that all required data elements, per Table 6.1, have been recorded. If any elements
are missing, then enter them into the database.
4. Transfer the samples to the freezer for long-term storage. Except during processing and
analysis stages, the samples must be stored frozen to less than or equal -20 ฐC.
5. Notify the EPA immediately about any problems involving sample integrity, conformity,
or inconsistencies as soon as possible following sample receipt and inspection.
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Table 6.1 Sediment Chemistry, Grain Size, and TOC Login: Required Data
Elements
Variable
SITE ID
SAMPLE
DATE COLLECT
Type
Character
Character
Date
ANALYSIS_TYPE
ARRIVAL_TEMP
CONDITION_CODE
Description
Site identification code
Sample number
Date that the field crew collected the sample
Character
Numeric
Character
Contaminant, TOC, or GRAIN SIZE
Temperature of sample upon arrival at the
laboratory
Condition codes describing the condition of
the sample upon arrival at the laboratory;
leave blank for control
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Variable
Type
COND COMMENT
Description
Character
Flag
OK
C
L
ML
Q
Definition
Sample is in good condition
Sample container is cracked
Sample or container is
leaking
Sample label is missing
Other quality concerns, not
identified above
Explanation for Q FLAG (if needed)
6.5 Laboratory Analysis: Requirements
The laboratory shall perform analysis of the sediment samples to determine the moisture content,
grain size, and concentrations of TOC, metals, mercury, pesticides, PAHs, and PCBs.
Table 6.2 identifies the storage requirements. Laboratories may choose to use any analysis
method, including those in Table 6.2, which measures the parameters to the levels of the method
detection limits identified in Table 6.3. In addition, the contaminant analysis method must meet
the precision and accuracy targets of 30% and 20%, respectively. For each batch of contaminant
samples, precision is assessed using the relative percent difference (RPD) between the matrix
spike (MS) and the matrix spike duplicate (MSD); and accuracy by the average percent recovery
(%Rs) between the matrix spike and matrix spike duplicate. Section 6.3.1 provides the equations
used to calculate the RPD and %Rs. The precision and accuracy targets for each batch of TOC
are both 10% and determined by the RPD of one sample and its duplicate (for precision) and the
analysis of Certified Reference Material (CRM; for accuracy). The grain size target precision is
10% as determined using a Laboratory Control Sample (LCS) (accuracy is not applicable).
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Table 6.2 Sediment Chemistry, Grain Size, and TOC: Analytical Methods
Storage
Requirements
Freeze samples with
maximum of -20ฐ C
Type
Metals (except Mercury)
Mercury
PCBs, Pesticides, PAHs
Methods that Meet the QA/QC
Requirements (any method
that meets the QA/QC
requirements is acceptable)
Extraction: EPA Method 3051A
Analysis: EPA Method 6020A14
EPA Method 245.715
Extraction: EPA Method 3540C
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14 For example, see:
Method 3051A "Microwave Assisted Acid Digestion of Sediments, Sludges, Soils, And Oils" retrieved
June 27, 2014 from http://www.epa.gov/osw/hazard/testmethods/sw846/pdfs/3051a.pdf: and
Method 6020A "Inductively Coupled Plasma-Mass Spectrometry" retrieved June 27, 2014 from
http://www.epa.gov/osw/hazard/testmethods/sw846/pdfs/6020a.pdf.
15 For example, see Method 245.7 "Mercury in Water by Cold Vapor Atomic Fluorescence Spectrometry, Revision
2.0" (EPA-821-R-05-001, February 2005), retrieved June 27, 2014 from
http://water.epa.gov/scitech/methods/cwa/bioindicators/upload/2007 07 10 methods method 245 7.pdf.
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Refrigerate at 4ฐ C
(do not freeze)
TOC
Grain Size
Analysis: EPA Method 8270D16
Lloyd Kahn Method17
Any method that reports the
determination as %silt and meets
QA/QC requirements
Table 6.3 Sediment Chemistry, Grain Size, and TOC: Required Parameters
Type
META
L
PCB
UNITS
% sand and
% silt/clay
mg/kg
dry weight
M9/9
(ppm)
dry weight
ng/g
(ppb)
Parameter
Grain Size
Total Organic Carbon (TOC)
Aluminum
Antimony
Arsenic
Cadmium
Chromium
Copper
Iron
Lead
Manganese
Mercury
Nickel
Selenium
Silver
Tin
Vanadium
Zinc
2,2',3,3',4,4',5,5',6,6'-
Decachlorobiphenyl
2,4'-Dichlorobiphenyl
2,2',3,3',4,4',5-Heptachlorobiphenyl
2,2',3,4',5,5',6-Heptachlorobiphenyl
2,2',3,4',5,5',6-Heptachlorobiphenyl
2,2',3,3',4,4'-Hexachlorobiphenyl
2,2',3,4,4',5'-Hexachlorobiphenyl
2,2',4,4',5,5'-Hexachlorobiphenyl
2,2',3,3',4,4',5,5',6-
Nonachlorobiphenyl
CAS
Number
not applicable
not applicable
7429-90-5
7440-36-0
7440-38-2
7440-43-9
7440-47-3
7440-50-8
7439-89-6
7439-92-1
7439-96-5
7439-97-6
7440-02-0
7782-49-2
7440-22-4
7440-31-5
7440-62-2
7440-66-6
2051-24-3
34883-43-7
35065-30-6
52663-68-0
35065-29-3
38380-07-3
35065-28-2
35065-27-1
40186-72-9
PCB
Number
(where
applicabl
e)
209
8
170
187
180
128
138
153
206
MDL
Targe
t
0.05%
0.01%
1500
0.2
1.5
0.05
5.0
5.0
500
1.0
1.0
0.01
1.0
0.1
0.3
0.1
1.0
2.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
16 For example, see:
Method 3540C "Soxhlet Extraction" retrieved June 27, 2014 from
http://www.epa.gov/osw/hazard/testmethods/sw846/pdfs/3540c.pdf: and
Method 8270D "Semivolatile Organic Compounds by Gas Chromatography/Mass Spectrometry (GC/MS)
retrieved June 27, 2014 from http://www.epa.gov/osw/hazard/testmethods/sw846/pdfs/8270d.pdf.
17 For example, the "Lloyd Kahn Method" developed by Lloyd Kahn at EPA Region II and retrieved from
www.ni.gov/dep/srp/guidance/rs/llovdkahn.pdf.
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Type
PEST
PAHs
UNITS
dry weight
ng/g
(ppb)
dry weight
ng/g
(ppb)
Parameter
2,2',3,3',4,4',5,6-Octachlorobiphenyl
2,3,3',4,4'-Pentachlorobiphenyl
2,2',4,5,5'-Pentachlorobiphenyl
2,3',4,4',5-Pentachlorobiphenyl
2,3,3',4,6'-Pentachlorobiphenyl
3,3',4,4',5-Pentachlorobiphenyl
2,2',3,5'-Tetrachlorobiphenyl
3,3',4,4'-Tetrachlorobiphenyl
2,2',5,5'-Tetrachlorobiphenyl
2,3',4,4'-Tetrachlorobiphenyl
2,2',5-Trichlorobiphenyl
2,4,4'-Trichlorobiphenyl
2,4'-DDD
2,4'-DDE
2,4'-DDT
4,4'-DDD
4,4'-DDE
4,4'-DDT
Aldrin
Alpha-BHC
Beta-BHC
Delta-BHC
Alpha-Chlordane
Gamma-Chlordane
Dieldrin
Endosulfan 1
Endosulfan II
Endosulfan Sulfate
Endrin
Endrin Aldehyde
Endrin Ketone
Heptachlor
Heptachlor Epoxide
Hexachlorobenzene
Lindane
Mi rex
Cis-Nonachlor
Oxychlordane
Trans-Nonachlor
Acenaphthene
Acenaphthylene
Anthracene
Benz(a)anthracene
Benzo(b)fluoranthene
Benzo(k)fluoranthene
Benzo(g,h,i)perylene
Benzo(a)pyrene
Benzo(e)pyrene
CAS
Number
52663-78-2
32598-14-4
37680-73-2
31508-00-6
38380-03-9
57465-28-8
41464-39-5
32598-13-3
35693-99-3
32598-10-0
37680-65-2
7012-37-5
53-19-0
3424-82-6
789-02-6
72-54-8
72-55-9
50-29-3
309-00-2
319-84-6
319-85-7
319-86-8
5103-71-9
5566-34-7
60-57-1
959-98-8
33213-65-9
1031-07-8
72-20-8
7421-93-4
53494-70-5
76-44-8
1024-57-3
118-74-1
58-89-9
2385-85-5
5103-73-1
26880-48-8
39765-80-5
83-32-9
208-96-8
120-12-7
200-280-6
205-99-2
207-08-9
191-24-27-2
50-32-8
192-9
PCB
Number
(where
applicabl
e)
195
105
101
118
110
126
44
77
52
66
18
28
MDL
Targe
t
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
10
10
10
10
10
10
10
10
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Type
UNITS
Parameter
Biphenyl
Chrysene
Dibenz(a,h)anthracene
Dibenzothiophene
2,6-Dimethylnaphthalene
Fluoranthene
Fluorene
lndeno(1 ,2,3-c,d)pyrene
1 -Methylnaphthalene
2-Methylnaphthalene
1 -Methylphenanthrene
Naphthalene
Perylene
Phenanthrene
Pyrene
2,3,5-Trimethylnaphthalene
CAS
Number
92-54-4
218-01-9
53-70-3
132-65-0
581-42-0
205-99-2
86-73-7
193-39-5
90-12-0
91-57-6
832-69-9
91-20-3
198-55-0
85-01-8
129-00-0
2245-38-7
PCB
Number
(where
applicabl
e)
MDL
Targe
t
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
6.6 Data Entry
Table 6.4 identifies the required data elements that laboratories must provide to EPA, preferably
in EPA's data template, available separately from EPA. If the laboratory applies its own QC
codes, the data transmittal should define the codes.
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Table 6.4 Sediment Chemistry, Grain Size, and TOC: Data Elements for Each
Sample
Variable
SITE ID
SAMPLE
ANALYSIS TYPE
REPEAT
DATE COLLECT
ARRIVAL TEMP
CONDITION_CODE
COND COMMENT
Type
Character
Character
Character
Numeric
Date
Numeric
Character
Character
Description
Site identification code or type of QC sample (e.g., LAB BLANK)
Sample number, LCS, QCCS, Blank, Matrix Spike, orCRM
Contaminant, TOC, or GRAIN SIZE
Duplicate
Date that the field crew collected the sample
Temperature of sample upon arrival at the laboratory
Condition codes describing the condition of the sample upon
arrival at the laboratory; leave blank for control
Flag
OK
C
L
ML
VT
VR
Q
Definition
Sample is in good condition
Sample container is cracked
Sample or container is leaking
Sample label is missing
Volume not sufficient for testing
Volume not sufficient for a retest, if required
Other quality concerns, not identified above
Explanation for Q FLAG (if needed)
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Variable
PARAMETER
CAS NO
LABNAME
METHOD
ANALYST
REVIEWER
INSTRUMENT
DATE
PROCESSED
QC_BATCH_LOT
HOLDING TIME
MATRIX
MDL
LRL
MOISTURE
MOIST UNIT
DILUTION
RECOVERY
RESULT
REASON
RESULT QUAL
UNIT
QC_CODE
COMMENT
Type
Character
Character
Character
Character
Character
Character
Character
Date
Character
Y/N
Character
Numeric
Numeric
Numeric
Character
Numeric
Numeric
Numeric
Character
Character
Character
Character
Character
Description
Analyte name
CAS Registry number
Laboratory name (abbreviation)
Laboratory method used
Last name or initials of person who performed the analysis
Last name or initials of the person who provided a separate
independent review of the data
Identification of instrument used for the analysis - provide
enough information to identify the particular instrument in the
laboratory
Date that the analysis started
Unique laboratory quality control lot numbers must be assigned
to each batch of samples. The lot number must associate each
batch of field samples to the appropriate laboratory control
sample, matrix spike, laboratory duplicate, method blank, and
CRM samples.
Analysis performed within holding time
Sediment (Water also is a permissible value if the laboratory
analyzes a very liquid sediment sample as water)
Lab method detection limit (based upon lab's historical data)
Lab reporting limit (based upon lab's historical data)
Moisture in the sample (value used by lab to convert wet units
to dry)
Unit used to report moisture (% or mg/kg)
Dilution of sample (blank or 1 if no dilution)
Only for appropriate QC samples
Concentration value
Reason for qualification in RESULT QUAL (usually blank)
Data qualifier (usually blank)
Unit of measurement for RESULT, MDL, and RL
Apply laboratory defined QC codes and describe in the
comments field. Provide set of laboratory's code as part of the
case narrative
Explain situation that created QC code, or any unusual aspects
of the analysis
6.7 Quality Measures
This section describes the quality assurance and quality control measures used to ensure that the
data will meet NCCA's requirements.
6.7.1 Assistance Visits
Assistance visits are intended to familiarize EPA with actual procedures being implemented by
different laboratories; and to ensure a clear and consistent understanding of procedures and
activities by both EPA and the laboratories. If EPA decides to conduct an assistance visit, a
qualified EPA scientist or contractor will administer a checklist based upon the steps described in
this chapter.
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6.7.2 QC Samples
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Once or twice during the performance period, the External QC Coordinator will provide one or
two identical sets of QC samples to all participating laboratories. Each set will contain up to five
QC samples. As determined by the External QC Coordinator, the QC samples may be synthetic;
aliquots of additional samples collected at NCC A sites; or reference samples obtained from an
organization such as the National Institute of Standards. Each laboratory will run the QC samples
following the same procedures used for the other samples. The External QC Coordinator will
compare the results to the expected value and determine consistency between laboratories (e.g.,
determine if one laboratory is consistently higher or lower than all others). Based upon the
evaluation, the External QC Coordinator may request additional information from one or more
laboratories about any unique laboratory practices that might account for differences between the
laboratory and others. The contractor shall analyze the external QC samples using the same
procedures as those for the field samples.
6.7.3 Summary of QA/QC Requirements
QC protocols are an integral part of all analytical procedures to ensure that the results are reliable
and the analytical stage of the measurement system is maintained in a state of statistical control.
The laboratory must conduct QC analyses for each batch of samples. Each batch shall consist of
no more than 20 samples. Unique laboratory quality control lot numbers must be assigned to
each batch of samples. The lot number must associate each batch of field samples to the
appropriate measures such as laboratory control sample, matrix spike, laboratory duplicate, and
method blank samples. Also, each laboratory QC samples (i.e., preparation and instrument
blanks, laboratory control sample (LCS), spike/duplicate, etc.) must be given a unique sample
identification. Table 6.5 provides a summary of the quality control requirements.
Table 6.5 Sediment Chemistry, Grain Size, and TOC: Quality control activities for
samples
Activity
Demonstrate competency for
analyzing sediment samples to
meet the performance measures
Check condition of sample when
it arrives.
Store sample appropriately.
While stored at the laboratory,
the sample must be kept at a
temperature <-20ฐ C except jars
for grain analyses are
refrigerated at 4ฐC.
Evaluation
Demonstration of competency
with sediment samples in
achieving the method detection
limits, accuracy, and precision
targets.
Sample issues such as cracked
container; missing label;
sufficient volume for test.
Check the temperature of the
refrigerator/freezer and
refrigerator per laboratory's
standard operating procedures.
Corrective Action
EPA will not approve any
laboratory for NCCA sample
processing if the laboratory
cannot demonstrate competency.
In other words, EPA will select
another laboratory that can
demonstrate competency for its
NCCA samples.
Assign appropriate condition
code identified in Table 6.4.
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.
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Activity
Evaluation
Corrective Action
Data analyst will consider
temperature deviations in
evaluating the data. He/she will
flag the deviations and determine
whether the data appear to be
affected and/or the data should
be excluded from the analyses.
Analyze sample within holding
time
The test must be completed
within the holding time of 1 year.
If the original test fails, then the
retest also must be conducted
within the holding time.
Perform test, but note reason for
performing test outside holding
time. EPA expects that the
laboratory will exercise every
effort to perform tests before the
holding time expires.
Perform once at the start of each
batch to evaluate the labeled
compound recovery (LCR) in a
Laboratory Control Sample
(LCS). This tests the
performance of the equipment.
Control limits for recovery cannot
exceed 100ฑ20%.
First, prepare and analyze one
additional LCS. If the second
blank meets the requirement,
then no further action is required.
If the second LCS fails, then
determine and correct the
problem before proceeding with
any sample analyses.
Perform once at the start of each
batch to evaluate the entire
extraction and analysis process
using a Method Blank
Control limits cannot exceed the
laboratory reporting level (LRL).
First, prepare and analyze one
additional blank. If the second
blank meets the requirement,
then no further action is required.
If the second blank fails, then
determine and correct the
problem (e.g., contamination,
instrument calibration) before
proceeding with any sample
analyses. Reestablish statistical
control by analyzing three blank
samples. Report values of all
blanks analyzed.
Check calibration immediately
before and immediately after the
sample batch (abbreviated as
QCCS for quality control check
sample)
Results must be ฑ10% of each
other or as specified in method
criteria
If calibration fails before analysis,
recalibrate and reanalyze QCCS
until it passes. If check fails after
all samples the batch have been
analyzed, verify the QCCS
reading. If the QCCS reading
fails a second time, then
reanalyze all samples in the
batch and report only the set of
results associated with the
acceptable QCCS reading. Also
report all QCCS readings for the
batch.
Compare results of one
laboratory duplicate sample (for
TOC) or matrix spike duplicate
sample (for contaminants) for
each batch (not required for grain
size)
Results must be within the target
precision goal in Section 6.5.
If both results are below LRL,
then conclude that the test has
passed. Otherwise, prepare and
analyze a split from different
sample in the batch. If the
second result is within the target
precision goal (see Section 6.5)
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Activity
Evaluation
Corrective Action
of the original sample, then
report the data and findings for
both QC samples. However, if
the two results differ by more
than the target precision goal,
review precision of QCCS
measurements for batch; check
preparation of split sample; etc.
and report evaluation and
findings in the case narrative.
Consult with the EPA HQ NCCA
Laboratory Review Coordinator
to determine if reanalysis of the
entire batch (at the laboratory's
expense) is necessary. If no
reanalysis is necessary, report
and quantify all samples in batch.
If reanalysis is necessary, then
report all QC sample and the 2nd
analysis of the batch. If the
second set also is unacceptable,
then assign a data code to each
sample in the batch.
Compare results of one matrix
spike sample per batch to
evaluate performance in matrix
(not required for TOC and grain
size)
Evaluate performance after the
first 3 batches; and then every
subsequent batch. Ideally,
control limits for recovery will not
exceed the target accuracy goal,
but this may not be realistic for all
parameters with this matrix.
If both the original and duplicate
results are below LRL, then
conclude that the test has
passed for the batch. Otherwise,
if any results are not within the
target accuracy goal for the first 3
batches, within 2 working days,
contact the EPA HQ NCCA
Laboratory Review Coordinator
to discuss method performance
and potential improvements.
After achieving acceptable
results or EPA's permission to
continue, perform the test for
every subsequent batch. For
each batch, report the results
from the original analysis and its
duplicate and their RPD for TOC;
the matrix spike, matrix spike
duplicate, RPD and %recovery
for contaminants.
Compare results of TOC Certified
Reference Material once per
each batch
Value must be within 10% of the
certified value.
If value is outside the acceptable
range, analyze a second CRM. If
the second CRM also is
measured outside the acceptable
range, then determine and
correct the problem (e.g.,
contamination, instrument
calibration) before reanalyzing all
samples in the batch.
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Activity
Evaluation
Corrective Action
Maintain the required MDL
identified in Section 6.5
Evaluate for each sample
If MDL could not be achieved,
then provide dilution factor or QC
code and explanation in the
comment field.
Participate in External Quality
Control
Evaluate QC samples provided
by the External QC Coordinator
Based upon the evaluation, the
External QC Coordinator may
request additional information
from one or more laboratories
about any deviations from the
Method or unique laboratory
practices that might account for
differences between the
laboratory and others. With this
additional information, the
External QC Coordinator will
determine an appropriate course
of action, including no action,
flagging the data, or excluding
some or all of the laboratory's
data.
Maintain completeness
Completeness objective is 95%
for all parameters.
Contact EPA HQ NCCA
Laboratory Review Coordinator
immediately if issues affect
laboratory's ability to meet
completeness objective.
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*Chapter2 provides contact information for the EPA HQ NCCA Laboratory Review Coordinator.
Laboratories under contract to EPA must contact the Task Order's Contracting Officer's Representative
(TOCOR) instead of the Laboratory Review Coordinator.
6.8 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 used
in the contaminant analyses and refrigerate those used for the grain size and TOC. 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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6.9 References
All references are from U.S. Environmental Protection Agency:
Method 245.7 "Mercury in Water by Cold Vapor Atomic Fluorescence Spectrometry, Revision
2.0" (EPA-821-R-05-001, February 2005), retrieved June 27, 2014 from
http://water.epa. gov/scitech/methods/cwa/bioindicators/upload/2007_07_10_methods_method_2
45_7.pdf.
Method 305 la "Microwave Assisted Acid Digestion of Sediments, Sludges, Soils, And Oils"
retrieved June 27, 2014 from
http://www.epa.gov/osw/hazard/testmethods/sw846/pdfs/3051a.pdf
Method 31 50A "Microwave Assisted Acid Digestion of Sediments, Sludges, Soils, and Oils,"
retrieved June 27, 2014 from
http://www.epa.gov/osw/hazard/testmethods/sw846/pdfs/3051a.pdf
Method 3540C Method 3540C "Soxhlet Extraction" retrieved June 27, 2014 from
http://www.epa.gov/osw/hazard/testmethods/sw846/pdfs/3540c.pdf
Method 6020 A "Inductively Coupled Plasma-Mass Spectrometry" retrieved June 27, 2014 from
http://www.epa.gov/osw/hazard/testmethods/sw846/pdfs/6020A.pdf oo
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Method 8270D "Semivolatile Organic Compounds by Gas Chromatography/Mass Spectrometry ^
(GC/MS) retrieved June 27, 20 1 4 from <
http://www.epa.gov/osw/hazard/testmethods/sw846/pdfs/8270D.pdf <<
O
Method 9171B "n-Hexane Extractable Material (FffiM) for Sludge, Sediment, And Solid H
Samples," retrieved June 27, 2014 from Q
http://www.epa.gov/osw/hazard/testmethods/sw846/pdfs/9071b.pdf ^
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7.0 WATER CHEMISTRY AND CHLOROPHYLL A
This chapter describes the analysis requirements for water quality samples. The purpose is to
determine concentrations of water quality parameters and chlorophyll a in water quality samples
collected in the 2015 NCCA and related studies. The laboratory shall perform analysis to
determine levels of ammonia (NHs), nitrate (NOs), nitrate-nitrite (NOs-NCh), total nitrogen
(TN), total phosphorous (TP) and ortho-phosphate (PO/O (also called soluble reactive phosphorus
(SRP), pH, conductivity and chlorophyll a found in coastal waters and Great Lakes. In addition,
the laboratory shall measure chloride (Cl) and sulfate (SO/t) levels in Great Lakes samples.
