S9L.
Region I, New England
U.S. Army Corps of
Engineers
New England District
REGIONAL IMPLEMENTATION MANUAL
for the
EVALUATION OF DREDGED MATERIAL
PROPOSED FOR DISPOSAL IN NEW
ENGLAND WATERS
Prepared by
U.S. EPA - NEW ENGLAND
and the
U.S. ARMY CORPS OF ENGINEERS,
NEW ENGLAND DISTRICT
September 2002
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9/5/02
SECTION PAGE
1. INTRODUCTION 1-1
2.. ADMINISTRATIVE REQUIREMENTS
2.1. Permit Requirements 2-1
2.2. Coordination 2-3
3. TIERED TESTING
3.1. Tier I - Existing Information 3-1
3.2. Tier II - WQ Criteria, TBP 3-1
3.3. Tier III - Biological Evaluation 3-1
3.4. Tier IV - Case-specific studies 3-1
4. SAMPLING METHODOLOGY
4.1. Development of a Sampling and Analysis Plan 4-1
4.2. Sample Collection 4-2
4.3. Sample Handling, Preservation and Storage 4-4
4.4. Sampling of Reference, Control Sediments and Water 4-5
4.5. Sample Documentation 4-7
5. PHYSICALJCHEMICAL TESTING OF SEDIMENTS
5.1. Initial Characterization of Sediment 5-1
5.2. Chemical Analysis of Sediment 5-2
5.3 Additional Physical Characterization of Sediment 5-4
5.4 Quality Control Measures 5-4
5.5 Data Reporting 5-6
6. WATER COLUMN EFFECTS EVALUATION
6.1 Tier II - Compliance with \f ter Qu I t Ci eria 6-1
6.2. Tier III - Water Column Toxicity 6-3
6.3 Quality Control Measures 6-4
6.4 Numerical Models for Initial Mixing Evaluations 6-5
7. BENTHIC EFFECTS EVALUATION
7.1. Tier Ill - Whole Sediment Toxicity 7-1
7.2. Tier Ill - Bioaccumulation Testing 7-3
7.3 Statistical Analysis 7-4
7.4 Quality Control Measures 7-5
7.5 Data Reporting 7-5
8. REFERENCES
9. APPENDICES
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LIST OF TABLES
TABLE PAGE
1. Parameters used for the physical characterization of sediments. 5-8
2. Metal contaminants-of-concern 1 analyticat methods-and-target - -
detection limits (dry weight) routinely analyzed in sediments. 5-9
3.Organic contaminants of concern, analytical methods and
target detection limits (dry weight) routinely analyzed in sediments. 5-10
4. Additional parameters used for the physical characterization of
sediments. 5-13
5. Required contaminants, recommended methods, target detection
levels and water quality criteria used in water quality criteria compliance
determination. 6-8
6. Organisms required for the water column bioassay. 6-10
7. Organisms required for the whole sediment toxicity and
bioaccumulation tests. 7-6
8. Chemical constituents and detection limits routinely used for
bloaccumulation evaluations of proposed dredged material. 7-7
9. Recommended statistical methods for biological testing. 7-10
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LIST OF FIGURES
FIGURE AFTER PAGE
1. Example Drawing of Area Proposed for Dredging 2-2
2. Generalized Coordination Procedure for Sediment Suitability
Determination 2-3
3. Generalized Tier Testing Process 3-1
4. Sediment Grain Size Gradation Graph 5-2
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LIST OF APPENDICES
I. Additional Priority Pollutants of Concern and Target Quantitation Limits
II. Quality ControlSummary-Sheets -
III. Forms for Atterberg Limits
IV. Procedures for Collection of Large Volume Water Samples
V. Sea Urchin Larval Toxicity Test Procedure
VI. Species-Specific Testing Conditions
VII. Pore Water Collection Procedure
VIII. Procedures for Addressing Ammonia Presence in Mysidopsis Sediment Toxicity Tests
(Elizabeth Southerlañd Memo to Mario P. Del Vicario, dated June 14, 1994)
IX. AED Laboratory Operating Procedure, Measurement of Total Lipids using Modified Bligh-
Dyer Method
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List of Abreviations
AED Atlantic Ecology Division, EPA Research Lab, Narragansett, Rhode Island
APHA American Public Health Association
ASTM American Society of Standards and Materials
CRM Certified Reference Material
CWA Clean Water Act
DM Dredged Material
DOA Department of the Army
ENG U.S. Army Engineering Form
EPA U.S. Environmental Protection Agency