In the following discussion, Sections 7.1, 7.2, and 7.3 summarize the procedure; health and
safely concerns; and definitions and required resources. Section 7.4 provides the steps for
acknowledging sample receipt. Sections 7.5 - 7.6 provide the minimum requirements that the
laboratory must meet in performing the analyses and the required data elements. Section 7.7
describes EPA's external review of laboratory operations and other quality measures. Section 7.8
identifies references used in developing the procedure.
7.1 Summary of the Procedure
This chapter describes the analysis of ammonia, nitrate-nitrite, total nitrogen, total phosphorous
and ortho-phosphate, nitrate, pH, conductivity and chlorophyll a, and chloride samples collected
for EPA's 2015 National Coastal Condition Assessment (NCCA). As described in Section 7.5,
unless otherwise contractually bound by other requirements, the laboratory may choose to use
any method that meets EPA's specifications for contamination measurements.
7.2 Health and Safety Warnings
The laboratory must require its staff to abide by appropriate health and safety precautions. In
addition to the laboratory's usual requirements such as a Chemical Hygiene Plan, the laboratory
must adhere to the following health and safety procedures: ^
">^
1. Laboratory facilities must properly store and dispose of solutions of weak acid. o1
J3
2. Laboratory personnel must wear proper personal protection clothing and equipment (e.g. <->
lab coat, protective eyewear, gloves). B
<
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 H
avoid contacting skin and mucous membranes with acid. If skin contact occurs, remove H
clothing immediately. Wash and rinse the affected skin areas thoroughly with large U
amounts of water.
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7.3 Definitions and Required Resources (Personnel, Laboratories, and Equipment)
This section provides definitions and required resources for using the procedure.
7.3.1 Definitions
The procedure uses the following terms:
Cl: Chloride
Detection Limit is the minimum concentration at which the analyte can be detected with
confidence. In other words, the outcome can be reported with confidence that it is greater
than zero (i.e., present in the sample) Also see "Sample-Specific Detection Limit."
Duplicates are defined as two aliquots of the same sample which are analyzed separately
using identical procedures. The results are used to evaluate the precision of the laboratory
analyses.
NARS: National Aquatic Resource Surveys. The National Coastal Condition Assessment
(NCCA) is part of the NARS program.
NARS Information Management System (NARS IM): The EVI system established to
support all surveys, including NCCA, in the NARS program. The EVI system is used to track
the samples from field collection to the laboratory.
NCCA: National Coastal Condition Assessment. Freshwater and coastal samples will be
collected during the field stage of NCCA.
Nib: Ammonia
NO3: Nitrate
a
NO3-NO2: Nitrate-nitrite ^
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Percent Recovery: Recovery is measured by comparing the concentrations of a sample split <3
into two parts; and one part is spiked with a known concentration value. G is the ^
concentration measured in the spiked part; C is the concentration measured in the unspiked Q
part; and s is the known concentration amount for the spike. The following equation is used ^
to calculate the percent recovery: >H
Cs- C ฃ
%Rs = x 100 %
ง
Relative Standard Deviation (RSD): The precision at each concentration is reported in H
terms of the RSD. To calculate the RSD, first calculate the standard deviation, S, as follows: ^
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5 =
1/2
where n is the number of replicate samples, C, is the concentration measure for the kth
sample, and C is the average concentration of the replicate samples. Then, RSD is calculated
as:
5
RSD = -= x 100
l>
Reporting Limit: A reporting limit is the point at which the measured value of the analyte
can be reported with confidence.
Sample-Specific Detection Limit: Most samples will have a sample-specific detection equal
to the method's detection limit. For diluted samples, the sample-specific detection limit will
be the product of the method's detection limit and the dilution factor. Typical values for the
dilution factors will be 10 or 100.
SO4: Sulfate.
Spiked Sample: See Percent Recovery definition for purpose of spiked samples.
SRP: Soluble Reactive Phosphorus (also called orthophosphate)
TN: Total nitrogen
TP: Total phosphorous
7.3.2 General Requirements for Laboratories
Expertise. To demonstrate its competency /expertise, the laboratory shall provide EPA with
performance data demonstrating their proficiencies in analyzing water quality samples. In
addition, the laboratory must provide 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.
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), Laboratory Quality Assurance Manuals,
QAPPs, and applicable Standard Operating Procedures (SOPs).
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To demonstrate its ongoing commitment, the person in charge of quality issues for the
organization shall sign the NCCA QAPP Certification Page.
7.3.3 Personnel
The procedure refers to the following personnel:
Laboratory Technician: This procedure may be used by any laboratory technician who
is familiar with the NCCA Quality Assurance Project Plan, and this procedure in the
NCCA Laboratory Operations Manual.
7.3.4 Equipment/Materials
The analytical method, selected by the laboratory, identifies the necessary equipment.
7.4 Sample Receipt
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. For each sampled site, the lab will receive the following
samples on wet ice:
One 250 ml amber bottle labeled 'CHEM' for water chemistry analyses
A filter in a 50 ml tube for chlorophyll a labeled 'CHLA'
Additionally, as a separate batch shipment the lab will receive 250 ml bottles labeled 'NUTS' for
dissolved nutrients analyses (either from the crews or from an EPA batching laboratory). Crews
and the batch lab will maintain these samples frozen but will ship overnight on wet ice.
The laboratory technician must inspect the samples promptly on receipt and:
1 . Log the samples into the National Aquatic Resource Survey Information Management
system (NARS-IM) within 24 clock hours. Alternatively, for shipments with a large
number of samples, the laboratory may email a spreadsheet with the sample login and
sample condition information to NARS-IM (see Chapter 2 for contact information).
3
2. Check that each shipping container has arrived undamaged. Check the temperature of one "o
of the samples in the cooler using a thermometer that reads to at least -20 ฐC (i.e., the P
expected temperature of frozen samples), or an infra-red (IR) temperature "gun" and <;
record the reading. Temperature of the wet ice shipments should be 4 ฐC or at less. >}
.Record the condition and temperature of the sample in the database using the codes in H
Table 7.1. |
3. Verify that all required data elements, per Table 7. 1, have been recorded in the NARS IM S
database. If any data elements are missing, then enter them into the database. p^
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a. Water chemistry aliquots are prepared following the requirements in Section 7.5
and then are stored in a refrigerator at 4ฐ C.
b. Chlorophyll-a filters to the freezer for no more than 30 days before analysis.
Except during processing and analysis stages, the filter must be stored frozen to
less than or equal -20 ฐC ฑ 2ฐ.
c. Dissolved nutrient samples are prepared following the requirements in Section 7.5
and then are stored in a refrigerator at 4ฐ C.
5. Notify the EPA immediately about any problems involving sample integrity, conformity,
or inconsistencies as soon as possible following sample receipt and inspection.
Table 7.1 Water Chemistry Login: Required Data Elements
Variable
SITE ID
SAMPLE
DATE COLLECT
ANALYSIS_TYPE
ARRIVAL_TEMP
CONDITION COD
E
COND COMMENT
Type
Character
Character
Date
Character
Numeric
Character
Character
Description
Site identification code
Sample number
Date that the field crew collected the sample
Water Chemistry or Chlorophyll a or Nutrients
Temperature of sample upon arrival at the laboratory
(CHEM, CHLA and NUTS sample will be on wet ice);
Condition codes describing the condition of the sample
upon arrival at the laboratory; leave blank for control
Flag
OK
C
L
ML
NF
Q
Definition
Sample is in good condition
Sample container is
cracked
Sample or container is
leaking
Sample label is missing
Sample is not at proper
temperature
Other quality concerns, not
identified above
Explanation for Q FLAG (if needed)
7.5 Preparation of Water Chemistry Aliquots
Figure 7.1 presents the sample preparation processing steps for the water chemistry indicators,
including filtering and acidifying.
For the dissolved nutrient (NUTS) sample, the laboratory technician:
1. Thaws the frozen sample.
2. Splits the sample into two aliquots as shown in figure 7.1.
3. Adds ultra-pure acid (H2S04, depending on the analytes, see Table 7.2) to one of the two
aliquots. Caps the bottle tightly and inverts the bottle several times to mix.
4. Stores all aliquots in a refrigerator at 4ฐC.
For the unfiltered, water chemistry (CHEM) sample, the laboratory technician
1. Thaws the frozen sample.
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2. Splits the sample into two aliquots as shown in figure 7.1.
3. Adds ultra-pure acid (H2S04,) to one aliquot of the unfiltered, CHEM sample. Caps the
bottle tightly and inverts the bottle several times to mix.
4. Stores all aliquots in a refrigerator at 4ฐC.
If the dissolved nutrient sample is compromised in some way, the laboratory technician will filter
a new sample from the water chem (CHEM) sample as follows:
1. Uses 0.4um pore size polycarbonate filters for all filtration.
2. Rinses vacuum filter funnel units thoroughly with reverse-osmosis (RO) or de-ionized
(DI) 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 DI
water through the filter, with vacuum pressure, to rinse the filter. Discard the rinse water.
3. Places 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 DI water before continuing.
4. After all filtered and unfiltered aliquots are collected, adds ultra-pure acid (tfeSO/t,
depending on the analyte, see Table 7.2) to the sample in the aliquot container. Cap
tightly and invert the bottle several times to mix.
5. Stores all aliquots in a refrigerator at 4ฐC.
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Sample Receipt
250 ml amber bottle (CHEM) and 250 ml bottle
(NUTS) (filtered in the field)
Inspect samples and complete tracking
form
^ '
[ NUTS Filtered in
I
Process Sample within 24 hours
the field ]
I
t
f \
HOPE bottle
Not acid washed
Store at 4 ฐC in
darkness
v /
A
Analyses
Chloride (28 days)
Nitrate (7 days)
Ortho-phosphate (7 days)
Nitrate-Nitrite (7 days)
(freshwater)Sulfate (28 days)
y .
HOPE bottle
Acid washed
Preserve with H2SO4
( ,
J
Analyses
Ammonia (28 days)
Nitrate-Nitrite (28 days)
(marine/brackish)
L. J
CHEM Not Filtered ]
|_
f \
HOPE bottle
Acid washed
Preserve with H2SO4
\ /
I
Analyses
Total Phosphorus (28
days)
Total Nitrogen (28 days)
^ J
y
f X
HOPE bottle
NOT Acid
washed store
in 4ฐC
\ /
V
r ">
Analyses
pH (3 days)
Conductivity (28
days)
Figure 7.1 Water Chemistry and Dissolved Nutrient Samples: Receipt and Holding
Times
Table 7.2 Water chemistry: acid preservatives added for various indicators
Indicators
Preservatives
H2S04Used for:
NH4
Total N
Total P
NO2-NO3
7.6 Water Chemistry and Chlorophyll a Analysis: Requirements
The laboratory shall perform analysis of the samples to determine the ammonia (NHs), chloride
and sulfate (Great Lakes only), nitrate-nitrite (NOs-NCh), total nitrogen (TN), total phosphorous
(TP) and ortho-phosphate, nitrate (NOs), and chlorophyll a. As an alternative to specifying
laboratory methods for sample analysis, NCCA uses a performance-based approach that defines
a set of laboratory method performance requirements for data quality as shown in Table 7.3.
Method performance requirements for this project identify the reporting limit, precision, and
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accuracy objectives for each parameter. NCCA is designating the reporting limit as the lowest
value that the laboratory needs to quantify (as opposed to just detecting the parameter in the
sample), and is the value of the lowest non-zero calibration standard that the laboratory must use.
EPA has set the value to double the long-term method detection limit (LT-MDL), following
guidance presented in Oblinger, Childress et al. (USGS, 1999)18.
NCCA expresses precision and accuracy objectives in both absolute and relative terms following
Hunt and Wilson (1986). The transition value is the value at which performance objectives for
precision and accuracy switch from absolute (< transition value) to relative (> transition value).
For pH, the objectives are established for samples with lower, midrange and higher pH levels.
For duplicate samples, NCCA estimates the precision 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.
Accuracy 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.
Table 7.4 summarizes the analytical methods used at the NCCA central laboratory (EPA ORD-
Corvallis). Other participating laboratories may use alternative analytical methods for each target
analyte as long as they can satisfactorily demonstrate the alternative method is able to achieve
the performance requirements as listed in Table 7.3. Appendix A identifies the information that
the laboratory should provide to the NCCA Laboratory Review Coordinator to use in
determining whether the laboratories meet the necessary requirements.
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18 If a laboratory has questions related to meeting the -LT-MDL, they may contact the NCCA Laboratory Review
Coordinator to discuss concerns.
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Table 7.3 Water Chemistry and Chlorophyll-a: Laboratory Method Performance
Requirements
Parameter
Ammonia (Nhb)
Chloride (Cl)
Conductivity
Nitrate-Nitrite
(NO3-NC-2)
pH (Laboratory)
Total Nitrogen
(TN)
Total
Phosphorous (TP)
and
ortho-Phosphate
Nitrate (NO3)
Sulfate (SCM)
Chlorophyll-a
Units
mg N/L
mg CI/L
|j,S/cm
at 25ฐC
mg N/L
Std
Units
mg N/L
mg P/L
mgN/L
mg/L
(og/L in
extract
Potential
Range
of Samples1
Oto 17
0 to 5, 000
1-66,000
0 to 360
(as nitrate)
3.5-10
0.1 to 90
Oto 22
(as TP)
0. to 360
0 to 5000
0.7 to 11, 000
Method
Detection
Limit
Objective2
0.01 marine
(0.7 ueq/L)
0.02 freshwater
0.20 (6 ueq/L)
1.0
0.01 marine
0.02 freshwater
N/A
0.01
0.002
0.01 marine
(10.1 ueq/L)
0.03 freshwater
0.5 freshwater
(10.4 ueq/L)
1.5
Transitio
n Value3
0.10
1
20
0.10
5.75, 8.25
0.10
0.02
0.1
2.5
15
Precision
Objective
4
ฑ0.01 or
ฑ10%
ฑ0.10 or
ฑ10%
ฑ2 or ฑ10%
ฑ0.01 or
ฑ10%
<5.75 or
> 8.25 =
ฑ0.07;
5.75-8.25 =
ฑ0.15
ฑ0.01 or
ฑ10%
ฑ0.002 or
ฑ10%
ฑ0.01 or
ฑ5%
ฑ0.25 or
ฑ10%
ฑ 1.5 or
ฑ10%
Accuracy
Objective
5
ฑ0.01 or
ฑ1 0%
ฑ0.10 or
ฑ1 0%
ฑ2 or ฑ 5%
ฑ0.01 or
ฑ1 0%
<5.75 or
>8.25
=ฑ0.15;
5.75-8.25
= ฑ0.05
ฑ0.01 or
ฑ1 0%
ฑ 0.002 or
ฑ1 0%
ฑ0.01 or
ฑ5%
ฑ0.25 or
ฑ1 0%
ฑ 1.5 or
ฑ1 0%
1 Estimated from samples analyzed at the EPA Western Ecological Division-Corvallis laboratory between
1999 and 2005
2 The method detection limit is determined as a one-sided 99% confidence interval from repeated
measurements of a low-level standard across several calibration curves.
3 Value for which absolute (lower concentrations) vs. relative (higher concentrations) objectives for
precision and accuracy are used.
4 For duplicate samples, precision is estimated as the pooled standard deviation (calculated as the root-
mean square) of all samples at the lower concentration range, and as the pooled percent relative
standard deviation of all samples at the higher concentration range. For standard samples, precision is
estimated as the standard deviation of repeated measurements across batches at the lower concentration
range, and as percent relative standard deviation of repeated measurements across batches at the higher
concentration range.
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For pH precision, the looser criteria applies to mid-range samples. For NCCA, that is less of a concern than the
ability to measure more acidic or basic samples accurately and precisely.
5 Accuracy is estimated as the difference between the measured (across batches) and target values of
performance evaluation and/or internal reference samples at the lower concentration range, and as the
percent difference at the higher concentration range.
Table 7.4 Water Chemistry and Chlorophyll-a: Analytical Methods Used by Central
Laboratory, EPA ORD-Corvallis)
Analyte
Nitrate+ Nitrite, as
N
Ammonia, as N
Total nitrogen
(TN)
Total phosphorus
(TP) and ortho-
Phosphate
Nitrate,
chloride, sulfate
Chlorophyll-a
(Chl-a)
pH (lab)
Specific
conductance @
25ฐC
Summary of Method19
Ion Chromatography (freshwater
samples)
OR
FIA automated colorimetric (cadmium
reduction for brackish samples)
FIA automated colorimetric (salicylate,
dichloroisocyanurate)
Persulfate Digestion; FIA Automated
Colorimetric Analysis (Cadmium
Reduction, sulfanilamide)
Persulfate Digestion; Automated
Colorimetric Analysis (molybdate,
ascorbic acid)
Ion Chromatography (Great Lakes
samples only)
Extraction 90% acetone analysis by
fluorometry
Automated, using ManSci PC-Titrate
w/ Titra-Sip autotitrator and Ross
combination pH electrode. Initial pH
determination for ANC titration
Electrolytic, Man-Tech TitraSip automated analysis
OR manual analysis, electrolytic
References20
EPA 300.6; SW-846
9056A; APHA4110B
EPA 353 2
APHA 4500-NOs-N-E
Lachat 10-1 07-04-1 -C
Lachat10-107-06-3-D
EPA353.2 (modified)
APHA 4500-N-C
(modified)
ASTMWK31786
U.S. EPA (1987)
Lachat 10-1 07-04-1 -C
(modified)
APHA 4500-P-E
USGS I-4650-03
U.S. EPA (1987)
Lachat 11 5-01 -1-B
(modified)
EPA 300.6; SW-846
9056A; APHA 411 OB
EPA 445.0 , EPA 446.0
EPA 150.6 (modified)
EPA 120.6
WRS SOP21
WRS 36A.O
(April 201 1
WRS 40A.5
(May 2011)
WRS 30A.4
(April 2011)
WRS 34A.5
(April 2011)
WRS 34A.5
(April 2011)
WRS 40A.5
(May 2011)
WRS71A.3
(April 2011)
WRS16A.O
(April 2011)
WRS 16A.O (April
2011)
WRS11A.4
(April 2011)
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19 FIA=Flow injection analysis. AAS=Atomic Absorption Spectrometry
20 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 (StandardMethods). ASTM=American Society of
Testing and Materials.
21 WRS= Willamette Research Station. References are to laboratory SOP being used at central laboratory. Available
upon request from the EPA HQ Laboratory Review Coordinator.
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7.7 Data Entry
Table 7.5 identifies the required data elements that laboratories must provide to EPA, preferably
in EPA's data template, available separately from EPA.
Table 7.5 Water Chemistry and Chlorophyll-a: Data Elements for Each Sample
Variable
SITE ID
SAMPLE
ANALYSIS TYPE
REPEAT
DATE COLLECT
ARRIVAL TEMP
CONDITION_CODE
COND COMMENT
PARAMETER
CAS NO
LABNAME
METHOD
ANALYST
REVIEWER
INSTRUMENT
DATE
PROCESSED
QC_BATCH_LOT
HOLDING TIME
MATRIX
MDL
LRL
DILUTION
RESULT
REASON
RESULT QUAL
UNIT
QC_CODE
Type
Character
Character
Character
Numeric
Date
Numeric
Character
Character
Character
Character
Character
Character
Character
Character
Character
Date
Character
Y/N
Character
Numeric
Numeric
Numeric
Numeric
Character
Character
Character
Character
Description
Site identification code or type of QC sample (e.g., LAB BLANK)
Sample number, LCS, QCCS, Blank, Matrix Spike, orCRM
Contaminant
Duplicate
Date that the field crew collected the sample
Temperature of sample upon arrival at the laboratory
Condition codes describing the condition of the sample upon
arrival at the laboratory; leave blank for control
Flag Definition
OK Sample is in good condition
C Sample container is cracked
L Sample or container is leaking
ML Sample label is missing
NF Sample is not at pro per temperature
Q Other quality concerns, not identified above
Explanation for Q FLAG (if needed)
Analyte name
CAS Registry number
Laboratory name (abbreviation)
Laboratory method used
Last name or initials of person who performed the analysis
Last name or initials of the person who provided a separate
independent review of the data
Identification of instrument used for the analysis - provide
enough information to identify the particular instrument in the
laboratory
Date that the analysis started
Unique laboratory quality control lot numbers must be assigned
to each batch of samples. The lot number must associate each
batch of field samples to the appropriate laboratory control
sample, matrix spike, laboratory duplicate, method blank, and
CRM samples.
Analysis performed within holding time
Water
Lab method detection limit (based upon lab's historical data)
Lab reporting limit (based upon lab's historical data)
Dilution of sample (blank or 1 if no dilution)
Concentration value
Reason for qualification in RESULT QUAL (usually blank)
Data qualif er (usually blank)
Unit of measurement for RESULT, MDL, and LRL
Apply laboratory defined QC codes and describe in the
comments field. Provide set of laboratory's code as part of the
case narrative
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Variable
COMMENT
Type
Character
Description
Explain situation that created
of the analysis
QC code, or any unusual aspects
7.8 Quality Measures
This section describes the quality assurance and quality control measures used to ensure that the
data will meet NCCA's requirements. QC protocols are an integral part of all analytical
procedures to ensure that the results are reliable and the analytical stage of the measurement
system is maintained in a state of statistical control. The laboratory must conduct QC analyses
for each batch of samples. Each batch shall consist of no more than 20 samples. Unique
laboratory quality control lot numbers must be assigned to each batch of samples. The lot
number must associate each batch of field samples to the appropriate measures such as
laboratory control sample, matrix spike, laboratory duplicate, and method blank samples. Also,
each laboratory QC samples (i.e., preparation and instrument blanks, laboratory control sample
(LCS), spike/duplicate, etc.) must be give a unique sample identification. Table 7.5 provides a
summary of the quality control requirements.
Table 7.5 Water Chemistry and Chlorophyll-a: Quality control activities for water
quality samples
QC Sample
Type and
Description
Demonstrate
competency
for analyzing
water
samples to
meet the
performance
measures
Check
condition of
sample when
it arrives.
Indicators
All
All
Description
Demonstration
of past
experience
with water
samples in
achieving the
method
detection
limits
Sample issues
such as
cracked
container;
missing label;
temperature;
adherence to
holding time
requirements;
sufficient
Frequency
Once
Once
Acceptance
Criteria
See Appendix
A
No sample
issues or
determination
that sample
can still be
analyzed
Corrective
Action
EPA will not
approve any
laboratory for
NCCA sample
processing if
the laboratory
cannot
demonstrate
competency. In
other words,
EPA will select
another
laboratory that
can
demonstrate
competency for
its NCCA
samples.
Lab determines
if the sample
can be
analyzed or
has been too
severely
compromised
(e.g.,
contamination).
Assign
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QC Sample
Type and
Description
Store sample
appropriately.
Analyze
sample within
holding time
Indicators
All
All
Description
volume for
test.
Check the
temperature of
the
refrigerator
per
laboratory's
standard
operating
procedures.
Frequency
Record
temperature of
sample upon
arrival at the
laboratory. Check
temperature of the
refrigerator/freezer
where samples
are stored at least
daily if using a
continuous
temperature
logger and twice
daily (once at
beginning of the
day and once at
the end) not using
a continuous
logger.
Acceptance
Criteria
While stored
at the
laboratory, the
sample must
be kept at a
maximum
temperature of
4ฐ C (for
aliquots
except
chlorophyll a)
and -20ฐ C for
the chlorophyll
a sample.