FWS U.S. Fish and Wildlife Service
CC/MS Gas Chromatography/Mass Spectroscopy
ITM Inland Testing Manual
LC5O Median Lethal Concentration
LPC Limiting Permissible Concentration
LIS Long Island Sound
LQAP Laboratory Quality Assurance Plan
MAS Marine Analysis Section, New England District, Corps of Engineers
MDL Method Detection Limit
MLLW Mean Lower Low Water
MLW Mean Low Water
MPRSA Marine Protection, Research and Sanctuaries Act
NAE New England District, U.S. Army Corps of Engineers
NMFS National Marine Fisheries Service
NOAA National Oceanic and Atmospheric Administration
NYDEC New York Department of Environmental Conservation
PAH Polycyclic Aromatic Hydrocarbon
PCB Polychiorinated Biphenyl
PQL Practical Quantitat ion Limit
PSEP Puget Sound Estuary Program
ppb parts per billion
ppm parts per million
pptr. parts per trillion
QNQC Quality AssurancelQuality Control
RIM Regional Implementation Manual
RL Reporting Limit
SAP Sampling and Analysis Plan
SIM Selected Ion Monitoring
SRM Standard Reference Material
TBP Theoretical Bioaccumulation Potential
TOC Total Organic carbon
TQL Target Quantitation Limit
USACE U.S. Army Corps of Engineers
WQC Water Quality Criteria
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1. INTRODUCTION
This Regional Implementation Manual (RIM) presents sediment testing guidelines and
reporting requirements for applicants who wish to obtain a Department of Army permit from
the New England District (NAE) of the U.S. Army Corps of Engineers (Corps) for dredging
and the open water disposal of dredged material projects as well as federal navigation
projects. This guidance is consistent with national guidance (described below) and has
been approved by the U.S. Environmental Protection Agency (EPA) and the Corps in
cooperation with the U.S. Fish and Wildlife Service. (FWS), National Marine Fisheries
Service (NMFS) and the various permitting and environmental resource agencies of the five
coastal New England states: Maine, New Hampshire, Massachusetts, Rhode Island and
Con necticut.
This manual implements the national testing guidelines under Section 103 of the Marine
Protection, Research and Sanctuaries Act (MPRSA) (33 USC 1401 et seq.) and Section
404 of the Clean Water Act (CWA) (33 Usc 1344 et seq.). The MPRSA governs (1) all
disposal projects in New England ocean waters (seaward of the territorial sea baseline),
and (2) all Federal disposal projects of any amount, and those non-Federal disposal
projects exceeding 25,000 cubic yards in size, in Long Island Sound. In addition, Section
404 of the Clean Water Act regulates the disposal of dredged and fill materials into waters
of the U.S. landward of the territorial sea baseline and fill material within the territorial sea.
The guidance and requirements specified in this document will be used by the regulatory
agencies for all disposal activities subject to both Section 103 of the MPRSA (40 CFR Parts
227.6 and 227.13) and Section 404 of the Clean WaterAct (40 CFR Parts 230.60 and 61).
The MPRSA requires that operations involving the transportation and discharge of dredged
materials in ocean waters are to be evaluated to determine the potential impact to the
marine environment. The proposed disposal must be evaluated through the use of criteria
published by the EPA in Title 40 of the Code of Federal Regulations, Parts 220-228 (40
CFR 220-228). In accordance with Subsection 227.27 (b) of the regulations, EPA and
Corps developed a testing manual to define procedures for evaluating the suitability of
dredged material for ocean disposal that are based upon the testing requirements in the
regulations. National guidance is provided in the document “Evaluation of Dredged
Material Proposed for Ocean Disposal Testing Manual” commonly known and hereafter
referred to as the “Green Book” (EPA/USACE, 1991). This document replaces the first
testing manual “Ecological Evaluation of Proposed Discharge of Dredged Material into
Ocean Waters” (EPA/USACE, 1977).