The test must
be completed
within the
holding time
specified in
the analytical
method.
Corrective
Action
appropriate
condition code
identified in
Table 7.1.
If at any time
samples are
warmer than
required, note
temperature
and duration
(either from
the
Lilt-
continuous
temperature
log or from
the last
manual
reading) in
comment
field. Lab
will still
perform
test. EPA
expects that
the laboratory
will exercise
every effort to
maintain
samples at the
correct
temperature.
Perform test in
all cases, 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.
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QC Sample
Type and
Description
Analyze
Laboratory/
Reagent
Blank
Analyze
Filtration
Blank
Determine
LT-MDL Limit
for Quality
Control
Check
Sample
(QCCS)
Analyze
Calibration
QCCS
Indicators
All
All dissolved
analytes
All
All
Description
ASTMTypell
reagent water
processed
through
filtration unit
Prepared so
concentration
is four to six
times the LT-
MDL objective
Frequency
Once per day prior
to sample analysis
Prepare once per
week and archive
Prepare filter
blank for each box
of 100 filters, and
examine the
results before any
other filters are
used from that
box.
Once per day
Before and after
sample analyses
Acceptance
Criteria
Control limits
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QC Sample
Type and
Description
Analyze
Laboratory
Duplicate
Sample
Analyze
Standard
Reference
Material
(SRM)
Indicators
All
When
available for
a particular
indicator
Description
Frequency
One per batch
One analysis in a
minimum of five
separate batches
Acceptance
Criteria
Control limits
< precision
objective
Manufacturers
certified range
Corrective
Action
replicate
samples)
analyzed since
the last
acceptable
QCCS
measurement.
If results are
below LRL:
Prepare and
analyze split
from different
sample
(volume
permitting).
Review
precision of
QCCS
measurements
for batch.
Check
preparation of
split sample.
Qualify all
samples in
batch for
possible
reanalysis.
Analyze
standard in
next batch to
confirm
suspected
inaccuracy.
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
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QC Sample
Type and
Description
Analyze
Matrix Spike
Samples
Use
consistent
units for QC
samples and
field samples
Indicators
Only
prepared
when
samples with
potential for
matrix
interferences
are
encountered
All
Description
Verify that all
units are
provided
consistently
within each
indicator.
Frequency
One per batch
Data reporting
Acceptance
Criteria
Control limits
for recovery
cannot exceed
100ฑ20%
For each
indicator, all
field and QC
samples are
reported with
the same
measurement
units
Corrective
Action
standard
measurements
that are
acceptable.
Qualify all
sample batches
analyzed since
the last
acceptable
reference
standard
measurement
for possible
reanalysis.
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).
If it is not
possible to
provide the
results in
consistent
units, then
assign a QC
code and
describe the
reason for
different units
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QC Sample
Type and
Description
Maintain
completeness
Indicators
All
Description
Determine
completeness
Frequency
Data reporting
Acceptance
Criteria
Completeness
objective is
95% for all
indicators
(useable with
or without
flags).
Corrective
Action
in the
comments field
of the
database.
Contact EPA
HQ NCCA
Laboratory
Review
Coordinator*
immediately if
issues affect
laboratory's
ability to meet
completeness
objective.
*Chapter2 provides contact information for the EPA HQ NCCA Laboratory Review Coordinator.
Laboratories under contract to EPA must contact the Task Order's Contracting Officer's Representative
(TOCOR) instead of the Laboratory Review Coordinator.
7.9 Sample and Record Retention
The laboratory shall retain:
1. The sample materials for a minimum of 1 year after collection. During this time, the
laboratory shall store the materials cold (e.g., 4 ฐ C) and in darkness. The lab shall retain
the sample materials from the 1 year point until the EPA publishes the final report at
ambient temperatures.
2. Original records, including laboratory notebooks 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.
7.10 References
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.
USEPA, 1987. Handbook of Methods for Acid Deposition Studies: Laboratory Analyses
for Surface Water Chemistry. EPA/600/4-87/026. U.S. Environmental Protection Agency,
Office of Research and Development, Washington D.C.
USEPA. 1997. Methods for the Determination of Chemical Substances in Marine and
Estuarine Environmental Matrices - 2nd Edition EPA No. 600-R-97-072. U.S.
Environmental Protection Agency, Office of Research and Development, Washington, DC,
retrieved June 30, 1997 from http://www.epa.gov/microbes/documents/marinmet.pdf
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USEPA. September 1997. Method 353.4 "Determination of Nitrate and Nitrite in Estuarine and
Coastal Waters by Gas Segmented Continuous Flow Colorimetric Analysis, Revision 2.0",
retrieved June 30, 2014 from http://www.epa.gov/microbes/documents/m353_4.pdf
USGS. 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 by Childress, Oblinger, et a/.,
retrieved June 30, 2014 from http://pubs.usgs.gov/of/1999/0193/report.pdf.
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.
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8.0 SEDIMENT TOXICITY TESTING
This chapter describes the analysis requirements for sediment toxicity testing. The purpose is to
assess the toxicity of sediment samples collected in the 2015 NCCA and related studies.
At each sampling site, the Field Operations Manual (FOM) instructs the crews to collect
sediment samples. The field crew then ships the samples on wet ice to the laboratory. If EPA
uses a batching laboratory, it will refrigerate the samples, before shipping on wet ice to the
analysis laboratory.
In the following discussion, Sections 8.1, 8.2, and 8.3 summarize the procedure; health and
safety concerns; and definitions and required resources. Section 8.4 provides the steps for
acknowledging sample receipt. Sections 8.5 - 8.6 provide the minimum requirements that the
laboratory must meet in performing the analyses and the required data elements. Section 8.7
describes EPA's external review of laboratory operations and other quality measures. Section 8.8
identifies references used in developing the procedure.
8.1 Summary of the Procedure
This chapter describes toxicity testing of sediment samples collected for EPA's 2015 National
Coastal Condition Assessment (NCCA). As described in Section 8.5, unless otherwise
contractually bound by other requirements, the laboratory may choose to use any method that
meets EPA's specifications.
8.2 Health and Safety Warnings
The laboratory must require its staff to abide by appropriate health and safety precautions. In
addition to the laboratory's usual requirements such as a Chemical Hygiene Plan, the laboratory
must adhere to the following health and safety procedures:
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. K
lab coat, protective eyewear, gloves). g
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must avoid inhalation, skin contact, eye contact, or ingestion. Laboratory personnel must >^
avoid contacting skin and mucous membranes with acid. If skin contact occurs, remove ^
clothing immediately. Wash and rinse the affected skin areas thoroughly with large HH
amounts of water. Q
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8.3 Definitions and Required Resources (Personnel, Laboratories, and Equipment) ฃ
This section provides definitions and required resources for using the procedure. Q
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8.3.1 Definitions
The procedure uses the following terms:
Replicates are defined as two or more aliquots of the same sample which are analyzed
separately using identical procedures. The results are used to evaluate the precision of the
laboratory analyses.
NARS: National Aquatic Resource Surveys. The National Coastal Condition Assessment
(NCCA) is part of the NARS program.
NARS Information Management System (NARS IM): The EVI system established to
support all surveys, including NCCA, in the NARS program. The EVI system is used to track
the samples from field collection to the laboratory.
NCCA: National Coastal Condition Assessment. Freshwater and coastal samples will be
collected during the field stage of NCCA.
%CONT_SURV: Average percentage of organisms that survived in the replicate test
chambers over the percent survival in control.
%REP_SURV: Percentage of organisms that survived in the test chamber for each set of
replicates.
8.3.2 General Requirements for Laboratories
Expertise. To demonstrate its expertise, the laboratory shall provide EPA with performance data
demonstrating their proficiencies in analyzing water quality samples. In addition, the laboratory
must provide 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. ง
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Quality assurance and quality control requirements. pq
To demonstrate its expertise in quality assurance and quality control procedures, the organization H
shall provide EPA with copies of the quality-related documents relevant to the procedure. ^
Examples include Quality Management Plans (QMP), QAPPs, and applicable Standard o
Operating Procedures (SOPs). X
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To demonstrate its ongoing commitment, the person in charge of quality issues for the H
organization shall sign the NCCA QAPP Certification Page. Q
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Preparation for the work
To demonstrate its preparation for the work, the laboratory shall provide documentation that it
has complied with the following control analyses prior to the start of any work.
1. The laboratory shall ensure that the water source for the overlying water has been
demonstrated to support survival, growth, and reproduction of the test organisms. The
laboratory shall provide information on how the laboratory maintains the quality of the
water used for the tests.
2. The laboratory shall ensure that the clean sediment is appropriate for the control tests.
The laboratory shall provide information about the sediment chemistry analysis and
explanation of how the control sediment was selected
3. The laboratory shall ensure that the organisms are healthy for the tests. The laboratory
shall provide the source of the organisms; historic information about the culturing; and
procedures for evaluating the condition and age of the organism and water quality upon
arrival. If the laboratory intends to purchase the organisms (i.e., instead of in-house
culturing), identify the commercial source; its shipping arrangements (e.g., test organisms
are shipped in well-oxygenated water in insulated containers to maintain temperature
during shipment); and evaluation upon arrival at the laboratory (e.g., measuring
temperature and dissolved oxygen of the water in the shipping containers to determine if
the organisms might have been subjected to low dissolved oxygen or temperature
fluctuations).
4. The laboratory shall complete a "non-toxicant" test of each new chamber before using the
chamber for NCCA samples. A "new" chamber is one that the laboratory has not
previously used for any sediment toxicity testing for any client (e.g., replacement
glassware). Ideally, although EPA is not requiring it, the laboratory will test freshwater
and marine samples in wholly separate chambers.
Test requirements: The test chambers contain control sediment (sometimes called
the negative control) and clean overlying water for the amphipod species to be
tested. Survival of the test organisms will demonstrate whether facilities, water,
control sediment, and handling techniques are adequate to achieve acceptable
species-specific control survival. For the test to be acceptable, survival at 10 days
must equal or exceed the survival requirements in QA/QC specifications in
Section 8.7.
8.3.3 Personnel
O
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Laboratory Technician: This procedure may be used by any laboratory technician who H
is familiar with the NCCA Quality Assurance Project Plan, and this procedure in the ฃ
NCCA Laboratory Operations Manual. O
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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 H
QC contractor must be dedicated to QA/QC functions, and thus, must not be a primary g
laboratory or a field sampling contractor for NCCA. The QC contractor is responsible for ง
complying with instructions from the External QC Coordinator; coordinating and paying 9
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for shipments of the performance samples to participating laboratories; comparing results
from the laboratories; and preparing brief summary reports.
8.3.4 Equipment/Materials
The analytical method, selected by the laboratory, identifies the necessary equipment.
8.4 Sample Receipt
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. The laboratory must inspect the samples promptly on
receipt. As samples arrive, the laboratory must:
1. Log the samples into the National Aquatic Resource Survey Information Management
system (NARS-EVI) within 24 clock hours. Alternatively, for shipments with a large
number of samples, the laboratory may email a spreadsheet with the sample login and
sample condition information to NARS-IM (see Chapter 2 for contact information).
2. Check that each shipping container has arrived undamaged. Check the temperature of one
of the samples in the cooler using a thermometer that measures temperatures between 0
ฐC (refrigerated samples are typically 4 ฐC) and 30 ฐC (ambient room temperature is
typically less than 26 ฐC), or an infra-red (IR) temperature "gun" and record the reading.
Field crews and the batching laboratory will ship sediment samples on wet ice. Record
the condition and temperature of the sample in the database using the codes in Table 8.1.
3. Verify that all required data elements, per Table 8.1, have been recorded. If any elements
are missing, then enter them into the database.
4. Transfer the samples to the refrigerator until ready for toxicity testing. Except during
processing and analysis stages, the samples must be stored at 4ฐC.
5. Notify the EPA immediately about any problems involving sample integrity, conformity,
or inconsistencies as soon as possible following sample receipt and inspection.
Table 8.1 Sediment Toxicity Login: Required Data Elements
FIELD
LAB ID
TYPE
DATE RECEIVED
SITE ID
VISIT NUMBER
SAMPLE ID
DATE
COLLECTED
FORMAT
Character
Character
MMDDYY
Character
Numeric
Numeric
MMDDYY
DESCRIPTION
Name or abbreviation for laboratory
Control or NCCA Sample
Date sample was received by lab; leave blank for control
NCCA site id as used on sample label; leave blank for control
Sequential visits to site (1 (or blank) or 2); leave blank for control
Sample id as used on field sheet (on sample label); leave blank
for control
Date sample was collected; leave blank for control
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FIELD
ARRIVAL_TEMP
CONDITION CODE
COND COMMENT
FORMAT
Numeric
Character
Character
DESCRIPTION
Temperature of sample upon arrival at the laboratory (it should
arrive on wet ice).
Condition codes describing the condition of the sample upon
arrival at the laboratory; leave blank for control
Flag
OK
C
L
ML
NF
VT
VR
HT
Q
Definition
Sample is in good condition
Sample container is cracked
Sample or container is leaking
Sample label is missing
Sample is not at proper temperature
Volume not sufficient for testing (VT)
Volume not sufficient for a retest, if required
Received outside holding time
Other quality concerns, not identified above
Explanation for Q FLAG (if needed)
8.5 Toxicity Testing: Requirements
The laboratory shall perform toxicity testing of sediment samples. Laboratories may choose to
use any analysis method using the required organisms oiHyalella azteca (freshwater) or
Leptocheirusplumulosus (marine). The laboratory's method must meet the quality requirements
in Section 8.7, including mean survival of the control's treatments must remain greater than or
equal to 80% and 90%, respectively. It is essential that the contractor require that all of its
laboratory technicians use the same procedures and meet the required quality elements. At a
minimum, the laboratory must:
1. Perform the procedures using the 10-day tests. Possible methods include those described
in the following documents:
a. Marine: Test Method 100.4 in EPA 600/R-94/02522 or ASTM E1367-0323
b. Freshwater: Test Method 100.1 in EPA 600/R-99/06424 or ASTM E170625
2. Test the following number of replicates for each sample and control:
a. Marine: 5 replicates with 20 organisms per replicate
b. Freshwater: 4 replicates with 10 organisms per replicate
3. Test no more than 10 samples and one control within each batch.
22 Chapter 11 in Methods for Assessing the Toxicity of Sediment-associated Contaminants with Estuarine and
Marine Amphipods, June 1994, retrieved from
http://water.epa.gov/polwaste/sediments/cs/upload/marinemethod.pdf.
23 American Society for Testing and Materials (ASTM). 2008. E1367-03 "Standard Guide for Conducting 10-Day
Static Sediment Toxicity Tests With Marine and Estuarine Amphipods." Annual Book of Standards, Water and
Environmental Technology, Vol. 11.05, West Conshohocken, PA.
24 Section 11 in Methods for Measuring the Toxicity and Bioaccumulation of Sediment-associated Contaminants
with Freshwater Invertebrates, Second Edition, March 2000, retrieved from
http://water.epa.gov/polwaste/sediments/cs/upload/freshmanual.pdf.
25 ASTM 2009 E1706. "Standard Test Method for Measuring the Toxicity of Sediment-Associated Contaminants
with Freshwater Invertebrates."
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4. Use the following organisms for the tests:
a. Marine: Leptocheirusplumulosus
b. Freshwater: Hyalella azteca
5. Select organisms for each batch of tests that are:
a. From the same culture;
b. Cultured at the same temperature as will be used for the tests;
c. (optional) EPA would prefer but does not require that the organisms are cultured
in the same water as that used for testing.
6. Use a water source (for the overlying water) demonstrated to support survival, growth,
and reproduction of the test organisms.
a. For marine sediments, 175 mL of sediment and 800 mL of overlying seawater
b. For freshwater sediments, lOOmL of sediment and 175mL of overlying freshwater
7. Use clean sediment for control tests.
8. Implement the following for exposure/feeding
a. For marine sediments, exposure is static (i.e., water is not renewed), and the
animals are not fed over the 10 d exposure period
b. For freshwater, exposure is renewed (i.e., 2 volumes a day) and the animals are
fed over the 10 day exposure period
9. Follow the following procedure for homogenization/sieving: Water above the sediment
is not discarded, but is mixed back into the sediment during homogenization. Sediments
should be sieved for marine samples (following the 10 day method) and the sieve size
should be noted. For freshwater samples, they should not be sieved to remove indigenous
organisms unless there is a good reason to believe indigenous organisms may influence
the response of the test organism. For freshwater samples, large indigenous organisms
and large debris can be removed using forceps and if sediments must be sieved, the
samples should be analyzed before and after sieving (e.g., pore-water metals, DOC, and
AVS) to document the influence of sieving on sediment chemistry (note sieve size).
Additional details are provided in the summary tables 8.2 and 8.3.
Table 8.2 Test Conditions for Conducting 10-d Sediment Toxicity Tests for marine
sediments
Parameter
1 . Test type:
2. Temperature:
3. Salinity
4. Light quality:
5. Illuminance:
6. Photoperiod:
7. Test chamber:
8. Sediment volume:
Conditions
Whole sediment toxicity test, static
25ฐCforl_. plumulosus
20%o
Wide-spectrum fluorescent lights
500 -1000 lux
24LOD
1 L glass beaker or jar with ~10 cm I. D.
175 ml (2 cm)
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9. Overlying water volume:
10. Renewal of overlying
water:
1 1 . Size and life stage of
amphipods:
12. Number of organisms
per chamber:
13. Number of replicate
chambers/treatment:
14. Feeding:
15. Aeration:
16. Overlying water:
17. Overlying water quality:
18. Test duration:
19. Endpoints:
20. Test acceptability:
800ml
None
L. plumulosus: 2-4 mm (no mature males or females)
20 per test chamber
5 (required)
None
Water in each test chamber should be aerated overnight before start of
test and throughout the test aeration at rate that maintains >90%
saturation of dissolved oxygen concentration
Clean sea water, natural or reconstituted water
Temperature daily; pH, ammonia, salinity, and DO at test start and
end.
10 d
Survival
Minimum mean control survival of 90%
Table 8.3 Test Conditions for Conducting 10-d Sediment Toxicity Tests for freshwater
sediments
Parameter
1 . Test type:
2. Temperature:
3. Light quality:
4. Illuminance:
5. Photoperiod:
6. Test chamber:
7. Sediment volume
8. Overlying water volume:
9. Renewal of overlying water:
10. Age of organisms:
1 1 . Number of organisms/
chamber:
12. Replicate
chambers/treatment:
13. Feeding:
14. Aeration:
15. Test duration:
16. Endpoint:
17. Test acceptability:
Conditions
Whole-sediment toxicity test with renewal of overlying water
23ฐฑ1ฐC
Wide-spectrum fluorescent lights
100 to 1000 lux
16L8D
300 ml high-form beaker
100mL
175 ml
2 volume additions/d; continuous or intermittent (e.g., 1 volume
addition every 12 h)
7- to 14-d old at the start of the test (1- to 2-d range in age)
10
4 required
YCT food, fed 1 .0 ml daily (1 800 mg/L stock) to each test
chamber.
None unless DO in overlying water drops below 2.5 mg/L
10d
Survival
Min. mean control survival of 80%.
8.6 Data Entry
Tables 8.3 and 8.4 identify the required data elements describing the test conditions and outcomes
for the replicates and batches. Laboratories must provide the data elements to EPA, preferably in
EPA's data template, available separately from EPA.
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Table 8.3 Sediment Toxicity Replicates: Laboratory method performance
requirements
FIELD
LAB ID
TYPE
SAMPLE ID
RETEST
CHAMBER ID
BATCH ID
REPLICATE
TEST TYPE
ORGANISM
NO_SURVIVED
%REP_SURV
REP COMMENT
%CONT_SURV
FORMAT
Character
Character
Numeric
Yor blank
Character
Character
Numeric
Character
Character
Numeric
Numeric
Character
Numeric
DESCRIPTION
Name or abbreviation for laboratory
Control or NCCA Sample
Sample id as used on field sheet (on sample label); leave blank
for control
Y for yes if the sample is being retested; blank if original test or
control
Identification code for test chamber
Identification code for batch
Replicate number: 1-5 for marine; 1-4 for freshwater
Marine or Freshwater
Leptocheirus plumulosus (marine) or Hyalella azteca (freshwater)
Number of organisms that survived out of 20 (marine) and 1 0
(freshwater)
Percentage of organisms that survived in the test chamber for the
replicate
Any comments about the test procedures or any abnormalities
Optional Field: Average percentage of organisms that survived in
the replicate test chambers over the percent survival in control.
Table 8.4 Laboratory method performance requirements for sediment toxicity
batches
FIELD
BATCH ID
BATCH_SAMPLES
TEST TYPE
ORGANISM
CONTROL
START DATE
END DATE
%SURV
BATCH_PASS
QC CODE
QC DESCRIPTION
SURV COMMENT
FORMAT
Character
Numeric
Character
Character
Character
MMDDYY
MMDDYY
Numeric
P/F
Character
Character
Character
DESCRIPTION
Identification code for batch
Number of NCCA samples in the batch (integer<1 0) excluding the
control
Marine or Freshwater
Leptocheirus plumulosus (marine) or Hyalella azteca (freshwater)
Source of control sediment
Date that the laboratory starts the test procedure for the batch
Date that the laboratory ends the test procedure for the batch
%Survival for the sample (or control) calculated using the
%REP SURV
Indicate if the batch passed (P) or failed (F) the QA/QC
requirements (e.g., control achieved required survival rates)
Laboratory assigned code for QC issues with the sample
Description of conditions associated with the QC_CODE
Any comments about the test procedures or any abnormalities
8.7 Quality Measures
This section describes the quality assurance and quality control measures used to ensure that the
data will meet NCCA's requirements.
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8.7.1 Assistance Visits
Assistance visits are intended to familiarize EPA with actual procedures being implemented by
different laboratories; and to ensure a clear and consistent understanding of procedures and
activities by both EPA and the laboratories. If EPA decides to conduct an assistance visit, a
qualified EPA scientist or contractor will administer a checklist based upon the steps described in
this chapter.
8.7.2 QC Samples
Once or twice during the performance period, the External QC Coordinator will provide one or
two identical sets of QC samples to all participating laboratories. Each set will contain up to five
QC samples. As determined by the External QC Coordinator, the QC samples may be synthetic;
aliquots of additional samples collected at NCC A sites; or reference samples obtained from an
organization such as the National Institute of Standards. Each laboratory will run the QC samples
following the same procedures used for the other samples. The External QC Coordinator will
compare the results to the expected value and determine consistency between laboratories (e.g.,
determine if one laboratory is consistently higher or lower than all others). Based upon the
evaluation, the External QC Coordinator may request additional information from one or more
laboratories about any unique laboratory practices that might account for differences between the
laboratory and others. The contractor shall analyze the external QC samples using the same
procedures as those for the field samples.
8.7.3 Summary of QA/QC Requirements
QC protocols are an integral part of all analytical procedures to ensure that the results are reliable
and the analytical stage of the measurement system is maintained in a state of statistical control.
The laboratory must conduct QC analyses for each batch of samples. Each batch shall consist of
no more than 10 samples. Unique laboratory quality control lot numbers must be assigned to
each batch of samples. The lot number must associate each batch of field samples to the
appropriate measures such as laboratory control samples. Table 8.5 provides a summary of the
quality control requirements.
Table 8.5 Quality control activities for sediment toxicity samples
Activity
Laboratory demonstrates
competency for conducting
sediment toxicity analyses
Check condition of sample when
it arrives.
Evaluation
EPA will review SOPs, lab
certifications, past performance
results, etc. as part of the lab
verification process.
Sample issues, such as cracked
or leaking container; missing
label; temperature; adherence to
Corrective Action
EPA will not approve any
laboratory for NCCA sample
processing if the laboratory
cannot demonstrate
competency. In other words,
EPA will select another
laboratory that can demonstrate
competency for its NCCA
samples.
Assign appropriate condition
code identified in Table 8.1.
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Activity
Evaluation
Corrective Action
holding time requirements;
insufficient volume for test.
Sample storage
All samples: 4 ฐC at arrival at
the laboratory (temperature
recorded at arrival) and while
stored at the laboratory.
Record temperature upon
arrival at the laboratory.
Check temperature of the
refrigerator where samples
are stored at least daily if
using a continuous
temperature logger and twice
daily (beginning and end of
day) if the lab does not have a
continuous logger. If
refrigerator is warmer than
required, note temperature
and duration (either from the
continuous temperature log or
from the last manual reading)
in comment field. Lab will
still perform test. EPA
expects that the laboratory
will exercise every effort to
maintain samples at the
correct temperature.
Holding Time
The test must be completed
within 8 weeks after sample
collection. If the original test
fails, then the retest also must
be conducted within the 8 weeks
after sample collection.
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.
Check that the organisms are
healthy before starting the test
Unhealthy organisms may
appear to be discolored, or
otherwise stressed (for example,
greater than 20 percent mortality
for the 48 hours before the start
of a test).
Don't start test using unhealthy
organisms.
Maintain conditions as required
in Section 8.3.
Check conditions (e.g.,
temperature, DO) each test day.
Record conditions in bench
sheet or in laboratory database.
Note any deviations in
comments field (Table 8.1). In
extreme cases, conduct a new
toxicity test for all samples
affected by the adverse
conditions.
Control survival rates
For a test of a batch of samples
to be considered valid, the
control's mean survival in
Hyalella and Leptocheirus
treatments must remain >80%
and >90%, respectively.
Data template includes a field to
record if a test passed or failed
the control requirements. If a
test fails, retest all samples in
the batch. Report both the
original and retest results. If
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Activity
Evaluation
Corrective Action
both tests fail, submit data to
EPA for further consideration.
Include comments in the data
template noting any particular
factors that may have caused
the test to fail twice.
*Chapter2 provides contact information for the EPA HQ NCCA Laboratory Review Coordinator.
Laboratories under contract to EPA must contact the Task Order's Contracting Officer's Representative
(TOCOR) instead of the Laboratory Review Coordinator.
8.8 Sample and Record Retention
The laboratory shall retain:
1. The sample materials, including vials until March 31, 2016 which will allow EPA with
time to review the data and contact the laboratory with any questions about the samples.
Until this time, the laboratory shall refrigerate the sediment samples. The laboratory shall
periodically check the sample materials for degradation.
2. Original records, including laboratory notebooks, 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.
8.9 References
American Society for Testing and Materials (ASTM). 2008. E1367-03 "Standard Guide for
Conducting 10-Day Static Sediment Toxicity Tests With Marine and Estuarine Amphipods."
Annual Book of Standards, Water and Environmental Technology, Vol. 11.05, West
Conshohocken, PA.
ASTM. 2009. E1706. "Standard Test Method for Measuring the Toxicity of Sediment-
Associated Contaminants with Freshwater Invertebrates.
United Stated Environmental Protection Agency (USEPA). 1994. Chapter 11 in Methods for
Assessing the Toxicity of Sediment-associated Contaminants with Estuarine and Marine
Amphipods, retrieved on March 13, 2014 from
http://water.epa.gov/polwaste/sediments/cs/upload/marinemethod.pdf.
USEPA. 2000. Section 11 in Methods for Measuring the Toxicity and Bioaccumulation of
Sediment-associated Contaminants with Freshwater Invertebrates, Second Edition, retrieved on
March 13, 2014 from http://water.epa.gov/polwaste/sediments/cs/upload/freshmanual.pdf.
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9.0 FISH TISSUE FILLET (GREAT LAKES)
Laboratory Methods incorporated in EPA OST Manuals/QAPP.
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10.0 MERCURY IN FISH TISSUE PLUGS
10.1 Summary of the Procedure
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 C of this LOM.
10.2 General Requirements for Laboratories
Competency. To demonstrate its competency, the laboratory shall provide EPA with
performance data demonstrating their proficiencies in analyzing water quality samples. In
addition, the laboratory must provide 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.
Also, the lab must provide a demonstration of past experience with fish tissue samples in
applying the laboratory SOP in achieving the method detection limit.
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 NCCA QAPP Certification Page.
10.2.1 Personnel
Laboratory Technician: This procedure may be used by any laboratory technician who
is familiar with the NCCA Quality Assurance Project Plan, and this procedure in the M
NCCA Laboratory Operations Manual. =s
10.2.2 Equipment/Materials H
The analytical method, selected by the laboratory, identifies the necessary equipment. E
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Because EPA initiates tracking procedures designed to recover any missing shipment, the
laboratory personnel responsible for tracking samples must start the following login steps within
24 clock hours of receiving a delivery.
1. Report receipt of samples in the NARS EVI sample tracking system (within 24 clock hours).
Alternatively, for shipments with a large number of samples, the laboratory may email a
spreadsheet with the sample login and sample condition information to NARS-EVI (see
Chapter 2 for contact information).
2. Inspect each sample THE SAME DAY THEY ARE RECEIVED:
a. Verify that the sample IDs in the shipment match those recorded on the:
i. Chain of custody forms when the batching laboratory sends the samples to the
microcystins laboratory; or
ii. Sample tracking form if the field crew sends the shipment directly to the State
laboratory.
b. Record the information in Table 9.1 into NARS IM, including the Condition Code for
each sample:
i. OK: Sample is in good condition
C: Sample container was cracked
L: Sample container is leaking
ML: Sample label is missing
VT: Volume not sufficient for testing
W: Sample is warm (>8ฐ), record the temperature in the comment field, and
perform the assay
Q: other quality concerns, not identified above.
c. If any sample is damaged or missing, contact the EPA HQ Laboratory Review
Coordinator to discuss whether the sample can be analyzed. (See contact information
in Chapter 2 of the Manual).
11.
iii.
iv.
v.
vi.
vn
3. Store samples in the freezer until sample preparation begins.
4. Maintain the chain of custody or sample tracking forms with the samples.
Table 9.1 Fish Tissue Plugs Login: Required Data Elements
FIELD
LAB ID
DATE RECEIVED
SITE ID
VISIT NUMBER
SAMPLE ID
DATE COLLECTED
CONDITION CODE
FORMAT
text
MMDDYY
text
numeric
numeric
MMDDYY
text
DESCRIPTION
Name or abbreviation for QC laboratory
Date sample was received by lab
NCCA site id as used on sample label
Sequential visits to site (1 or 2)
Sample id as used on field sheet (on sample
label)
Date sample was collected
Condition codes describing the condition of the
sample upon arrival at the laboratory.
Flag | Definition
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FIELD
CONDITION
COMMENT
FORMAT
text
DESCRIPTION
OK
C
L
ML
VT
W
Q
Sample is in good condition
Sample container is cracked
Sample or container is leaking
Sample label is missing
Volume or mass not sufficient for
testing (VT)
Sample is warm (>8ฐ)
Other quality concerns, not identified
above
Comments about the condition of the sample. If
the condition code=W then provide the
temperature
10.4 Quality Measures
This section describes the quality assurance and quality control measures used to ensure that the
data will meet NCCA's requirements. Tables 9.2 and 9.3 provide a summary of the
measurement data quality objectives and quality control requirements.
10.4.1 Assistance Visits
Assistance visits are intended to familiarize EPA with actual procedures being implemented by
different laboratories; and to ensure a clear and consistent understanding of procedures and
activities by both EPA and the laboratories. If EPA decides to conduct an assistance visit, a
qualified EPA scientist or contractor will administer a checklist based upon the steps described in
this chapter.
10.4.2 QC Samples
Once or twice during the performance period, the External QC Coordinator will provide one or
two identical sets of QC samples to all participating laboratories. Each laboratory will run the
QC samples following the same procedures used for the other samples. The External QC
Coordinator will compare the results to the expected value to determine whether the values are
within expected ranges. The contractor shall analyze the external QC samples using the same
procedures as those for the field samples.
Table 9.2 Measurement data quality objectives
Variable or Measurement
Mercury
MDL
0.47 ng/g
Quantitation
Limit
5.0 ng/g
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Table 9.3 Quality Control
Activity
Demonstrate competency
for analyzing fish samples
to meet the performance
measures
Check condition of sample
when it arrives.
Store sample
appropriately. While
stored at the laboratory,
the sample must be kept
at a maximum
temperature of-20ฐC.
Analyze sample within
holding time
Maintain quality control
specifications from
selected method/SOP
(that meets the
measurement data quality
objectives)
Maintain the required MDL
Use consistent units for
QC samples and field
samples
Maintain completeness
Evaluation/Acceptance
Criteria
Demonstration of past experience
with fish tissue samples in
applying the laboratory SOP in
achieving the method detection
limit
Sample issues, such as
punctures or rips in wrapping;
missing label; temperature;
adherence to holding time
requirements; sufficient volume
for test. All samples should arrive
at the laboratory frozen.
Check the temperature of the
freezer per laboratory's standard
operating procedures.
The test must be completed
within the holding time (i.e., 1
year). If the original test fails,
then the retest also must be
conducted within the holding
time.
Data meet all QC specifications
in the selected method/SOP.
Evaluate for each sample
Verify that all units are provided
in wet weight units and
consistently
Completeness objective is 95%
for all parameters.
Corrective Action
EPA will not approve any
laboratory for NCCA sample
processing if the laboratory
cannot demonstrate competency.
In other words, EPA will select
another laboratory that can
demonstrate competency for its
NCCA samples.
Assign an appropriate condition
code.
Record temperature of sample
upon arrival at the laboratory. If
at any other time, samples are
warmer than required, note
temperature and duration in
comment field.
Perform test, but note reason for
performing test outside holding
time. EPA expects that the
laboratory will exercise every
effort to perform tests before the
holding time expires.
If data do not meet all QC
requirements, rerun sample or
qualify data. If the lab believes
the data are to be qualified
without rerunning sample, the lab
must consult with the EPA
Survey QA Lead before
proceeding.
If MDL could not be achieved,
then provide dilution factor or QC
code and explanation in the
comment field.
If it is not possible to provide the
results in the same units as most
other analyses, then assign a QC
code and describe the reason for
different units in the comments
field of the database.
Contact the EPA Survey QA
Lead immediately if issues affect
laboratory's ability to meet
completeness objective.
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11.0 FECAL INDICATOR: ENTEROCOCCI
Laboratory methods incorporated into EPA ORD Manuals/QAPP.
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APPENDIX A: LABORATORY REMOTE EVALUATION FORMS
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NCCA 2015 Document Request Form - Chemistry Laboratories
EPA and its state and tribal partners will conduct the 2015 National Coastal Condition
Assessment. NCCA is a survey of the nation's coastal waters and Great Lakes. It is designed to
provide statistically valid regional and national estimates of the condition of coastal waters and
the Great Lakes. Consistent sampling and analytical procedures ensure that the results can be
compared across the country.
As part of the 2015 NCCA, 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 2015
NCCA.
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 laboratories will be required to complete the following forms and check the specific
parameter in which your laboratory will be conducting an analysis for the 2015 NCCA:
D Water Chemistry and chlorophyll a (all of the analytes identified in the LOM and QAPP)
D Microcystin
D Mercury in Fish Tissue Plugs
D Sediment Chemistry ^
D Grain Size g
D Total Organic Carbon (TOC) ฃ
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indicator: ;>
D A signature on the attached Laboratory Signature Form indicates that your laboratory ^
will follow the quality assurance protocols required for chemistry labs conducting H
analyses for the 2015 NCCA. S
D A signature on the Quality Assurance Project Plan (QAPP) and the Laboratory 2
Operations Manual (LOM) Signature Form indicates that you will follow both the QAPP 5
and the LOM. g
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If you have not been approved within the last 5 years through the laboratory verification Q
process for the water chemistry indicator, in order for us to determine your ability to CQ
participate as a laboratory in the NCCA, we are requesting that you submit the following _i
documents (if available) for review:
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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):
D A copy of your laboratory's accreditations and certifications if applicable (i.e. NELAC,
ISO, state certifications, NABS, etc.).
D An updated copy of your laboratory's QAPP and Laboratory Quality Assurance Manuals
D Standard Operating Procedures (SOPs) for your laboratory for each analysis to be
performed (if not covered in 2015 NCCA LOM).
D Documentation attesting to experience running all analytes for the 2015 NCCA, including
chlorophyll a.
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Laboratory Signature Form - Chemistry Laboratories
I _ certify that the laboratory,
located in _ , will abide by the following
standards in performing the following data analysis and reporting for the 2015
National Coastal Condition Assessment (NCCA).
This applies to the _ chemistry indicator.
1.) Use procedures identified in the 2015 NCCA Laboratory Operations
Manual (or equivalent). If using equivalent procedures, please provide the
procedures and obtain approval from EPA.
2.) Read and abide by the 2015 NCCA Quality Assurance Project Plan
(QAPP) and related Standard Operating Procedures (SOPs).
3.) Have an organized IT tracking system in place for recording sample
tracking and analysis data.
4.) Provide Quality Control (QC) data for internal QC check, on a quarterly
basis.
5.) Provide data using the template provided on the NARS Sharefile.
6.) Provide data results in a timely manner. This will vary with the type of
analysis and the number of samples to be processed. Sample data must be
received no later than May 1, 2016 or as otherwise negotiated with EPA.
7.) Participate in a laboratory technical assessment or audit if requested by
EPA NCCA 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, for
water chemistry. <
Signature _ Date
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NCCA 2015 Document Request Form - Biology Laboratories
EPA and its state and tribal partners will conduct the 2015 National Coastal Condition
Assessment. NCCA is a survey of the nation's coastal waters and Great Lakes. It is designed to
provide statistically valid regional and national estimates of the condition of coastal waters and
the Great Lakes. Consistent sampling and analytical procedures ensure that the results can be
compared across the country.
As part of the 2015 NCCCA, 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 2015
NCCA.
The first step of this assessment process will involve the review of your laboratory's certification
and/or documentation. Subsequent actions may include (if needed): reconciliation exercises
and/or a site visit.
All laboratories will be required to complete the following forms and check the specific
parameter in which your laboratory will be conducting an analysis for the 2015 NCCA:
D Mercury in Fish Plugs
D Benthic Macroinvertabrates
D Sediment Toxicity
GO
If your laboratory has been previously approved within the last 5 years for the specific ง
parameters: O
D A signature on the attached Laboratory Signature Form indicates that your laboratory ฃ
will follow the quality assurance protocols required for biology laboratories conducting O
analyses for the 2015 NCCA. 5
D A signature on the Quality Assurance Project Plan (QAPP) and the Laboratory R
Operations Manual (LOM) Signature Form indicates you will follow both the QAPP and ^
the LOM. g
H
If you have not been approved within the last 5 years through the laboratory verification 9
process for the specific parameters, in order for us to determine your ability to participate w
as a lab in the NCCA, we are requesting that you submit the following documents (if ?
available) for review: p4
D Documentation of a successful quality assurance audit from a prior National Aquatic ^
Resource Survey (NARS) that occurred within the last 5 years. ^
D Documentation showing participation in previous NARS for this particular indicator. 2
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D Documentation of NABS (or other) certification for the taxonomists performing analyses
(if applicable).
D An updated copy of your Laboratory's QAPP and Laboratory Quality Assurance
Manuals.
D Standard Operating Procedures (SOPs) for your lab for each analysis to be performed (if
not covered in 2015 NCCA LOM).
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Laboratory Signature Form - Biology Laboratories
I _ certify that the laboratory
located in _ , will abide by the following standards in
performing biology data analysis and reporting for the 2015 National Coastal Condition
Assessment (NCAA).
This applies to the _ biological indicator.
Use procedures identified in the 2015 NCCA Lab Operations Manual (or equivalent). If using
equivalent procedures, please provide the procedures and obtain approval from EPA.
Read and abide by the 2015 NCCA Quality Assurance Project Plan (QAPP) and related Standard
Operating Procedures (SOPs).
Have an organized IT tracking system in place for recording sample tracking and analysis data.
Use taxonomic standards outlined in the 2015 NCCA Laboratory Operations Manual.
Participate in taxonomic reconciliation exercises during the field and data analysis season, which
include conference calls and other laboratory reviews.
Provide Quality Control (QC) data for internal QC checks, including for sorting, on a monthly
basis.
Provide data using the template provided on the NARS Sharefile.
Provide data results in a timely manner. This will vary with the type of analysis and the number
of samples to be processed. Sample data must be received no later than May 1, 2016 or as
otherwise negotiated with EPA. Samples results for independent taxonomic QC described in the 5
LOM and QAPP must be provided to EPA prior to final datasets to allow for reconciliation to w
take place. PH
Participate in a Laboratory technical assessment or audit if requested by EPA NCCA staff (this %
may be a conference call or on-site audit). P
Agree to utilize taxonomic nomenclature and hierarchical established for NCCA 2015. ^
Signature _ Date H
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APPENDIX B: TARGET FISH SPECIES FOR WHOLE FISH ANALYSES
Table B.1 Northeast region primary and secondary marine target species - whole
body fish tissue collection (Ecofish)
FAMILY NฐRT
Ictaluridae
Moronidae
Paralichthyidae
Pleuronectidae
Sciaenidae
Sparidae
FAMILY
HEAST REGION PRIMARY I
SCIENTIFIC NAME
Ameiurus catus
Ictalurus punctatus
Morone americana
Paralichthys dentatus
Pseudopleuronectes americanus
Cynoscion regalis
Sciaenops ocellatus
Stenotomus chrysops
;COFISH TARGET SPECIES
COMMON NAME
White catfish
Channel catfish
White perch
Summer flounder
Winter flounder
Gray weakfish
Red drum
Scup
EAST REGION SECONDARY ECOFISH TARGET SPEC
SCIENTIFIC NAME COMMON NAME
Achiridae Trinectes maculatus \ Hogchoaker
Anguillidae
Atherinopsidae
Batrachoididae
Ephippidae
Moronidae
Mugulidae
Pomatomidae
Sciaenidae
Serranidae
Triakidae
Triglidae
Anguilla rostrata
Menidia menidia
Opsanus tau
Chaetodipterus faber
Morone saxatilis
Mugil cephalus
Pomatomus saltatrix
Bairdiella chrysoura
Menticirrhus saxatilis
Centropristis striata
Mustelus canis
Prionotus carolinus
Prionotus evolans
American eel
Atlantic silverside
Oyster toadfish
Atlantic spadefish
Rock fish
Black mullet
Bluefish
Silver perch
Northern kingfish
Black sea bass
Smooth dogfish
Northern searobin
Striped searobin
FISH PLUG LIST*
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
IES
FISH PLUG LIST*
Secondary
Secondary
Secondary
GO
w
GO
GO
I I
PH
W
H-l
o
* Indicates whether species also occurs in the primary or secondary fish plug list
Table B.2 Southeast region primary and secondary marine target species - whole
body fish tissue collection (Ecofish)
.
Paralichthyidae
Sparidae
BJBBI^B
Cichlidae
Haemulidae
UTHEAST REGION PRIMARY E
SCIENTIFIC NAME
Ariopsis felis
Bagre marinus
Paralichthys albigutta
Paralichthys dentatus
Paralichthys lethostigma
Cynoscion arenarius
Cynoscion nebulosus
Cynoscion regalis
Leiostomus xanthurus
Lagodon rhomboides
THEAST REGION SECONDARY
SCIENTIFIC NAME
Tilapia mariae
Haemulon aurolineatum
Bairdiella chrysoura
Menticirrhus americanus
COFISH TARGET SPECIES
COMMON NAME
Hardhead sea catfish
Gafftopsail sea catfish
Gulf flounder
Summer flounder
Southern flounder
Sand weakfish (or seatrout)
Speckled trout
Gray weakfish
Spot croaker
Pinfish
ECOFISH TARGET SPECIES
COMMON NAME
Spotted tilapia
Tomtate
Silver perch
Southern kingfish
FISH PLUG LIST*
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
FISH PLUG LIST*
O
PH
GO
s
o
w
CH
GO
K
GO
I I
PH
H
s
PQ
X
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National Coastal Condition Assessment 2015
Version 2.0, July 2015
Laboratory Operations Manual
Page 135 of 178
Serranidae
Centropristis striata
Black sea bass
* Indicates whether species also occurs in the primary or secondary fish plug list
Table B.3 Gulf region primary and secondary marine target species - whole body
fish tissue collection (Ecofish)
axmvm
Paralichthyidae
Sparidae
i
Diodontidae
Gerreidae
Haemulidae
Ictaluridae
Lepisosteidae
Lutjanidae
Sciaenidae
Serranidae
Triglidae
GULF REGION PRIMARY ECOF
SCIENTIFIC NAME
Ariopsis felis
Eagre marinus
Paralichthys albigutta
Paralichthys dentatus
Paralichthys lethostigma
Cynoscion arenarius
Cynoscion nebulosus
Cynoscion regalis
Leiostomus xanthurus
Micropogonias undulatus
Sciaenops ocellatus
Lagodon rhomboides
3ULF REGION SECONDARY EG
SCIENTIFIC NAME
Caranx hippos
Chloroscombrus chrysurus
Chilomycterus schoepfii
Eucinostomus gula
Orthopristis chrysoptera
Ictalurus furcatus
Lepisosteus oculatus
Lutjanus griseus
Pogonias cromis
Diplectrum formosum
Prionotus scitulus
rISH TARGET SPECIES
COMMON NAME
Hardhead sea catfish
Gafftopsail sea catfish
Gulf flounder
Summer flounder
Southern flounder
Sand weakfish (or seatrout)
Speckled trout
Gray weakfish
Spot croaker
Atlantic croaker
Red drum
Pinfish
DFISH TARGET SPECIES
COMMON NAME
Crevallejack
Atlantic bumper
Burrfish
Silver jenny
Pigfish
Blue catfish
Spotted gar
Gray snapper
Black drum
Sand perch
Leopard searobin
FISH PLUG LIST*
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
KaHSi d Mici IKJ^B
GO
w
GO
GO
I I
PH
W
H-l
o
* Indicates whether species also occurs in the primary or secondary fish plug list
Table B.4 Western region primary and secondary marine target species - whole
body fish tissue collection (Ecofish)
Atherinopsidae
Cottidae
Cynoglossidae
Embiotocidae
Gasterosteidae
Paralichthyidae
Pleuronectidae
Sciaenidae
Serranidae
/ESTERN REGION PRIMARY ECOFISH TARGET SPECIES
SCIENTIFIC NAME
Atherinops affinis
Leptocottus armatus
Oligocottus rimensis
Symphurus atricaudus
Cymatogaster aggregata
Embiotoca lateralis
Gasterosteus aculeatus
Paralichthys californicus
Citharichthys sordidus
Citharichthys stigmaeus
Isopsetta isolepis
Parophrys vetulus
Psettichthys melanostictus
Platichthys stellatus
Genyonemus lineatus
Paralabrax nebulifer
COMMON NAME FISH PLUG LIST*
Lopsmelt silverside
Pacific staghorn sculpin
Saddleback sculpin
California tonguefish
Shiner perch
Striped seaperch
Lhree-spined stickleback
California flounder
Pacific sanddab
Speckled sanddab
Butter sole
English sole
Pacific sand sole
Starry flounder
White croaker
Barred sand bass
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
O
PH
GO
s
o
w
CH
GO
K
GO
I I
PH
H
s
PQ
X
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National Coastal Condition Assessment 2015
Version 2.0, July 2015
Laboratory Operations Manual
Page 136 of 178
Paralabrax maculatofasciatus
Spotted sand bass
,ION SECONDARY ECO. .