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Under the CWA, the testing described here will assist the Corps of Engineers in making the
factual determinations regarding the effect of the discharge on the aquatic ecosystem and
compliance with the 404(b)(1) guidelines (40 CFR Parts 230.10 and 230.11). It implements
the manual uEvaluation of Dredged Material Proposed for Discharge in Waters of the U.S. -
Testing Manual (hereafter known as the “Inland Testing Manual” (or ITM)) (EPA/USACE
1998) as specified in 40 CFR Parts 230.60 and 230.61.
The 1991 Green Book and 1998 ITM provide new and improved testing methods and
contain revised guidance that reflects the regulatory experience gained since the 1977
testing manual was published. The Green Book, ITM and the companion quality
assurance/quality control (QAIQC) manual (EPNUSACE 1995) provide guidance on the
tiered-testing approach, sampling methodology, testing procedures, statistical methods and
QNQC. The purpose of the national guidance is to provide a framework to insure
consistency with the national ocean disposal regulatory program.
This Regional Implementation Manual (RIM) of the 1991 Green Book and 1998 ITM applies
the national guidance to the New England area, providing additional guidance agreed upon
by EPA Region I and NAE in cooperation with the above listed agencies. It replaces the
previous regional manual entitled “Guidance for Performing Tests on Dredged Material to
be Disposed of in Open Waters” (EPNNAE, 1989) which implemented the 1977 national
guidance document. This current manual provides needed supplementary guidance on:
permit application requirements, data and reporting requirements, a list of the contaminants
of concern, species for biological testing and specific procedural requirements agreed upon
by state and federal agencies. This will avoid unnecessary confusion and possible delays
or expenses through the submission of improper data. The reader should be aware that
this document does not attempt to duplicate or replace the detailed information contained
in the Green Book, ITM or the QA/QC manual except where noted. However, this
document is designed to be used in conjunction with these manuals providing additional
information or clarification when needed. Specific references to appropriate sections are
provided.
New and more advanced testing procedures and guidelines are continually being
developed and refined by the research and development laboratories of the EPA and
USACE. In addition, ongoing monitoring of designated disposal sites in New England
waters under the NAE Disposal Area Monitoring System (DAMOS) program can provide
effects-based feedback to the regulatory agencies allowing them to make more refined,
environmentally sensitive and efficient decisions regarding the acceptability of open water
disposal of dredged material. It can also provide the necessary information on whether any
site-specific criteria may be needed for a particular disposal site. As a result, this document
will be revised as needed to incorporate any necessary modifications of the testing
guidance.
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All application information, as discussed in the following sections, should be submitted by
applicants to the NAE Corps of Engineers office in Concord, Massachusetts. The Corps
will supply copies of the information to the other Federal agencies including EPA, FWS and
NMFS. Note that applicants are required to contact the appropriate state regulatory agency
directly. The applicant should know that additional information may be required on a
project by project basis.
Questions about this manual should be directed to:
U.S. Army Corps of Engineers
New England District
Regulatory Division
698 Virginia Road
Concord, MA 01742-275 1
(978) 318-8335
or
U.S. Environmental Protection Agency
Region I - New England
Office of Ecosystem Protection (CWQ)
One Congress Street, Suite 1100
Boston, MA 02114-2023
(617) 918-1553
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2.0 ADMINISTRATIVE REQUIREMENTS
2.1 General
When applying for a Department of the Army permit to dispose of dredged material into
open water, the applicant will be required by the NAE’s Marine Analysis Section to
provide the information indicated below. This information represents the first of four
information tiers used in the evaluation of dredged material. As discussed in Section 3,
it is possible that the evaluative processes in the remaining tiers (including biological
testing of the materials proposed for dredging) would be necessary.