TICIC NAME COMMON NAME FISH PLUG LIST"
Echinodermata/
Toxopneustidae
Batrachoididae
Chimaeridae
Embiotocidae
Paralichthyidae
Pleuronectidae
Sciaenidae
Tripneustes gratilla
(Hawaii ONLY;
Porichthys notatus
Porichthys myriaster
Hydrolagus colliei
Amphistichus argenteus
Xystreurys liolepis
Pleuronichthys guttulatus
Microstomus pacificus
Lepidopsetta bilineata
Lyopsetta exilis
Umbrina roncador
Collector urchin
Plainfin midshipman
Specklefin midshipman
Spotted ratfish
Barred surfperch
Fantail sole
Diamond turbot
Dover sole
Rock sole
Slender sole
Yellowfin croaker
Secondary
Secondary
Secondary
* Indicates whether species also occurs in the primary or secondary fish plug list.
Table B.5 Great Lakes primary and secondary target species - whole body fish
tissue collection (Ecofish)
Catostomidae
Cyprinidae
Gasterosteidae
Ictaluridae
Gadidae
Osmeridae
Percidae
Percopsidae
Salmonidae
Sciaenidae
GREAT LAKES PRIMARY ECOF
Moxostoma macrolepidotum
Ambloplites rupestris
Lepomis gibbosus
Lepomis macrochirus
Micropterus dolomieu
Pomoxis annularis
Pomoxis nigromaculatus
Cottus bairdii
Cottus cognatus
Couesius plumbeus
Cyprinus carpio
Pimephales notatus
Esox lucius
Esox masquinongy
Gasterosteus aculeatus
Neogobius melanostomus
Proterorhinus marmoratus
Ameiurus nebulosus
Ictalurus punctatus
Noturus flavus
Lota lota
Morone americana
Morone chrysops
Osmerus mordax
Gymnocephalus cernuus
Perca flavescens
Percina caprodes
Sander canadensis
Sander vitreus
Percopsis omiscomaycus
Coregonus artedi
Coregonus clupeaformis
Oncorhynchus gorbuscha
Oncorhynchus kisutch
Oncorhynchus mykiss
Oncorhynchus tshawytscha
Salvelinus namaycush
Aplodinotus grunniens
rISH TARGET SPECIES
^^ซMiWi W g i g FJ" 1 ^^B
Shorthead redhorse
Rock bass
Pumpkinseed
Bluegill
Smalhnouth bass
White crappie
Black crappie
Mottled sculpin
Slimy sculpin
Lake chub
Common carp
Bluntnose minnow
Northern pike
Muskellunge
Lhree-spined stickleback
Round goby
Lubenose goby
Brown bullhead
Channel catfish
Stonecat
Burbot
White perch
White bass
American/ rainbow smelt
Ruffe
Yellow perch
Logperch
Sauger
Walleye
Lrout-perch
Cisco/ lake herring
Lake whitefish
Pink salmon
Coho salmon
Rainbow trout
Chinook salmon
Lake trout
Freshwater drum
^BaHSi a Mici iH^^B
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
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National Coastal Condition Assessment 2015
Version 2.0, July 2015
Laboratory Operations Manual
Page 137 of 178
GREAT LAKES SECONDARY ECOFISH TARGET SPECIES
Catostomidae
Centrarchidae
Clupeidae
Cyprinidae
Esocidae
Fundulidae
Ictaluridae
Salmonidae
SCIENTIFIC NAME
Catostomus catostomus
Catostomus commersonii
Moxostoma anisurum
Micropterus salmoides
Alosa pseudoharengus
Dorosoma cepedianum
Cvprinella spiloptera
Luxilus cornutus
Notropis stramineus
Esox niger
Fundulus diaphanus
Fundulus majalis
Ameiurus melas
Prosopium cylindraceum
Salmo trutta
Salvelinus fontinalis
Salvelinus fontinalis x namaycush
COMMON NAME
Longnose sucker
White sucker
Silver redhorse
Largemouth bass
Alewife
American gizzard shad
Spotfin shiner
Common shiner
Sand shiner
Chain pickerel
Banded killifish
Striped killifish
Black bullhead
Round whitefish
Brown trout
Brook trout
Splake
Secondary
Secondary
* Indicates whether species also occurs in the primary or secondary fish plug list
GO
W
GO
GO
I I
PH
W
H-l
o
O
PH
GO
s
o
w
CH
GO
K
GO
I I
PH
H
s
PQ
X
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National Coastal Condition Assessment 2015 Laboratory Operations Manual
Version 2.0, July 2015 Page 138 of 178
APPENDIX C: EXAMPLE SOPS FOR MERCURY IN FISH TISSUE PLUG
ANALYSES
GO
w
GO
o
&
h-i
fin
GO
GO
GO
I I
PH
o
PH
M
CH
O
GO
w
h-1
CH
X
w
o
X
-------
4Seurofins
Frontier Global Sciences
Document Title:
Mercury in Water by Oxidation, Purge & Trap
__a_nd_CV-AFS (EPA Method 1631, RevE)
Eurofins Document Reference:
EFGS-SOP-137-R02
Eii irofi rts
Refererifee
EFGS-SOP-137-R02
Revision
6/17/2013
Status
Final
FGS-SOP-137.02
Level 3
SOP
L.O c 81 ,;Di6c Umi nl/jG
NA
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"' ...'!' :'' ':} ''.:. -.- ..-:,.,:",-.' ,-v .':'..
:aH4::f;:?;;o'{,:f:^;::;/VK:^Eiii;
:l^P^||;|S:;f|l:ff
Ryan Nelson
Dave Wundertich and Patrick Garcia-Strickland
-------
C-yeuroflns j
Frontier Etobaf Sciences
Document Title:
Mercury in Water by Oxidation, Purge & Trap
__and CV-AFS (EPA Method 1631, Rev E)
Eurofins Document Reference:
EFGS-SOP-137-R02
Table of Contents
1 Revision Log; , 4
2 Reference: , , . 4
3 Cross Reference: ,.,, , 5
4 Purpose: ,, , - 5
5 Scope: , . , . 5
6 Basic Principles: , , 5
7 Reference Modifications: Q
8 Definitions: , 0
9 Interferences: g
10 Safety Precautions, Pollution Prevention and Waste Handling:.. , g
11 Personnel Training and Qualifications:, 10
12 Sample Collection, Preservation, and Handling: , .10
13 Apparatus and Equipment: -)-!
14 Reagents and Standards: ., 1-j
15 Calibration: , , ..__ U
16 Procedure: ,..,,.. -jg
17 Calculations:... , _ 21
18 Statistical Information/Method Performance: ,.,,. 22
19 Quality Assurance/Quality Control: , 22
20 Corrective Action , 24
21 List of Attachments , ,.,.,_ 25
Table 1; QC Requirements for Total Mercury. , ,, 26
Appendix A: Example - Standard Operating Procedure Training Record ....27
-------
K^eurofins [
I Frontier Global Sciences
Document Title:
Mercury in Water by Oxidation, Purge & Trap
and CV-AFS (EPA Method 1631, Rev E)
Eurofins Document Reference:
EFGS-SOP-137-R02
Approvals:
Prepared by:
Date:
/I 3
Approved by:
B
~~**u<~~ P
L A^-i a /JL.J..JJ
Date:
Approved by:
Date:
-------
%'fieurofins (
Frontier Gioba' Sciences
Document Title:
Mercury in Water by Oxidation, Purge & Trap
and CV-AFS (EPA Method 1631, Rev E)
Eurofins Document Reference:
EFGS-SOP-137-R02
1 Revision Log:
. ^^flpfesfei^y^g
Cover
AH
13.1, 13.2
14.8
14.9
15.2-15.4
16.7
>?il ^ififiitiifep? ^ .::::y;^mm;-+ >;./,>;
Required change
Formatting requirement per LOM
SOP-LAB-201
Required
Required
Required
Required
Required
?;Oh(anifcS;2^;^^^^^^
Changed company name from Frontier Global Sciences to
Eurofins Frontier Global Sciences
Reformatted document to new corporate specifications.
Added hardware and software components
Updated mercury standard prep
Updated standard and reaqent documentation procedures
Updated calibration information
Added instrument maintenance and troubleshooting
2 Reference:
EPA Method 1631, Revision E: Mercury in Water by Oxidation, Purge and Trap, and
Cold Vapor Atomic Fluorescence Spectrometry, 2002.
Method 1669, "Method for Sampling Ambient Water for Determination of Metals at
EPA Ambient Criteria Levels," U.S. Environmental Protection Agency, Office of Water,
Office of Science and Technology, Engineering and Analysis Division (4303), 401 M
Street SW, Washington, DC 20460, April 1995 with January 1996 revisions.
Bloom, N.S.; and Tsalkitzis, E. Standard Operating Procedure FGS-012 Determination
of Total Mercury in Aqueous Media (Modified EPA Method 1631). Frontier
GeoSciences inc., Quality Assurance Manual 1995.
Bloom, N.S.; Ultra-Clean Sample Handling, Environmental Lab 1995, March/April, 20.
Bloom, N.S.; Horvat M., and Watras CJ. Results of the Internationai Mercury
Speciation Intercomparison Exercise. Wat Air Soil Pollut. 1995, 80, 1257.
Bloom, N.S.; Crecelius, E.A. Determination of Mercury in Seawater at Sub-nanogram
per Liter Levels.Mar.Chem.1983, 14, 49.
Bloom, N.S.; Crecelius, E.A. Distribution of Silver, Lead, Mercury, Copper, and
Cadmium in Central Puget Sound Sediments Mar. Chem 1987, 21, 377-390.
Bloom, N.S.; Fitzgerald, W.F. Determination of Volatile Mercury Species at the
Picogram Level by Low-Temperature Gas Chromatography with Cold-Vapor Atomic
Fluorescence Detection. Anal. Chem. Acta. 1988, 208,151,
Cossa, D.; Couran, P. An International Intercomparison Exercise for Total Mercury in
Seawater. App.Organomet. Chem. 1990, 4, 49,
Fitzgerald, W.F.; Gill, G.A. Sub-Nanogram Determination of Mercury by Two-Stage
Gold Amalgamation and Gas Phase Detection Applied to Atmospheric Analysis Anal
Chem. 1979, 15, 1714.
Gill, G.A.; Fitzgerald, W.F. Mercury Sampling of Open Ocean Waters at the Picogram
Level Deep Sea Res. 1985, 32, 287.
2.12 EPA Method 30.B, Determination of total vapor phase mercury emissions from coal-
fired combustion sources using carbon sorbent traps.
2.1
2.2
2.3
2.4
2.5
2.6
2.7
2.8
2.9
2.10
2.11
-------
4>*eurofins
frontier Global Sciences
Document Title:
Mercury in Water by Oxidation, Purge & Trap
and CV-AFS (EPA Method 1631, Rev E)
Eurofins Document Reference:
EFGS-SOP-137-R02
2,13 Chemical Hygiene Plan, Eurofins Frontier Global Sciences, current version.
2.14 National Environmental Laboratory Accreditation Conference, NELAC Standard
September 8, 2009.
2.15 Department of Defense Quality Systems Manual for Environmental Laboratories,
prepared by DoD Environmental Quality Workgroup, Final Version 4.2, October 2010. '
3 Cross Reference:
Document
Document Title
SOP FGS-003
Pipette Verification, Calibration and Maintenance
SOP FGS-007
Cleaning of Sampling jEquipment and Bottles
SOP FGS-008
Ultra Clean AqueousjSampie Collection
SOP FGS-012
Oxidation of Aqueous Samples for Total Mercury Analysis
SOP FGS-061
Gold Trap Construction
I nil -l-l-l-lil-lll-l-ll-l-l-l-l-lll ^^^===^^
Standard Operating Procedure Training Record
SOP FGS-094, App F
Waste Disposal Procedure for Client Sample Waste
SOP FGS-099
SOP FGS-121
Determination of Total Mercury by Flow Injection AFS (Mod 1631EJ
SOPFGS-155
Calibration of Voiumetric Dispensers
4 Purpose;
4,1
This SOP is designed to ensure that alt reproducible traceable procedures in EPA
1631 are followed in the standardization of the total mercury analyzers and in the
analysis of samples for total mercury, as well as to establish the limits wherein data will
be considered acceptable.
5 Scope:
5.'
5.2
This Standard Operating Procedure (SOP) describes a method for the determination of
total mercury (Hg) in filtered and unfiltered water by oxidation, purge and trap,
desorption, and cold vapor atomic fluorescence spectrometry (CVAFS).
This method is designed for the determination of mercury in the range of 0.5-40 ng/L
(ppt). Application may be extended to higher levels by selection of a smaller sample
size, as long as the instrument value (intensity) remains within the calibration curve.
5.3 The Control Limits are established from EPA 1631 E.
6 Basic Principles:
6.1
6.2
6.3
For analysis of aqueous samples, an aliquot of oxidized sample is neutralized with
hydroxylamine-hydrochloride {NH2OH-HCI) to destroy free halogens, and added to a
bubbler,
Stannous chloride (SnCi2) is added to the bubbler to reduce the Hg(ll) to volatile Hg(0),
and the bubblers are sealed with Keck clips. Blanked gold traps are placed at the end
of soda-lime pre-traps. The bubbler is purged with nitrogen (N2) for 20 minutes. All gas
that flows into the bubbler should only leave the system through the soda-iime pre-trap
and then the gold trap,
The gaseous mercury amalgamates to the gold traps, which are removed and
individually placed in the analytical train. The gold trap is heated, thus releasing the
mercury into the argon gas stream flowing into the instrument.
-------
Jyeurofins
Frontier Global Sciences
Document Titie:
Mercury In Water by Oxidation, Purge & Trap
and CV-AFS (EPA Method 1631, Rev E)
Eurofins Document Reference:
EFGS-SOP-137-RG2
7 Reference Modifications:
7.1 There were no significant modifications to this method,
8 Definitions;
8.1
8.2
8,3
Analytical Duplicate (AD): A representative sample (that yielded a result within the
calibration curve) is analyzed a second time during the analytical run. The second
analysis should be at the same aliquot as the original.
Analytical Run - The continuous analysis of one or more batches during the same 12
hour-shift. Each analytical day requires a minimum five-point calibration curve, ICV, at
least 3 IBLs, and CCV/CCB every ten runs. An analytical day must conclude with a
CCV/CCB.
Analytical Spike and Analytical Spike Duplicate (AS/ASD): A representative sample is
selected and spiked, with a dilution of the primary source, during the analytical run, at
a target concentration of 1-5X the ambient concentration of the sample. These QC
samples are used to indicate sample matrix effects on the anaiyte of interest. Non-
detectable samples are spiked at 1 - 5 x of the MRL/PQL
Batch: 20 client samples or less grouped for preparation. See Quality Assurance
Section for batch requirements.
Calibration Standards (CAL) - a series of standards that will be used to calibrate the
instrument, made from a primary source stock standard. A calibration blank plus at
least five different concentrations are required, beginning with one at PQL
concentration.
Certified Reference Material (CRM) - a standard of known composition that is certified
by a recognized authority and representing a sample matrix. It is used to verify the
accuracy of a method.
Continuing Calibration Blank (CCB): An instrument blank that is used to monitor the
ambient blank concentration after the Continuing Calibration Verification (CCV).
Continuing Calibration Verification (CCV): An aliquot of standard from the same source
as the calibration standard, at a value of 20ng/L (2.0ng in ~100mL bubbler water). This
standard is analyzed after every 10 analytical runs, and determines whether the
instrument is maintaining calibration.
8.9 Continuing Demonstration of Capability (CDOC)
8.10 Control Limit (CL) - the limit of the range of acceptability for the quality control
samples
8.4
8.5
8.6
8.8
8.11
8.12
Equipment Blank (EB): Reagent water processed through the sampling devices and
placed in a sample container prior to using the equipment to collect samples and used
to demonstrate that the sampling equipment is free from contamination,
Field Blanks (FB): A sample of reagent water placed in a sample container in the field
and used to demonstrate that samples have not been contaminated by sample
collection or transport activities. EPA-1631E recommends the analysis of at least one
field blank per 10 samples collected at the same site at the same time. Analyze the
blank immediately before analyzing the samples in the batch.
-------
Frontier Gtoba! Sciences
Document Title:
Mercury in Water by Oxidation, Purge & Trap
and CV-AFS (EPA Method 1631, Rev E)
Eurofins Document Reference:
EFGS-SOP-137-R02
8.13
8.14
8,16
8.17
8.18
8.19
8.22
8.23
8,24
8.25
Initial Calibration Verification (ICV): A standard that is prepared from a secondary
source stock standard with a value of 15ng/L (1.5ng in ~1QOmL bubbler). This standard
is run immediately following the calibration curve and verifies instrument calibration. It
is always followed by the IBLs.
Initial Blank Level (IBL): An instrument blank that is used to demonstrate the ambient
blank concentration of the instrument. One per bubbler is needed at the beginning of
the analytical run.
8.15 Initial Demonstration of Capability (IDOC).
Laboratory Control Sample (LCS and LCSD) or Quality Control Sample (QCS): A
sample (and duplicate) containing a known concentration of mercury that is used to
monitor complete method performance. The preferred LCS is a matrix matched
Certified Reference Material (CRM), but a blank spike meets the requirement also. In
LIMS, the LCS is always referred to as a Blank Spike (BS), whether it is matrix
matched or not
Limit of Detection (LOD) - equal to MDL and verified on a quarterly/annual basis,
depending on the preparation, by spiking within three times the established LOD and
showing a positive result on the instrument.
Limit of Quantitation (LOQ) - equal to POL and verified on a quarterly/annual basis,
depending on the preparation, by spiking within 2 times the LOQ and showing a
recovery between 70 - 130%.
LIMS: Laboratory information Management System. Computer software used for
managing samples, standards, and other laboratory functions.
8.20 May: This action, activity, or procedural step is optional.
8.21 May Not: This action, activity, or procedural step is prohibited .
Matrix Spike (MS) and Matrix Spike Duplicate (MSD): A representative sample is
selected and spiked with a dilution of the primary source at a known concentration.
The MS and MSD are run through the entire analytical process just as the samples
are. These QC samples will indicate sample matrix effects on the analyte of interest.
Method Blank (MBLK) or Preparation Blank (PB): For waters, reagent water that is
prepared and analyzed in a manner identical to that of samples. For digested solids,
preparations blanks consist of the same reagents used to digest the samples, in the
same volume or proportion and are carried through the complete sample preparation
and analytical procedure. Boiling chips are used as a blank matrix for solids.
Preparation blanks are referred to as BLK in LIMS.
Method Detection Limit (MDL): A limit derived from 40 CFR, Part 136, Appendix B.
This method produces a defined value that is the minimum concentration that can be
measured and reported with a 99% confidence that the analyte concentration is
greater than zero from a given matrix.
Method Duplicates/Method Triplicates (MD/MT): A second or third separate sample
dilution, taken from the same source sample, prepared and analyzed in the laboratory
separately. An MSD may be used as a duplicate.
-------
Jyeurofins
Document Title;
Mercury in Water by Oxidation, Purge & Trap
and CV-AFS (EPA Method 1631, Rev E)
Eurofins Document Reference:
EFGS-SOP-137-R02
8.26 Reagent water: 18 MQ minimum, reagent water starting from a pre-purified (distilled,
Reverse Osmosis, etc.) source.
8.27 Must: This action, activity, or procedural step is required.
8.28 Ongoing Precision and Recovery (OPR): A dilution of a secondary source resulting in
an instrumental concentration of 5.0 ng/L mercury.
8.29 PM: Project Manager.
8.30 Practical Quantitation Limit (PQL), Method Reporting Limit (MRL): The minimum
concentration that can be reported quantitatively. The PQL is often described as 1-10
times higher than MDL. Eurofins Frontier defines the PQL as the lowest concentration
that can achieve 70-130% recovery for 10 replicate sample preparations, tn LI MS, the
PQL is referred to as the MRL.
8.31 Primary Source: The stock standard used to make the calibration standard. Procedural
Method: A method where standards and samples are run through the analytical
procedure exactly the same. By NELAC definition, this SOP is a procedural method.
8.32 Secondary Source: The stock standard used to make the OPR standard.
8.33 Shall: This action, activity, or procedure is required,
Should: This action, activity, or procedure is suggested, but not required.
8.34
8.35
8.36
8.37
Stock Standard Solution (SSS) - a standard of analyte that is purchased from a
certified source for the preparation of working standards.
Total mercury: As defined by this method, al! bromine monochloride-oxidizable
mercury forms and species found in aqueous solutions. This includes, but is not limited
to, Hg(ll), Hg(0), strongly organo-complexed Hg(ll) compounds, adsorbed particutate
Hg{P), and several tested covatently bound organomercuriais {i.e. CH3HgCI, (CH3)2Hg,
and C6H5HgOOCCH3). The recovery of mercury bound within microbia! cells may
require additional preparation steps (i.e. UV oxidation, or oven digestion).
Travel or Trip Blank (TB): A sample of reagent water placed in a sample container in
the laboratory and used to demonstrate that samples have not been contaminated by
transport activities.
9 Interferences;
9.1
9.2
Gold and iodide are known interferences. At a mercury concentration of 2.5 ng/L and
at increasing iodide concentrations from 30 to 100 mg/L, test data have shown that
mercury recovery will be reduced from 100 to 0 percent. At iodide concentrations
greater than 3 mg/L, the sample should be pre-reduced with SnCI2 (to remove brown
color immediately prior to analysis) and additional or more concentrated SnCI2 should
be added to the bubbler containing sample. If samples containing iodide
concentrations greater than 30 mg/L are analyzed, it may be necessary to clean the
analytical system with 4N HCi after the analysis,
Water vapor has the potential to create recovery interferences. To prevent interference
from water, ensure that soda-lime pre-traps and gold traps remain dry.
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10.1
10.2
9.3 The presence of high concentrations of silver and/or gold can cause SnCI2 to
precipitate out of solution and adhere to the bubbler walls. High concentrations of
these metals can sometimes be found in the matrix spike samples from the digestion
sets that are shared with the trace metals group. When analyzing digestates where the
matrix spike samples have been spiked with silver or gold, the matrix-spiked samples
must not be used for mercury analysis. Instead, an alternate matrix spike and matrix
spike duplicate (MS/MSD) should be prepared and analyzed. If this is not possible, an
Analytical Spike/Analytical Spike Duplicate (AS/ASD) must be analyzed on the
ambient sample,
10 Safety Precautions, Pollution Prevention and Waste Handling;
Personnel will don appropriate laboratory attire according to the Chemical Hygiene
Plan. This includes, but is not limited to, laboratory coat, safety goggles and nitrite
gloves under clean gloves.
The toxicity or carcinogenicity of reagents used in this method has not been fully
established. Each chemical should be regarded as a potential health hazard and
exposure to these compounds should be as low as reasonably achievable. Chemists
should refer to the MSDS (Material Safety Data Sheets) for each chemical they are
working with.
10.2.1 Note; Use particular caution when preparing and using BrCI, as it releases
extremely irritating, corrosive fumes similar in effect to free chlorine. Always
handle this reagent in an approved fume hood
Ail personnel handling environmental samples known to contain or to have been in
contact with human waste should be immunized against known disease-causative
agents. Eurofins Frontier will reimburse the expense of Hepatitis A and B
immunizations for any laboratory staff member who desires this protection.
Hydrochloric acid: Very hazardous in case of skin contact (corrosive, irritant,
permeator), of eye contact (irritant, corrosive), of ingestion. Slightly hazardous in case
of inhalation (lung sensitizer). Non-corrosive for lungs. Liquid or spray mist may
produce tissue damage particularly on mucous membranes of eyes, mouth and
respiratory tract. Skin contact may produce burns. Inhalation of the spray mist may
produce severe irritation of respiratory tract, characterized by coughing, choking, or
shortness of breath. Severe over-exposure can result in death. Inflammation of the eye
is characterized by redness, watering, and itching. Skin inflammation is characterized
by itching, scaling, reddening, or, occasionally, blistering. For more information see
MSDS.
10.5 See Eurofins Frontier Global Sciences Chemical Hygiene Plan (CHP) for general
information regarding employee safety, waste management, and pollution prevention,
10.6 Pollution prevention information can be found in the current Eurofins Frontier Global
Sciences Chemical Hygiene Plan (CHP), which details and tracks various waste
streams and disposal procedures.
10.7 All laboratory waste is accumulated, managed, and disposed of in accordance with all
federal, state, and local laws and regulations. Any waste generated by this procedure
should be disposed of according to SOP FGS-099 "Waste Disposal Procedure for
10.3
10.4
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Client Sample Waste," which provides instruction on dealing with laboratory and client
waste,
11 Personnel Training and Qualifications:
11.1 An analyst must perform an initial demonstration of capability (IDOC) that includes four
replicates of a secondary source before being qualified to analyze samples without
supervision. Continuing DOC will be maintained and monitored via performance on
CRMs and other QC samples, as well as obtaining acceptable results on proficiency
testing exercises.
The analyst/laboratory technician must have read this SOP and other relevant SOPs
and have the training documented on the applicable form(s). The analysis may be
questioned on SOP by supervisor(s) and/or trainers.
Training is documented by the employee and supervisor, and is kept on file in the QA
Office. The employee must read, understand, and by signing the training document,
agree to perform the procedures as stated in all Standard Operating Procedures
(SOPs) related to this method.
Reading of the SOP must be documented on the correct form such as "Standard
Operating Procedure Training Record," Appendix F in FGS-094, the last page of this
SOP, Appendix A "Standard Operating Procedure Training Record" or a similar
document."
All employees must also, on a yearly basis, read the Quality Manual (QM), and
complete the yearly Ethics training.
All training documents including IDOCs, CDOCs, SOP reading, Initial QA orientation,
and Ethics training are stored by the Quality Assurance Manager in the employees
training file for ten years after the employee is no longer working for Eurofins Frontier
Global Sciences.
11.7 Chemical Safety Training, Compressed Gas Training, Chemical Hygiene Plan
documentation, and Shipping of Hazardous goods, are stored by the Health and
Safety Officer for ten years after the employee is no longer working for Eurofins
Frontier Global Sciences.
12 Sample Collection, Preservation, and Handling;
12.1 Aqueous samples are collected in rigorously cleaned fluoropolymer (e.g. Teflon) or
PETG bottles and caps (as described in FGS-007 "Cleaning of Sampling Equipment
and Bottles for Mercury Analysis"). Certified clean glass bottles with fluoropolymer lids
may be used if mercury is the only analyte of interest.
12.1.1 Aqueous samples are preserved upon receipt with 0.2N BrCI that has tested
low in mercury. Samples are typically preserved to 1% BrCI v/v, but may
require further oxidation due to high levels of organic matter or mercury. Refer
to FGS-012 "Oxidation of Aqueous Samples for Total Mercury Analysis" for
oxidation of aqueous samples. Samples requiring greater than 10% BrCI must
have a method blank prepared at the time of preservation. Preservation levels
should be limited to 1%, 2%, 3%, 5%, 10%, and 100%.
11.2
11.3
11.4
11.5
11.6
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12.2
12.1.2 Preservation levels other than 1% are written on the LiMS label of the sample
bottle. Preservation levels are also documented in the LIMS bench sheet by
adjusting the initial and final volumes. For example, a sample preserved at 2 %
BrCI must say "2" on the LIMS label, and have an initial volume of 100mL and a
final volume of 102mL in the bench sheet,
All samples should be collected utilizing clean techniques, so as not to cross-
contaminate samples with mercury. See FGS-008 "Ultra Clean Aqueous Sample
Collection" and EPA Method 1669 for aqueous sample techniques.
13 Apparatus and Equipment:
13.1 LIMS - Element, version 5.85 or higher; Computer - Windows XP, 7 or 8
13.2
13,3
13.4
13.5
13.6
13.7
13.8
13.9
Tehran 2500 Atomic Fluorescence Spectrophotometer (AFS) or equivalent; A high
sensitivity AFS Detector (IDL<1pg) with a required wavelength of 253.7 nm and
associated software,
Flow meter/needle valve: A unit capable of controlling and measuring gas flow to the
cold vapor generator at 200-500 mL/min.
Teflon Fittings: Connections between components and columns are made using Teflon
FEP tubing and Teflon friction fit tubing connectors.
Soda-Lime pre-trap: A 10cm x 0.9cm diameter Teflon tube containing 2-3 g of reagent
grade, non-indicating 8-14 mesh soda-lime (Ca(OH)2+NaOH) aggregates, packed
between portions of silanized glass wool. This trap is purged of mercury by placing it
on the output of a clean cold vapor generator and purging it with -3-5% HCI and -600
ML of SnCI2 for approximately 20 minutes with N2 at 40 mL/min.
Cold-vapor generator (bubbler): A 150 ml, tall, flat-bottom borosilicate flask with
standard taper 24/40 neck, fitted with a sparger having a coarse glass frit which
extends to within 0.2 cm of the flask bottom.
Gold Traps: Made from 12 cm lengths of 6 mm OD quartz tubing, with a 4-way crimp
3.0 cm from one end. The tube is filled with approximately 2.5 cm of 20/40 mesh gold-
coated quartz sand, the end of which is then plugged with quartz wool. Gold-coated
sand traps are heated to 450-500ฐC (the coil should have a barely visible red glow
when the room is darkened) with a coil consisting of 75 cm of 24-gauge nichrome wire
at a potential of 10 VAC. Potential is applied and finely adjusted with an auto-
transformer. Refer to SOP FGS-061 regarding the construction of gold traps used for
total mercury analysis.
Agilent Integrator Recorder or equivalent: Any multi-range chart recorder or integrator
with 0.1-5.0 mV input and variable speeds is acceptable. Data capture software may
also be used.
Pipettes: Calibrated variable pipettes with a range of 5 uL - 10 mL Used to make
solutions and sample dilutions. Pipettes are to be calibrated weekly according to SOP
FGS-003andFGS-155.
14 Reagents and Standards:
All reagents, except those made daily, must be entered into LIMS
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14.1 Reagent Water: 18-MO ultra pure deionized water starting from a pre-purified (distilled,
R.O., etc.) source is used. To remove any remaining trace metals and organics, an
activated carbon cartridge is placed between the final ion exchange bed and the 0.2-
um filter. Reagent water used in the mercury lab is checked weekly for total mercury
concentrations, and must test below 0.25ng/L.
14-2 Hydrochloric Acid (HCI): Concentrated (36-38% weight basis). Must be traee-metaf
purified and reagent grade. HCI is typically monitored through performance of the BrCI.
Sometimes it will be necessary to test the HCI directly. To do so, add 1 mL, using a
calibrated pipette, of HCI to approximately 1QQmL of purged bubbler water. Enter 1mL
as aliquot in the Excel spreadsheet. Do not prep blank correct. Analyze one replicate
per bottle. This reagent should test below 5.0 ng/L. This solution is considered stable
until the expiration date on the bottle, set by the manufacturer.
14.3 0.2N Bromine Monochloride (BrCI):
14.3.1 37.5 g of KBr is added to a 2.5-L bottle of concentrated HCI (pre-analyzed and
found to be below 0.25 ng/L Hg). The bottle is then inverted in a fume hood to
mix the acid and KBr. The solution then sits overnight allowing for the KBr to be
dissolved.
14.3.2 27.5 g of KBrO3, certified to be low in Hg, is slowly added to the acid. When all
of the KBrO3 has been added, the solution should have gone from yellow to red
to orange.
14.3.3 Loosely cap the bottle, and allow to sit for 30 minutes in a fume hood before
tightening the lid. Once capped invert bottle to make sure all of the solids goes
into solution. CAUTION: This process generates copious quantities of free
halogens (CI2, Br2, BrCI) which are released from the bottle. Add the
KBrO3 SLOWLY and in a well operating fume hood.
14.3.3,1 To test the BrCI, add 1 mL, using a calibrated pipette, of the BrCI to a
prep blank vial containing approximately 4 mL reagent water. Add 200 uL
Hydroxylamine-HCI to the vial; pour the entire contents into a bubbler
containing approximately 100 mL of purged water. Assume a 100 mL
aliquot in the Excel spreadsheet. This reagent must test below 0.20ng/L.
Do not prep blank correct. Analyze one replicate per bottle.
14.3,3.2 The expiration time for this reagent is set by default to six months in
LIMS. There is no suggested holding time in EPA method 1631E,
therefore the holding time can be extended, as long as the primary
reagent has not expired. The mercury concentration of the BrCI is
monitored through the preparation of water preparation blanks.
14,4 Hydroxylamine hvdrochlortde: dissolve 300g of NH2OH-HCI in reagent water and bring
the volume up to 1L. This solution may be purified by the addition of 1mL SnCI2
solution and purging overnight at 500mL/min with mercury-free N2. The working
reagent is a 25% solution that is made by adding one part reagent water to one part
50% hydroxylamine hydrochloride. This reagent must test below 0.25ng/L.
14.4.1 To test the Hydroxylamine-HCI (NH2OH-HCI), add 1 mL of the 50% reagent,
using a calibrated pipette, to approximately 100 mL of purged bubbler water!
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Assume a 100 ml aliquot in the Excel spreadsheet. This reagent must test
below 0.20 ng/L. Do not prep blank correct. Analyze one replicate per bottle.
14.4.2 The expiration time for this reagent is set by default to six months in LIMS.
There is no suggested holding time in EPA method 1631E; therefore the
holding time can be extended, as long as the primary reagent has not expired,
14.5 Stannous Chloride (SnCI2): Weigh out 500 g SnCI2 using a calibrated balance that also
has been verified for the day. Dissolve with three 100 ml aliquots of concentrated HCI
and transfer to a 1L I-CHEM glass bottle, which contains approximately 300 ml of
reagent water. Bring this solution up to approximately 1 L of volume and purge
overnight with mercury-free N2 at 500 ml/mm to remove all traces of mercury. Store
tightly capped. The working reagent is a 25 % solution that is made by adding one part
reagent water to one part 50 % stannous chloride.
14.5.1 To test the Stannous Chloride (SnCI2), add 1 ml of the 50% reagent, using a
calibrated pipette, to approximately 100 ml of purged bubbler water. Assume a
100 ml aliquot in the spreadsheet. This reagent must test below 0.20 ng/L. Do
not prep blank correct. Analyze one replicate per bottle.
14.5.2 The expiration time for this reagent by default is set to six months in LIMS.
There is no suggested holding time in EPA method 1631E; therefore the
holding time can be extended, as long as the primary reagent has not expired.
Argon Grade 4.7 or better {ultra high-purity grade): Argon that has been further purified
by the removal of mercury using a gold trap that is located in line between the gas
output and the analyzer gas input.
Nitrogen Grade 4.5 (standard laboratory grade): Nitrogen that can be further purified of
mercury using a gold trap that is located in line between the gas output and bubbler
14.8 Preparation of Total Mercury Standard Solutions:
14.6
14.7
14.8.1
Mercury standard solutions are prepared in ultra clean volumetric glassware
and gravimetrically calibrated pipettes. Resulting solutions must be stored in
glass or Teflon bottles and preserved to at least 2 % BrCI. All working
standards must be tested prior to use.
14.8.1.1
New working standards and standard dilutions are tested prior to use,
Three reps of the new standard are analyzed in the same run as three
reps of the current NIST 1641D standard. Analyze 200 pi of the NIST
1641D and assume 100 ml in the bubbler. The mean percent recovery of
the three standards should be ฑ5 % (95-105 %) of the true value and also
within 5 % of the average NIST 1641D recovery (e.g. If the average of
NIST 1641D recovery is 97 %, the range for the standard is 95-102 %). If
the standard does not test within this control limit, it is retested. If it still
does not meet the control limit, it is discarded and remade, unless
otherwise approved by the Quality Assurance Officer, NOTE: When
making serial dilutions to create various standard levels; the lowest
concentration may be used to test any of the higher concentration steps
(for example: if a 1Qng/mL calibration standard is created from a
1000ng/mL spiking standard, only the 10ng/mL standard requires testing.
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14.8.2
14.8.3
If the 1Qng/mL standard passes, then both standards are considered to
be passing within the control limits.)
Total Mercury Stock Standard Solution (Stock); Certified mercury standard
purchased from High Purity Standards (1000 |jg/mL (1 000 000 ng/mL) primary
source) or Absolute Standards (100 |jg/mL (100 000 ng/mL) secondary
source), or any equivalent standard.
Total Mercury Spiking Standard Solutions (Spiking Standard): Spiking
standards are made from either the primary or secondary sources.
14.8.3.2
14.8.3.3
14.8.3.1 To make standards, use an ultra clean volumetric flask and a calibrated
pipette. Add reagent water until flask is about half full. Add 2 % 0.2N BrCI
and the specific spike volume noted below (these volumes may be
changed as long as ratio and resulting concentration remains the same).
Bring up to the mark with reagent water and mix well prior to testing.
When spiking samples, no more than 200 ML of any spiking standard is
added to the sample to minimize effects on volume, it is also
recommended that staff pipette no less than 25 uL If possible, minimize
headspace during standard storage. Expiration date is currently set at
6 months or when the stock standard expires, whichever is shorter.
100,000 ng/mL Spiking Standard: Made from the Primary Stock Standard
(High Purity, or equivalent vendor). Dilute 10 mL of the stock standard to
100 mL of reagent water containing 2 % BrCI. (Can also be made by
preserving Secondary Stock Standard to 2% BrCI).
10,000 ng/mL Spiking Standard: If made from the Primary Stock Standard
(High Purity, or equivalent vendor). Dilute 1.0 mL of the stock standard to
100 mL of reagent water containing 2 % BrCI. If made from Secondary
Stock Standard, dilute 10mL of stock standard to 100mL with reagent
water containing 2% BrCI.
14.8.3.4 1,000 ng/mL Spiking Standard: If made from the Primary Stock Standard
(High Purity, or equivalent vendor). Dilute 0.250 mL of the stock standard
to 250 mL RO water containing 2 % BrCf. If made from Secondary Stock
Standard dilute 2.5mL of stock standard to 250mL with RO water
containing 2% BrCI.
14.8.3,5 100 ng/mL Spiking Standard: Made from a stock standard or dilution of a
stock standard with a concentration of 100,000 ng/mL. Ditute 0.100 mL of
the 100,000 ng/mL dilution to 100 mL of reagent water containing 2 %
BrCI. Expiration date is currently set at 3 months or when the stock
standard expires, whichever is shorter.
14.8.4 Calibration Standard (10 ng/mL): Must be made from a dilution of the Primary
Stock Standard (High Purity, or equivalent vendor). Typically made by diluting
0.5mL of a 10,000 ng/mL Primary Spiking Standard to 500 mL of reagent water
containing 2 % BrCI. Transfer to glass or Teflon bottle. The calibration
standard is considered stable for three months or until the stock standard
expires.
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14.8.5
14.8.6
14.8.7
14.8.8
14.8.9
Calibration Standard (1 ng/mL): Must be made from a dilution of a Primary
Stock Standard. Typically made by diluting 1.0mL of a 100 ng/mL Primary
Spiking Standard to 100mL with Reagent water containing 2% BrCL
Initial Calibration Verification (ICV): A 10 ng/mL ICV solution is prepared using
the Secondary Stock Standard (Absolute Standards, or equivalent vendor).
Use 0.100 mL (100 uL) of the Secondary Stock Standard to 1000 mL Milli-Q
containing 2 % BrCI. Transfer to one 1000 ml glass or Teflon bottle. The ICV
standard is considered stable for three months or until the stock standard
expires, it is recommended to alternate expiration date with the CAL standard.
Continuing Calibration Verification (CCV): For CCV analysis, use 200 (jL of the
10 ng/mL CAL standard {documented in LIMS as SEQ-CAL3), The True Value
is 20 ng/L.
Certified Reference Material (CRM) for Total Mercury in Water: A 1.5679 mg/L
solution (1,557 mg/kg at a density of 1.007 g/mL) is prepared by adding a 5,0
mL of CRM NIST 1641d (from ampoule) into a 1000 mL flask containing of
reagent water. This solution is diluted to 1000 mL, and an additional 10 mL of
0.2N BrCI is added, resulting in a final volume of 1010 mL, Preparing the
solution in this manner makes a 1:200 dilution of the stock CRM. This solution
is considered stable for one year, or until the stock standard expires. Results
are corrected for the additional 1 % BrCI in the analysis Excel spreadsheet and
in LIMS.
Ongoing Precision and Recovery (OPR) for "Strict" 1631 E: A 5.0 ng/L solution
is prepared by adding 100 pL of the 100 ng/mL secondary spiking standard into
2000 mL reagent water. An additional 1 % BrCI (20 mL) of BrCI is added, so
that the final volume is 2020 mL. This standard is analyzed at 100 mL at the
instrument, and preparation blank corrected exactly in the same manner as
samples
14.9 Documentation of Standards and Reagents:
14.9.1 Standards and Reagents are documented in LIMS upon receipt or creation. A
LIMS generated label is affixed to each standard and reagent that has the
name of the solution, the person who prepared or received it, the date it was
prepared or received, and the expiration date.
14.9.2 Each bottle of standard must be labeled with the following: the date of receipt
or creation, the initials (or name) of who entered the standard into LIMS, the
concentration and analyte, the expiration date and the LiMS ID. This
information must also appear on the certificate of analysis of stock standards.
14.9.3 Stock standards and CRMs are logged into LIMS upon receipt by Shipping and
Receiving (S&R) or the Quality Assurance department (QA). These do not
require testing, provided there is a Certificate of Analysis on file in QA. When
receiving a solid CRM, QA shall generate a work order in LIMS for total solids
analysis.
14.9.4 For all standards, L!MS documentation must include the following: a description
of the standard, department, expiration date of the standard (not to exceed the
expiration of the parent standard), the name of the person who made (or
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received) the standard or reagent, the date it was prepared (or received), final
volume, a reference date (date entered into LIMS), concentration units (ug/mL),
the vendor and vendor lot. The solvent lot is used to document the Lot Number
or LIMS ID of the BrCI that was used. In the comments section, the analyst
must enter the sequence and applicable results for documentation of standard
testing. Other notes may be entered in here as well. The correct parent
standard must be noted, as well as the amount used, Analytes are entered
individually from the list. LIMS will calculate the true value of the standard
based on the amount of the parent used and the final volume. Click the
appropriate radio button under Standard type. A Spike Mix is a standard that is
used in a bench sheet, and a Calibration standard is a standard used only in
sequences. A Reference Standard is a Certified Reference Material (CRM).
The standard must not be used until it has passed control limits and is
approved by the mercury supervisor, mercury laboratory manager, or QA for
use.
14.9.4.1 If the new standard is a calibration standard, a separate standard ID must
be created for each calibration point based on the final concentration in
the sequence (example: THg CAL1 0.10 ng orTHg CAL2 0.50 ng). These
are given the same expiration as the standard they are made from, and
will need to be generated every three months as each new working
calibration standard is made and tested.
14,9.4,2 To generate new "CAL" standards in LIMS, go to the Laboratory drop
down menu and select Standards. Open the current CAL1 standard and
click "Copy". Update the appropriate information, including the Prepared
Date, Expiration Date, Prepared By, and the Reference Date. For these
standards, which are to be used in the sequence, the final volume is
equal to the assumed aliquot in the bubbler (100 mL). Check that the
vendor lot is correct. Remove the old (expired) parent standard. Choose
the new parent standard, and enter the amount of standard added to the
bubbler for that calibration point. All depleted or expired standards are
moved into the Expired Standards Department once they are no longer
being used.
14.9.4.3 Each bottle of standard must be labeled with the following: the date of
receipt or creation, the initials (or name) of who entered the standard into
LIMS, the concentration and analyte, the expiration date and the LIMS ID.
This information must also appear on the certificate of analysis of stock
standards.
Neat reagents are logged into LIMS with a unique identifier upon receipt by
Shipping and Receiving Department and given a default expiration of 3 years,
unless otherwise noted by the manufacturer.
Working reagents are prepared by the analyst, logged into LIMS and assigned
a unique identifier. Reagents entered into LIMS must have the information
listed in section 14.9.2. In addition the parent neat reagents are added by their
unique identifier and the amount of each reagent is entered. It is not necessary
to enter anaiytes from the list for reagents. The Solvent Lot is not applicable to
working reagents. The radio button must be clicked to Reagent. If the reagent
14.9.5
14.9.6
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requires testing, it must test clean prior to using. Ail reagents used during
analysis and prep should be added to bench sheet,
14.9.7 Depleted or expired standards and reagents are segregated and removed from
use.
15 Calibration:
15.1 The analyst should label the strip chart/integrator printout with the corresponding
dataset ID as well as print and sign their name. For strip chart printouts, the analyst
should label the baseline ratios accordingly (usually X=1 and X=20) and label with the
analysis day start time and strip chart drum speed (usually 1 mm/min). The analyst
should note the end time as well. If using an integrator, the date and time should be
checked and corrected if necessary.
15.2 The calibration sequence determines the range of sample concentrations that are
reportable. The calibration sequence starts with a 5-point curve using the total mercury
calibration standard solution. The five points are: Q.05ng (0,50 ng/L), 0.10 ng (1.00
ng/L), 0.50 ng (5.00 ng/L), 2.00 ng (20.00 ng/L), and 4.00 ng (40.0 ng/L). An ICV/OPR
and IBLs (one for every bubbler used are analyzed immediately following the standard
curve.
15.2.1
15.2.2
15.2.3
Using the 10 ng/mL calibration standard, add 5 uL, 10 uL, 50 uL, and 200 uL to
the bubblers sequentially from the left to right. Add 300 uL SnC!2 to the
bubblers and seal bubbler tops using Keck Clips.
Place blanked gold traps securely at the end of soda-lime traps (pinched
section of gold trap closest to the soda-lime trap). Purge bubblers with N2 for a
minimum of 20 minutes,
Attach individual gold traps to the analytical, train and burn in sequential order,
Peaks produced should be labelled, as well as recorded in the Excef
spreadsheet in real time.
15.3 For the second round, add 400 fjl of the 10 ng/mL mercury calibration standard to the
first bubbler. Add 50uL of the 10 ng/mL ICV(OPR) standard to the second bubbler (5.0
ng/L). The third and fourth bubblers are used for the first and second IBLs and nothing
should be added to these bubblers. To ensure that nothing is added, keep it sealed
with a Keck Clip. Add 300 uL SnCI2 to ail bubblers except the fourth and seal bubbler
tops with Keck Clips.
15.4 For the third round, use the first and second bubbler to finish the IBLs needed for
1631. The third and fourth bubbler can be used for the first portion of the batch. If the
curve does not pass or needs to be investigated any batch portions analyzed in this
round will need to be reanalyzed.
15.5 Once the instrument is calibrated and the ICV/IBLs are analyzed and judged to be in
control, the instrument is operational. The sample concentrations must fall within the
range of the calibration standards or be diluted and reanalyzed.
15.6 The purge efficiency of the bubbler system is 100 % and is independent of volume at
the volumes used in this method. Calibration of this system is typically performed using
units of mass. For purposes of working in concentration, the volume is assumed to be
100 mL.
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Document Title: _ ,. _ , _ ,
Mercury in Water by Oxidation, Purge & Trap E^ ' r Q "^ซ onf^
and CV-AFS (EPA Method 1631, Rev E) tr-U5-bUP-'W-KtL>
15,7 This completes the instrument calibration for total mercury analysis.
16 Procedure:
16.1 When analyzing on the Tekran 2600, follow the procedure in EFGS-121 while still
adhering to the QA/QC criteria of this method.
16.2 Pre-analysis and Organization:
16.2.1 Prior to analyzing samples it is imperative to reference LiMS for all project
specific information, such as QC requirements, suggested dilutions, project
manager information, and specifics regarding spike levels.
16.2.2 The analyst should then locate samples and check the work order in LIMS for
notes about specific project requirements.
16.2.3 The analyst should compare the sample IDs to the work order and see that the
samples are accounted for, and notify the project manager of any
discrepancies in analysis required, sample identification, etc.
16.2.4 All mercury analyses receive a unique dataset identifier. This is comprised of
the instrument type and number, the date and the calibration number for that
day. The format is as follows: THg8-091218-1, where "THg "refers to a total
mercury analysis; "8" refers to the analyzer number 8; 091218 refers to the
date (December 18, 2009 in the YYMMDD format); and "1" refers to the first
calibration of the day,
The sequence number is assigned by LIMS when the data gets imported into
LIMS. The alpha-numeric code is based on the following format: 3B02001,
where the 3 refers to the year (2013), the "B" is the month (A= January,
B=February...L=December), "02" is the day of the month (February 2nd) and'
the final 3 digits is the nth sequence created on that particular year/month/day
combination.
16.2.5 In general, the analyst should organize their samples in the order listed on the
bench sheet. The first samples analyzed should be the preparation blanks,
then the LCS if analyzing solid samples, followed by actual samples. If
possible, run total and dissolved samples side by side to facilitate verification
that total concentration is greater than dissolved concentration. See QA
section.
16.2.6 All samples specified as being High QA should be analyzed prior to any
Standard QA projects that are being analyzed on the same instrument on the
same day. However, if concentrations are known, analyze samples with low
concentrations prior to samples with high concentrations
16.3 instrument Start Up:
16.3.1 Begin blanking gold traps. To do this, attach one trap at a time to the analytical
train and burn to the instrument. Ensure the Argon, is flowing at appropriate
levels (-25-40 mL/min). The pinched portion of the gold trap should be on the
left (closest to the analytical trap). Continue to burn traps in sequential order.
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Mercury in Water by Oxidation, Purge & Trap
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Eurofins Document Reference:
EFGS-SOP-137-R02
16.3.2 Rinse out the bubbler three times with reagent water and fill with about 100 ml
of reagent water. Using a pre-purged pipette, add 3-5 ml HCI. Initially add 600
uL of SnCl2,
16.3.3 Prepare one soda-lime trap for each bubbler. To prepare soda-lime traps, hold
soda-lime between two glass wool plugs in a Teflon tube. Cap the tubes with
Teflon plugs and attach to the bubbler. Once the soda-lime traps have been
attached, the bubbler system (soda lime trap and bubbler water/acid/ SnCI2)
must purge for a minimum of 20 minutes before beginning the instrument
calibration sequence.
16.4 Analyzing Aqueous Samples;
16.4.1 All aqueous samples should be preserved with BrCI according to FGS-012 at
least 24 hours prior to analysis. In the event a sample requires further oxidation
prior to analysis, additional BrCi is added and the sample should not be
analyzed for at least 12 additional hours. In special cases where rush turn-
around-time is required and an oxidation period of less than 24 hours may be
used, a heated oven digestion procedure can be utilized.
16.4.2 While bubbling and burning the standard curve, the analyst should prepare a
minimum of three BrCI method blanks (BLK) at 1% BrCi. Add 1 mL BrCI and
200 uL hydroxyiamine hydrochloride (NH2OH-HCI) to each bubbler. The aliquot
is assumed to be 100 ml. Any sample requiring an increased amount of
reagent must be accompanied by at least one method blank that includes an
identical amount of reagent,
16.4,3 After the instrument calibration sequence, preparation blanks and the
LCS/LCSD are analyzed.
16.4.4 All known field, equipment, and trip blanks should be analyzed before any other
sample types, usually after the BLKs. Aliquots of 100 ml should be analyzed,
provided there is adequate collected sample volume. Sample aliquot sizes of
125 ml can be analyzed upon request by the project manager.
16.4.5 For all waters, select the appropriate dilution (refer to L1IV1S, historical data
etc.).
16.4.5.1 For sample aliquots of 25 uL to 10.0 ml, use calibrated pipettes to
dispense the aliquots directly into bubbler. Due to minimal amounts of
BrCI in aliquots of 10 ml or less, NH2OH-HCI is not added. It is highly
recommended that the analyst should not pipette less than 25 uL A
dilution of the sample should be made to allow a larger aliquot to be
analyzed.
16.4.5.2 For sample aliquots greater that 10 mL, gravimetrically weigh out the
selected volume (ฑ0.2 g) into a clean 125 mL Teflon bottle. Once quantity
is weighed out, neutralize BrCI with 200 uL NH2OH-HCi no more than five
minutes prior to adding the sample to bubblers. The sample should turn
from a yellowish color to a clear/cloudy solution, depending on the matrix.
16.4.6 If the material is a seawater or highly dense liquid, it may be necessary to
account for the density if the aliquot is gravimetrically determined. Density
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checks can be performed at the time of analysis to determine if further
determinations are necessary,
16.4.7 The procedure for analysis is similar to that of the caiibration. Samples to be
analyzed are pipetted or poured into the bubbler (one sample per bubbler)
along with 300[jL SnCI2. Bubbler tops are sealed with Keck Clips to ensure
nominal sample leakage. Blanked gold traps are securely placed at the end of
the soda-lime trap. Purge bubblers with N2 for a minimum of 20 minutes,
remove gold traps, and sequentially place in the analytical train. Burn individual
traps to analyzer, labeling resulting peaks with corresponding sample in real
time,
16.4.7,1 Sample IDs, aliquot volume, BrCI percentage (group ID), peak
height/peak area, and dilution factor {if applicable) associated with each
sample should be entered into the THg Waters Template Excel
spreadsheet.
16.4.7.2 While purging one set of samples, the analyst should begin preparing the
next round of water samples in the same fashion to maximize efficiency,
16,5 End of analysis close-down procedure:
16,5.1 Turn off gas flow.
16.5.2 Carryout all end of day cleaning and restocking tasks.
16,6 The analytical data is compiled into an Excel file. The data is then copied and pasted
into an Excel template that is LIMS compatible,
16.7 Maintenance and Troubleshooting
16,7,1 ISSUE: No peaks at all
16.7.1.1 Ensure that the system is powered.
16.7.2 ISSUE: Low sensitivity
16,7.2.1 Make sure that you have freshly changed soda lime in the soda lime trap,
and that it is from a good source.
16,7.2.2 Do not use old calibration standards to calibrate the system.
16.7.2.3 Ma/ce sure you are running fresh SnC/2 solution.
16.7.2.4 Make sure that your stock Hg standard has not expired and is from a
reliable source and that it is not compromised.
16.7.2,5 Check the lamp voltage
16.7.3 ISSUE: High blanks
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Document Title:
Mercury in Water by Oxidation, Purge & Trap
and CV-AFS (EPA Method 1631, Rev E)
Eurofins Document Reference:
EFGS-SOP-137-R02
16,7,3,1 Check reagent (including water) quality
16.7.3.2 Check for system contamination
16.7.4 ISSUE: Nonlinearity of the calibration curve
16.7.4.1 Check and investigate high blanks.
16.7,4.2 Contaminated and expired soda lime. Change soda lime,
16.7.4.3 Mate sure your calibration standards are fresh and properly prepared.
17 Calculations:
17.1 Average all instrument blanks (PHX) using the peak area values from the TekMDS
software. Subtract the average (IB) from the peak area for each standard and sample.
17.2 Calculate the calibration factor (CFX) for mercury in each of the five standards using
the mean instrument-blank-subtracted peak area and the following equation:
CFX=PAX~IB/CX
Where:
17.2.1 PAx=peak area (or peak height) for mercury in standard
17.2.2 IB= mean peak height (or peak area) for mercury in bubbler blank
17.2.3 Cx=mass in standard analyzed (ng/L)
17.2.4 CFx=Calibration Factor of each concentration
17,2.4.1 Average the five calibration factors to establish mean value: CF(Avg)
(units/ng/L).
17.3 Sample results are then corrected for the average peak area values of at least three
preparation blanks (PBs), unless otherwise requested. This result is shown as the
Initial Result on the Excel spreadsheet and in LIMS.
17.4 Total Mercury in Water:
instrument Value (ng/L) = (Peak Height - BB) / CF(Avg)
Final Result (ng/L) = [(Instrument Value x DF) - (BLK)] x (Vf A/i)
Where:
17.4,1 CF(avg} = average calibration factor for curve (in units/ng/L).
17.4.2 BB = average bubbler blank peak area or peak height (in units)
17.4.3 Vf = Final volume of sample (in mL) from bench sheet.
17.4.4 Vj = initial volume of sample analyzed in mL prior to addition of BrCI.
17.4.5 DF - Dilution Factor - takes into account any instrumental dilution of the
sample
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17.4.6 BLK = average of the preparation blanks in ng/L.
17.5 A linear regression can be used as alternate calibration. A linear regression will not
change values significantly. If linear regression is used, the correlation coefficient (R)
must be >0.995.
18 Statistical Information/Method Performance:
18.1 The Method Detection Limit (MDL) is determined according to 40 CFR Part 136
Section B. Ten replicates {9 degrees of freedom) spiked 3-10 times the expected MDL
are run. The standard deviation (s) is taken from the resulting data and the MDL is
calculated as follows: MDL=2.821*s. This value should not be interpreted as the
method reporting limit.
18.2 The Practical Quantitation Limit (PQL) is the reporting limit for this method and is
included as the lowest calibration point (2003 NELAC regulation 5.5.5.2.2,1.h.3). The
PQL is determined by running ten samples with a concentration that will produce a
recovery of 70-130 %. The PQL is referred to as the Method Reporting Limit (MRL) in
LIMS.
18.3 Using clean handling techniques and reagents tested low for Hg content, the LOD
value for Total Hg in water is typically less than 0.2 ng/L, while the PQL is 0.50 ng/L.
18.4 Current LODs, LQQs, MDLs, and PQLs are stored at: Cuprum\General and
Admin\Quality Assurance\MDLs & PQLs.
19 Quality Assurance/Quality Control:
19.1 A minimum of three preparation blanks and one LCS/LCSD (preferably NIST 1641d),
must be analyzed per preparation batch. The upper control limit for each preparation
blank is equal to the PQL.
19.2 Matrix Spikes: One Matrix Spike/Matrix Spike Duplicate (MS/MSD) must be performed
for every 10 samples. The recovery of the MS/MSD must be between 71%-125%
recovery, and the Relative Percent Difference (RPD) below 24%. If an MS/MSD is out
of control, the analyst should investigate to identify the source of the failure. The MS
and MSD may be used as duplicates. Some failures may be qualified using QA
Qualification Flow Charts (Appendix A).
19.2.1 For aqueous samples, the MS/MSD is spiked at 1 to 5 times the ambient
concentration, with 0.25 ng, in the bubbler, being the minimum spiking level.
Sample aliquots for the MS/MSD should be the same as the ambient sample
aliquot, if sufficient sample volume exists. Spikes are added to the split aliquots
for volumes of 10mL or greater. For less than 1QmL aliquots, spikes are added
directly to the bubbler. NEVER ADD SPIKE DIRECTLY TO THE ORIGINAL
SAMPLE VESSEL UNLESS OTHERWISE STATED,
19.3 Matrix Duplicates - One Matrix Duplicate (IVID) may be analyzed for every batch of 20
samples. Upon request, a Matrix Triplicate (MT) may be performed. The MSD may
serve as the M.D if necessary. The Relative Percent Difference (RPD) and the
Relative Standard Deviation (RSD) of duplicate samples must be less than 24%. Some
failures may be qualified using QA Qualification Flow Charts.
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Mercury in Water by Oxidation, Purge & Trap
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Eurofins Document Reference:
EFGS-SOP-137-R02
19.3.1 For aqueous samples, analyze the parent, duplicate and triplicate at the same
dilution,
19.4 Laboratory Control Standard (LCS) or Quality Control Sample (QCS): For every batch
of samples, at least one LCS is processed and analyzed. The recovery of the LCS
must be within 80-120% for the aqueous NiST 1641d. An LCS Duplicate (LCSD)
should accompany the LCS.
19.4.1 A Certified Reference Material (CRM) is the preferred LCS, but a Blank Spike
may serve as an LCS if an appropriate CRM does not exist. The spiking level is
based on client request, historical data, or a default of mid-curve. A duplicate
blank spike must also be prepared as an LCSD.
19,5 Ongoing Precision and Recovery (OPR): An OPR must be analyzed at the beginning
and end of each analytical batch, or at the end of each 12-hour shift. The recovery of
the OPR must be within 77-123% to be considered in control,
19.6 All calibration standards must be traceable to the original standard source. The
calibration curve must be established at the beginning of the analytical run. It must
include at least five different concentrations, with the lowest concentration equal to the
PQL. The average response factor of each calibration standard is used to calculate the
sample values. The RSD of the response factors must be less than 15% of the mean
or the calibration fails.
19.7 ICV and CCV control limit is 77-123%. The CCV is analyzed every 10 analyses, and at
the end of an analytical run. CCBs are always analyzed after the CCVs,
19.8 Field Blanks: To be compliant with EPA 1631, clients must submit a field blank for
each set of samples (samples collected from the same site at the same time, to a
maximum of 10 samples),
19.8.1 If no field blanks are submitted by the client, their data will be flagged with "FB-
1631." "Required equipment/field/filter blank not submitted by the client. The
sample has been analyzed according to 1631E, but does not meet 1631E
criteria,"
19.9 Method or Preparation Blanks (BLK): Method blanks are used to demonstrate that the
analytical system is free from contamination that could otherwise compromise sample
results. Method blanks are prepared and analyzed using sample containers, labware,
reagents, and analytical procedures identical to those used to prepare and analyze the
samples.
A minimum of three 1 % BrCI method blanks per analytical batch are required.
Any sample requiring an increased amount of reagent must be accompanied
by at least one method blank that includes an identical amount of reagent.
If the result for any 1 % BrCI method blank is found to contain >0.50 ng/L Hg
(0.25 ng/L for DOD), the system is out of control. Mercury in the analytical
system must be reduced until a method blank is free of contamination at the
0.50 ng/L level.
For method blanks containing more than 1% BrCI, the control limit is equal to
0.50 ng/L multiplied by the final preservation percentage of BrCI. For example,
for a method blank preserved to 2 % BrCI, the control limit for the blank is 0.50
19.9.1
19.9.2
19.9,3
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Mercury in Water by Oxidation, Purge & Trap
and CV-AFS (EPA Method 1631, Rev ฃ}
Eurofins Document Reference:
EFGS-SOP-137-R02
ng/L * (102/101), or 0.50 ng/L. For 3% BrCI the control limit is
(103/101 )*0.50ng/L, or0.51ng/L
19.10 Instrument Blanks (IBL): A minimum of three instrument blanks must be analyzed with
each analytical batch. To analyze an instrument blank, attach a clean gold trap to the
bubbler. Purge and analyze as previously described and determine the amount of Hg
remaining in the system.
19.10.1 An instrument blank must be performed on all bubblers used during the
analytical run (normally four, but three at a minimum).
19.10.2 If the instrument blank is found to contain more than 0,50ng/L, the system is
out of control. The problem must be investigated and remedied and the
samples run on that bubbler must be reanalyzed. If the blanks from other
bubblers contain less than 0.50 ng/L, the data associated with those bubblers
remain valid, provided that all other QC criteria are met.
19.10.2.1.1 The mean result for all instrument blanks must be <0.25ng/L with
a standard deviation of 0.10 ng/L.
19,11 The analytical day must close with a CCV/OPR/CCB,
19,12 Because the method is done in real-time, it is EFGS' position that a single non-
compliant QC sample result does not automatically invalidate a data set. Alt data
points that can be explained and rerun with a passing result can be qualified. If the
source of error cannot be corrected for a QC standard that day, none of the data can
be validated, in the event that the system becomes out of control during the analysis
day, all results bracketed between valid QC data points shall still be considered valid
(CCV, OPR, CCB, etc),
19.13 The Control Limits are established from EPA 1631E.
20 Corrective Action
20.1
20.2
20.3
20.4
The data is reviewed as in the QC section (or matrix specific QC section) for all
parameters that pass specific requirements. If the data does not meet QC
requirements it is qualified or submitted for reruns. Data may be qualified (based on
scientific peer review) by the Group Supervisor, Project Manager, Lab Manager or QA
Officer.
Control Chart data is generated through LIMS to monitor the performance of the CCV,
LCS, MS, and MSD. This is done by the QA department.
Due to the real-time nature of the CVAFS method, failures must be investigated as
they happen. If the source of the problem can be identified, and corrected, the samples
may be rerun. If source of problem cannot be isolated, see the Senior Analyst, Group
Supervisor, or Laboratory Manager for instructions.
The Senior Analyst, Group Supervisor, Laboratory Manager, or QA Officer must be
informed if QC fails. It is also advisable to always alert the Project Managers,
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Mercury in Water by Oxidation, Purge & Trap
and CV-AFS (EPA Method 1631, Rev E)
Eurofins Document Reference:
EFGS-SOP-137-R02
21 List of Attachments
Table 1: QC Requirements for Total Mercury
Appendix A: Example - Standard Operating Procedure Training Record
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Document Title:
Mercury in Water by Oxidation, Purge & Trap
and CV-AFS (EPA Method 1631, Rev E)
Euroflns Document Reference:
EFGS-SOP-137-R02
Tab!ฉ 1: QC Requirements for Total Mercury
Initial Calibration Verification (ICV)
Continuing Calibration Verification (CCV)
Ongoing Precision and Recovery (OPR)
Initial Calibration Blank (ICB)/ Continuing
Calibration Blank (CCB)
Laboratory Control Standard (LCS) or Quality
Control Standard (QCS)
Calibration Curve RSD (Referred to as "Corr.
RSD CF" in Excel spreadsheet).
Lowest Calibration Point
1% BrCi Method Blank (BLK)
Matrix Duplicate (MD) and Analytical Duplicate
(AD)
Matrix Spike and Matrix Spike Duplicate
(MS/MSD) ; Analytical Spike (AS) and
Analytical Spike Duplicate (ASD)
77-123% Recovery
77-123% Recovery
77-123% Recovery
Individually, IBL and CCB <0.50ng/L,
but the mean of all the IBLs shall be
<0.25ng/L with a standard deviation of
0.10ng/L
80-120% Recovery for NIST1641d
and 75-125% for all other CRMs,
RSD<24%
RSD of Calibration Response Factor
<15%
75-125%
Less than 0.50ng/L (0.25ng/L for DOD
projects) (individually)
< 24% RPD
71-125% Recovery
< 24% RPD
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Document Title:
Mercury in Water by Oxidation, Purge & Trap
and CV-AFS (EPA Method 1631, Rev E]
Eurofins Document Reference:
EFGS-SOP-137-R02
Appendix A: Example - Standard Operating Procedure Training Record
By signing this document, I the employee, certifies to have read, understood and agreed to follow
the test method and quality procedure as described in this procedure.
Reading of SOP FGS-137.02:
Mercury in Water by Oxidation, Purge & Trap and CV-AFS (EPA Method 1631, Rev E.
SOP name and Revision number
Employee name (print)
Employee name (sign)
Date:
Supervisor name (sign)
Date:
Initial SOP Training (leave blank if not applicable)
Initial reading of method and training
1. Read method
2. Observe the method
3. Detailed review of method and associated literature
4. Supervised practice of method with trainer
5. Unsupervised practice of the method with trainer
6. Review of work with trainer and/or peer-review
7, IDOC to determine precision and accuracy
8. Determination of blanks
Initials
Date
Supervisor
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%y eiirafitis |
! Frontier Gioba! Sciences
Document Title:
Digestion Of Tissues for Total Mercury Eurofins Document Reference:
Analysis Using Nitric Acid and Sulfuric EFGS-SOP-01 1 -R05
Acids (70:30)
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FGS-SOP-011.05
Level 3
SOP
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Ryan Nelson
Dave Wunderlich and Patrick Garcia-Strickiand
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eurofins
Document Title:
Digestion of Tissues for Total Mercury
Analysis Using Nitric Acid and Sulfuric
Acids (70:30)
Eurofins Document Reference:
EFGS-SOP-Q11-RQ5
Table of Contents
1 Revision Log: ,, _ _ __ 4
2 Reference: ,, , , , , .....,,,, 4
3 Cross Reference: , , __ _ 4
4 Purpose: , , , , _ 4
5 Scope: _ __.__ 4
6 Basic Principles: ,.,....., 5
7 Reference Modifications: , ,.., 5
8 Definitions: , 5
9 Interferences: , ,,,,, Q
10 Safety Precautions, Pollution Prevention and Waste Handling: Q
11 Personnel Training and Qualifications: _ 7
12 Sample Collection, Preservation, and Handling: _g
13 Apparatus and Equipment: , _ __Q
14 Reagents and Standards: , _ g
15 Procedure: _____ ^0
16 Calculations: , , _____ -JQ
17 Statistical Information/Method Performance: , _,i
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Document Titfe:
Digestion of Tissues for Total Mercury
Analysis Using Nitric Acid and Suifuric
Acids (70:30)
Eurofins Document Reference:
EFGS-SOP-011-RQ5
Approvals:
Prepared by:
Date:
^Itoift
Approved by: : :L-
Date:
Approved by:
Date:
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v^eurofins j
Document Title:
Digestion of Tissues for Total Mercury
Analysis Using Nitric Acid and Sulfurie
Acids (70:30)
Eurofins Document Referenca;
EFGS-SQP-011-R05
1 Revision Log:
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14.3, 14.4
17.3
18.2-18.5
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Changed company name from Frontier Global Sciences
Eurofins Frontier Global Sciences.
Reformatted document to new corporate specifications.
Updated spiking ievefs for the matrix spike
Updated max contamination levels of reagent acids
Replaced MDL with LOD
Updated QC limits
Incorporated QA MOC 201 1-007
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to
2 Reference;
2.1
2.2
2.3
Chemical Hygiene Plan, Eurofins Frontier Global Sciences, current version.
EPA Method 1631, Revision E: Mercury in Water by Oxidation, Purge and Trap, and
Cold Vapor Atomic Fluorescence Spectrometry, 2002.
National Environmental Laboratory Accreditation Conference, NELAC Standard
September 8, 2009,
Department of Defense Quality Systems Manual for Environmental Laboratories,
prepared by DoD Environmental Quality Workgroup, Final Version 4.2, October 2010
2.4
3 Cross Reference:
Document
SOP FGS-003
SOP FGS-008
SOP FGS-038
SOP FGS-094, App F
SOP FGS-099
SOP FGS-121
SOP FGS-155
Document Title
Pipette Verification, Calibration and Maintenance
Ultra Clean Aqueous Sample Collection
Data Review and Validation
^tandard Operating Procedure Training Record
Waste Disposal Procedure for Client Sample Waste
Determination of Total Mercury in Various Matrices by
Fluorescence Spectrometry (EPA Method 1631E)
Flow Injection Atomic
Calibration of Volumetric Dispensers
4 Purpose;
4.1 The purpose of this Standard Operating Procedure (SOP) is to describe the method for
digesting biological tissue samples prior to analysis by CV-AFS for total mercury.
5 Scope:
5.1 This method is for the preparation, of biological tissue samples for the determination of
total mercury at concentrations less than 1 ng/g. Through the analysis of smaller
digestate aliquots, contaminated tissues of up to 10,000 ng/g can be directly
measured. Using clean handling techniques and low-level reagents, the typical
detection limit for samples prepared by this method is less than 1 ng/g.
5.2 Total mercury, as defined by this method, is all HNO3/H2S04/BrCI-oxidizable mercury
forms and species found in tissue matrices. This includes, but is not limited to, Hg(ll),
Hg(O), HgS, strongly organo-complexed Hg(II) compounds, adsorbed particulate Hg,
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4>*eurofins j
Document Title:
Digestion of Tissues for Total Mercury
Analysis Using Nitric Acid and Su If uric
Acids (70:30)
Eurofins Document Reference:
EFGS-SOP-011-R05
and several covatently bound organo-mercurials (i.e., CH3HgCl, (CH3)2Hg, and
C6H5HgOOCCH3).
6 Basic Principles:
6.1
Samples are collected using clean sample handling protocols into commercially
available clean glass containers with Teflon-lined caps (i.e., l-Chem glass jars) or 125
ml or 250 ml HOPE jars. Freezing (< -15ฐC) preserves tissue samples until sample
preparation is performed.
6.2
6.3
A subsampie of homogenized sample is digested with 10 ml of 70:30 HNO3/H2SO4.
The digested sample is diluted up to 40 mL with 10% (v/v) BrCL
7 Reference Modifications:
7.1 No significant modifications were made to this method.
8 Definitions;
8.1 Batch - no more than 20 client samples grouped for preparation. 3 Preparation Blanks,
1 CRM or 1 LCS/LCSD (or BS/BSD) set and 1 MD are prepared per every 20 samples;
1 MS/MSD set is prepared for every 10 samples.
8.2 Celsius (C), conversion of Celsius to Fahrenheit: (C * 1.8) + 32.
8,3 Fahrenheit (F), conversion of Fahrenheit to Celsius: (F - 32) * 5/9.
8.4 Method Detection Limit (MDL) - the limit derived from an exercise as described in 40
CFR, Part 136, Appendix B. The exercise produces a defined value that is the
minimum concentration that can be measured and reported with 99% confidence that
the analyte concentration is greater than zero from a given matrix.
8.5 Certified Reference Material (CRM) - a standard of known composition that is certified
by a recognized authority and representing a sample matrix. It is used to verify the
accuracy of a method.
8.6 Laboratory Control Sample {LCS) and Laboratory Control Sample Duplicate (LCSD), is
a sample containing known concentrations of the analytes of interest that is taken
through the entire preparation and analysis process in the same manner as the
samples to monitor complete method performance. A Certified Reference Material
(CRM) is preferred as the LCS, but a blank spiked sample also meets the requirement.
8.7 Preparation Blank (BLK) - Method blanks consist of the same reagents used to digest
the samples, in the same volume or proportion, and are carried through the complete
sample preparation and analytical procedure. Teflon boiling chips are added to the
preparation blanks.
8.8 Matrix Duplicate (MD) - a representative sample is selected and digested in the same
manner. This QC sample will indicate sample homogeneity on the analytes of interest
8.9 Matrix Spike (MS) and Matrix Spike Duplicate (MSD) - a representative sample is
selected and spiked with a secondary source at two to five times the ambient
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Document Title:
Digestion of Tissues for Total Mercury
Analysis Using Nitric Acid and Sulfuric
Acids (70:30)
Eurofins Document Reference:
EFGS-SOP-011-R05
concentration or at two to five times the MRL, whichever is greater. These QC samples
will indicate sample matrix effects on the analytes of interest,
8.10 May: This action, activity or procedure is optional.
8.11 May Not: This action, activity or procedure is prohibited.
8.12 Shall: This action, activity or procedure is required.
8,13 Should: This action, activity or procedure is suggested, but is not required.
9 Interferences:
9.1 Due to the high levels of halogens (i.e., iodine) typically found in tissue digestates, it is
recommended that aliquots of no more than 5.0 ml_ of the digestate be analyzed.
Otherwise, soda-lime traps may be overloaded and the gold traps may lose the ability
to amalgamate and retain mercury,
9.2 The high acidity and halogen levels that are found in tissue digestates necessitate the
changing of the bubbler water after every 10 ml of digestate analyzed. Failure to do
so can lead to low recoveries that would be reflected in the analysis of QC samples.
10 Safety Precautions, Pollution Prevention and Waste Handling:
10.1 Personnel will don appropriate laboratory attire according to the Chemical Hygiene
Plan. This includes, but is not limited to, laboratory coat, safety goggles, and nitrile
gloves under clean gloves.
10.2 The toxicity or carcinogentcity of reagents used in this method has not been fully
established. Each chemical should be regarded as a potential health hazard and
exposure to these compounds should be as low as reasonably achievable. Chemists
should refer to the MSDS (Material Safety Data Sheets) for each chemical they are
working with.
10.2.1 Note: Use particular caution when preparing and using BrCi, as it releases
extremely irritating, corrosive fumes similar in effect to free chlorine. Always
handle this reagent in an approved fume hood.
10,2.2 Note: Use particular caution when preparing and using the Nitric/Sulfuric Mixture.
Always handle this reagent in an approved fume hood.
10.3 AH personnel handling environmental samples known to contain or to have been in
contact with human waste should be immunized against known disease-causative
agents. Eurofins Frontier will reimburse the expense of Hepatitis A and B
immunizations for any laboratory staff member who desires this protection.
10.4 Nitric acid (HNO3): Corrosive. Strong oxidizer. Contact with other material may cause a
fire. Causes eye and skin burns. May cause severe respiratory tract irritation with
possible burns. May cause severe digestive tract irritation with possible burns. For
more information see MSDS.
10.5
Sulfuric acid (H2SO4): Corrosive. Causes eye and skin burns. May cause severe eye
irritation with possible burns. May cause severe respiratory tract irritation with possible
burns. May cause severe digestive tract irritation with possible burns. Cancer hazard.
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4?eurofitis j
Document Title:
Digestion of Tissues for Total Mercury
Analysis Using Nitric Acid and Sulfuric
Acids (70:30)
Eurofins Document Reference:
EFGS-SOP-011-R05
Animal studies suggest this acid may cause fetal effects. May cause kidney damage.
May cause lung damage. May be fatal if inhaled. Hygroscopic. Strong oxidizer.
Contact with other material may cause a fire. For more information see MSDS.
10.5.1
Eyes: Get medical aid immediately. Do NOT allow victim to rub or keep eyes
closed. Extensive irrigation with water is required (at least 30 minutes).
10.5.2 Skin: Get medical aid immediately. Flush skin with soap and water for at least 15
minutes while removing contaminated clothing and shoes. Wash clothing before
reuse. Destroy contaminated shoes.
10.6 See Eurofins Frontier Global Sciences Chemical Hygiene Plan (CHP) for general
information regarding employee safety, waste management, and pollution prevention.
10.7 Pollution prevention information can be found in the current Eurofins Frontier Global
Sciences Chemical Hygiene Plan (CHP), which details and tracks various waste
streams and disposal procedures.
10.8 All laboratory waste is accumulated, managed, and disposed of in accordance with all
federal, state, and local laws and regulations. Any waste generated by this procedure
should be disposed of according to SOP FGS-099 "Waste Disposal Procedure for
Client Sample Waste," which provides instruction on dealing with laboratory and client
waste.
11 Personnel Training and Qualifications:
11,1 An analyst must perform an initial demonstration of capability (IDOC) that includes four
replicates of a secondary source before being qualified to analyze samples without
supervision. Continuing DOC will be maintained and monitored via performance on
CRMs and other QC samples, as well as obtaining acceptable results on proficiency
testing exercises.
11,2 The analyst/laboratory technician must have read this SOP and other relevant SOPs
and have the training documented on the applicable form(s). The analyst may be
questioned on SOP by supervisor(s) and/or trainers.
11.3 Training is documented by the employee and supervisor, and is kept on file in the QA
Office. The employee must read, understand, and by signing the training document,
agree to perform the procedures as stated in all Standard Operating Procedures
(SOPs) related to this method.
11.4 Reading of the SOP must be documented on the correct form such as "Standard
Operating Procedure Training Record," Appendix F in FGS-094, the last page of this
SOP, Appendix A "Standard Operating Procedure Training Record" or a similar
document"
11.5 All employees must also, on a yearly basis, read the Quality Manual (QM), and
complete the yearly Ethics training.
11,6 All training documents including IDOCs, CDOCs, SOP reading, Initial QA orientation,
and Ethics training are stored by the Quality Assurance Manager in the employees
training file for ten years after the employee is no longer working for Eurofins Frontier
Global Sciences.
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frontier GSofoal Sciences
Document Title:
Digestion of Tissues for Total Mercury
Analysis Using Nitric Acid and Sulfuric
Acids (70:30)
Eurofins Document Reference:
EFGS-SOP-011-RG5
11.7 Chemical Safety Training, Compressed Gas Training, Chemical Hygiene Plan
documentation, and Shipping of Hazardous goods, are stored by the Health and
Safety Officer for ten years after the employee is no longer working for Eurofins
Frontier Global Sciences.
12 Sample Collection, Preservation, and Handling:
12,1 Samples must be collected in accordance with established ultraclean sampling
techniques (see FGS-008 "Ultra Clean Aqueous Sample Collection"). Samples may
be in commercially available clean glass containers with Teflon-lined caps (i.e., l-Chem
glass jars), or 125 ml or 250 ml HOPE jars.
12.2 Tissue sample preservation - The tissue sample must be frozen in the sampling
container at less than -15ฐC or freeze-dried and stored at room temperature. The
holding time for tissue samples is 1 year.
12.3 Just prior to digestion, samples are thawed and if necessary homogenized. The
sample is well mixed to ensure the most representative sample possible.
13 Apparatus and Equipment:
13.1 LIMS ~ Element, version 5.85 or higher; Computer - Windows XP, 7 or 8
13.2 40 ml or 20 ml l-Chem Vials: Borosiiicate glass, series 300 viais with Teflon-fined
septa in lids. The size used depends on the amount of sample available. The vials are
volumetrically accurate to ฑ 0.5 ml when filled such that the meniscus is just to the
bottom of the vial neck. The person performing the preparation should verify this.
13.3 Hot plate: A hot plate with the ability to achieve and maintain a temperature of 75 ฐC.
13.4 Pipettors: All-plastic, pneumatic, fixed volume and variable pipettes in the range of 5
to 10 mL Pipettes are to be calibrated weekly according to SOP FGS-003 and
13.5
FGS-155.
Clean hood.
13.6 Analytical Balance: A laboratory analytical balance capable of weighing to ฑ 1 mg, with
documented calibration.
13.7 Calibrated thermometer: Submerged in water in a 20 ml i-Chem vial. This vial is
placed on the hotplate during the digestion process. The analysts must record the
actual digestion temperature and the serial number of the thermometer used in the
digestion logbook.
13.8 Sample Digestion Log.
13.9 Stainless steel tools for homogenization
13.10 Tissue Homogenization Log.
13.11 Disposable spatula.
13.12 Teflon boiling chips.
13.13 Teflon reflux cap to fit the 40 ml and 20 ml l-Chem vials.
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4? eu refills |
Document Title:
Digestion of Tissues for Total Mercury
Analysis Using Nitric Acid and Sulfuric
Acids (70:30)
Eurofins Document Reference:
EFGS-SOP-011-R05
14 Reagents and Standards:
14.1 Reagent Water: 18 MQ ultra-pure deionized water starting from a pre-purified (distilled,
R.O., etc.) source. As a final mercury and organic removal step, the activated carbon
cartridge on the 18-MQ system is placed between the final ion exchange bed and the
0.2 Mm filter.
14.2 Nitric Acid (HNOs): Trace metal purified reagent-grade HNO3 is pre-analyzed and lot
sequestered. Several brands (Baker, Fisher, Omnitrace) have been found to have lots
with acceptably low levels of trace metals. This reagent should be from a lot number
that has been previously tested to be low for the analytes of interest. This reagent shall
be entered into LIMS and the expiration date is set to the same as the manufacturer's
expiration date.
14.3 Sulfuric acid (H2SO4) - Trace metal purified reagent-grade H2SO4 is pre-analyzed to <
50 ng/L Hg and lot sequestered before purchase. This reagent shall be entered into
the LIMS and is considered stable until the expiration date on the bottle (set by the
manufacturer,
14.4 Nitric/Sulfuric Acid Mixture: Carefully add 300 ml of pre-analyzed, (ow mercury (< 50
ng/L) concentrated sulfuric acid to 700 mL of pre-analyzed, low mercury concentrated
nitric acid to a pre-marked Teflon bottle. Stir constantly. This reagent shall be entered
into the LIMS with an expiration date of six months. CAUTION: THIS MIXTURE
BECOMES VERY HOT AND EMITS CAUSTIC FUMES.
14,5
14.6
Potassium Bromide (KBr), neat: this reagent is pre-certified by the vendor to be low in
mercury and is entered into the LIMS with a five year expiration date.
Potassium Bromate (KBrO3), neat: this reagent is pre-certified by the vendor to be low
in mercury and is entered into the LIMS with a five year expiration date.
14.7 0.2N Bromine Monochloride (BrCI):
14.7.1
14.7.2
14.7.3
37.5 g of KBr is added to a 2.5 L bottle of concentrated HCl (pre-analyzed and
below 5 ng/L Hg). The bottle is inverted in a fume hood to mix the acid and KBr.
The solution sits overnight, allowing the KBr to dissolve,
27.5 g of KBrOS (certified to be low in Hg) is slowly added to the acid. As the
KBrO3 is added, the solution should go from yellow to red to orange.
CAUTION: This process generates copious quantities of free halogens {CI2,
Br2, BrCI) which are released from the bottle. Add the KBrO3 SLOWLY in a
well operating fume hood,
Loosely cap the bottle and allow to sit for 30 minutes (in a fume hood) before
tightening. Once tightly capped, invert bottle to make sure all of the solids go into
solution.
14.7.4 This reagent shall be entered into the LIMS with a six month expiration date.
14.8 10% (v/v) of 0.2N BrCI: 200 mL of 0.2N BrCI is diluted up to 2,0 L with reagent water in
a clean, empty HCl bottle. This bottle is fitted with a 10 mL repipettor. The expiration
time for this reagent is set by default to six months in the LIMS.
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4>*eurof!n$
Document Title:
Digestion of Tissues for Total Mercury
Analysis Using Nitric Acid and Suifuric
Acids (70:30)
Eurofins Document Reference:
EFGS-SOP-011-R05
15 Procedure;
15.1
If needed, the sample is dissected and homogenized with acid-washed stainfess steel
tools.
15.11 The process used for homogenization, number of samples, work order number,
client name, and initials of the technician are entered into the Tissue
Homogenization Log,
15.2 Weigh at least a 0.5 g aliquot (but not more than 0.65 g) for common and unknown
samples, and up to 1.0 g ฑ 0.025 g for low-level or large-grain samples. This aliquot is
placed into a 40 ml l-Chem glass vial.
15.2.1 tf limited sample is available, use 20 ml_ glass vials and drop the initial mass of
the samples to 0.25g ฑ 0.025 g.
15.2.2 It is imperative that all biological tissue samples are thoroughly homogenized.
The importance of representativeness cannot be understated.
15.2.3 Batch requirements for this digestion limit the number of samples to 20. In each
batch, there must be three method blanks (BLKs), a Blank Spike and Blank Spike
Duplicate (BS/BSD) that is preferably a Certified Reference Material (CRM) or a
Laboratory Control Spike (LCS, prepared at 8 ng/g), a Matrix Duplicate (MD), and
a Matrix Spike and Matrix Spike Duplicate (MS/MSD).
10.0 ml of 70:30 (v/v) HN03/H2SO4 solution is pipetted in and the sample is swirled.
Note: 5,0 ml of 70:30 (v/v) HA/O/H2SO4 solution is used for limited samples prepared
in 20 mL vials (15.2.1),
15.3
15.4 The vial is placed on a hot plate operating at 75ฑ5ฐC with a Teflon reflux can in place
instead of the vial's lid. An aluminum rack id often used to keep the vials from tipping
over while on the hot plate.
15.4.1 A calibrated thermometer submerged in water is placed in a 20 ml l-Chem vial.
This l-Chem vial with a calibrated thermometer is placed on the hot plate during
the digestion process. The analysts must record the actual digestion
temperature and the serial number of the thermometer used in the digestion
logbook.
15.5 After the samples start to reflux, the samples are heated at 75ฑ5ฐC for an additional 2
hours or until all organic matter is dissolved.
15.6 The samples are allowed to cool and are diluted to 40 ml {or to 20 mis for limited
sample digestions as described in 15.2.1) with a 10% (v/v) solution of 0.2N BrCI,
capped with their respective lids, and are thoroughly shaken. Sample digestates
should be allowed to settle prior to an aliquot being taken for analysis.
15.7 Analysis for total mercury is according to Eurofins Frontier SOP FGS-121.
16 Calculations:
16.1 This preparation procedure does not involve calculations.
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^eurofins i
Document Title:
Digestion of Tissues for Total Mercury
Analysis Using Nitric Acid and Sulfuric
Acids (70:30)
Eurofins Document Reference;
EFGS-SOP-011-R05
17 Statistical information/Method Performance:
17.1 Method Detection Limit (MDL) and Practical Quantitation Limit (PQL) studies are
based on 40 CFR 136, Appendix B. The MDL and PQL must be performed for each
analyte/rnatrix/preparation combination.
17.2 The Practical Quantitation Limit (PQL) is the reporting limit for this method and is
included as the lowest calibration point (2003 NELAC regulation 5.5.5.2.2.1.h.3). The
PQL is determined by running ten replicate samples with a concentration that wilt
produce a recovery of 70-130% for most analytes, but the recovery requirements are
analyte dependent. The PQL is referred to as the Method Reporting Limit (MRL) in
LIMS.
17.3 The current LOD value for Total Hg in tissue prepared by the Nitric and Sulfuric Acids
(70:30) Digestion is 0.16 ng/g, while the PQL is 0.8 ng/g.
17.4 Current LODs and PQLs are stored at: \Genera! and Admin\Quality Assurance\MDLs
& PQLs.
18 Quality Assurance/Quality Control:
18,1 Maximum Sample Batch Size: 20 samples.
18.2 Preparation Blanks: Minimum of three per batch. Each preparation blank must be less
than one-half the PQL for the method.
18.2.1 The preparation blanks are prepared with a similar mass of Teflon boiling chips
as the samples, with the same reagents, and put through the same preparation
process as the samples.
18,3 Certified Reference Material (CRM, representing the sample matrix when commercially
available); a Laboratory Control Spike (LCS) and Laboratory Control Spike Duplicate
(LCSD) prepared at 8 ng/g is used when a suitable CRM is not available: One per
batch in duplicate. The control limits are 77-123% recovery.
18.4 Matrix Duplicate (MD) Sample: One per batch. The control limit for the RPD is < 24%.
18.5 Matrix Spike/Matrix Spike Duplicate (MS/MSD) Samples: One set per 10 samples. The
control limits are 71-125% recoveries and an RPD of < 24%.
18.6 Follow the flow charts in SOP FGS-038 "Data Review and Validation" to determine if
any QC falling outside the established control limits can be qualified.
18.7 All of the quality control limits for the analysis method are included on the "Data
Review Checklist
18.7.1 The data review checklists are located at: \\cuprum\General and Admtn\Quality
Assurance\Data Review\Current Data Review Checklists.
19 Corrective Action:
19.1 Limiting the source of contamination/error in the preparatory stage can decrease QC
problems during analysis. Limiting such contamination/error sources may include:
cleaning all digestion tools in a 10% HCI solution, ensuring all samples are thoroughly
homogenized, changing gloves whenever appropriate, flushing repipettors at least
Revision: 5
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Document Title:
Digestion of Tissues for Total Mercury
Analysis Using Nitric Acid and Sulfuric
Acids (70:30)
Eurofins Document Reference:
EFGS-SOP-011-R05
three times before dispensing into vials and, in general, following ultra-clean
procedures.
19.2 A failing QC point does not necessary fail the entire dataset. If upon analysis a QC
sample is out of control, some investigation must be performed to assess if the
difficulties are related to matrix effects. The cause and method of determining the set's
failure must be documented on the checklist and in the MMO notes, and the Group
Supervisor shall be informed. See SOP FGS-038 "Data Review and Validation" for
flow charts regarding analytical issues,
19.3 Additional corrective actions are listed in the SOP for total mercury analysis (Eurofins
Frontier SOP FGS-121).
20 List of Attachments
Appendix A: Example - Standard Operating Procedure Training Record
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Document Title:
Digestion of Tissues for Total Mercury
Analysis Using Nitric Acid and SuSfurie
Acids (70:30)
Eurofins Document Reference:
EFGS-SOP-011-R05
Appendix A: Example - Standard Operating Procedure Training Record
By signing this document, I the employee, certifies to have read, understood and agreed to follow
the test method and quality procedure as described in this procedure.
Reading of SOP EFGS-011.05:
Digestion of Tissues for Total Mercury Analysis Using Nitric and Suifuric Acids (70:30).
SOP name and Revision number
Employee name (print)
Employee name (sign)
Date:
Supervisor name (sign)
Date:
Initial SOP Training (leave biank if not applicable)
Initial reading of method and training
1. Read method
2. Observe the method
3. Detailed review of method and associated literature
4. Supervised practice of method with trainer
5. Unsupervised practice of the method with trainer
6. Review of work with trainer and/or peer-review
7. IDOC to determine precision and accuracy
8. Determination of blanks
Initials
Date
Supervisor
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