Additional guidance on preparing applications can be found in the most recent edition of
NAE’s GUIDE FOR PERMIT APPLICANTS Useful general application information,
application forms, and sample project plans are available in the book (also available on
the NAE website at Www.nae.usace.army.mil). Contact the Regulatory Reception
Center to ensure you have the most current copy, phone number 978-318-8338.
Upon receipt of a permit application or pre-application inquiry, NAE will assign a
Regulatory Division Permits Project Manager who will serve as the applicant’s point of
contact throughout the review process. Information required for review by the Corps
and coordinating Federal and State agencies include, but are not limited to the following
data.
1. A statement describing why the proposed dredging is required, if it is “new, or
improvement” or “maintenance” dredging, and the area (square feet) and volume (cubic
yards) of material to be dredged. If the project is comprised of several “segments”(e.g.,
marina basin and an entrance channel), volumes and square footage information should
be provided separately for each. The volume estimate(s) should include The :- xirnum
estimate overaepth. Cuuent and proposed water depths should be descnbed based on
mean low water.
2. Alternative dispos.al locations with information in sufficient detail to evaluate their
potential for use. This would include a comprehensive survey of potential upland,
beneficial use and aquatic sites and information utilized in their evaluation.
3. The date when the project was last dredged and any previous sediment and
biological effects test data for this or nearby projects which would aid in typifying project
sediments. In the absence of any previous test data, a description of the bottom
material should be provided (e.g., rock, sand, vegetated, etc.).
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4. Information on locations of outfalls, non-point sources of contaminants and any
recent contaminant spills must also be included as described in Chapters 2 and 8 of the
ITM (EPNACE 1998). These data may be obtained from sources such as State water
pollution control agencies (e.g., Department of Environmental Protection), U.S. Coast
Guard and harbormasters. The sources of all information must be properly
documented.
5. Two legible copies of 8.5” X 11.0” drawings (Figure 1) including plan views and cross
sections of the area to be dredged with the following information noted:
-Depth of dredging referenced to MLW (mean low water) or MLLW ()
-Depth of proposed overdredge indicated
-Existing depths referenced to MLW or MLLW
-Square footage of the area to be dredged
-Outfall locations (industrial discharges, etc.)
-Non-point sources of contaminants (parking lots, oil storage tanks, hazardous
waste, etc.)
-Proposed and historical sampling locations (if appropriate)
-Readily identifiable landmarks and a project locus insert
6. Type of dredging equipment to be used (clamshell, hydraulic, etc.) and any unique
handling procedures to be used, such as a sealed clamshell or special runback controls.
7. Proposed dredged material disposal site. A locus sheet and detailed plan view
should be provided for each upland disposal site. The plans must include information
on any significant resources near the proposed site, limits of regulated areas if
applicable (e.g., wetlands and waterbodies), and for open water sites, whether the site
is a containment (i.e., depositional) or dispersive site.
If beach nourishment is proposed, plans should include high tide line, mean high water,
and mean low water, as well as a delineation of any vegetated areas and/or resource
areas in the vicinity (e.g., shellfish beds). Grain size information of the beach sediment
will likely be required for beach nourishment proposals, for comparison to the dredged
material, to insure they are compatible.
8. Dewatering site. If the material is to be dewatered, the following information should
be provided: a description of the site; a location map; plan view and cross section of the
dewatering site; calculations used to determine the capacity of the dewatering area; and
details of the methods to be used to control runback.
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FIG RE 1.
Example Drawing of Ares Propoaod for Dredging
DREDaNG PR IoIJ y
PERFORMED UNDER
COC PERMiT NO. CT..NHAV O..3 4 6
.J ::
2
2
21.4 4t.
- : :; Ill
WALKWAY
I DOCK
(a &O0LPHP4S
1•:•:-:•:•:•:•:•:•:•: -:•:•: : .y .‘: :•: - :•:•: :•:•: .:.:.: .: .y
, :::::::::::: ::::::::
/ / /e. .t
$
*1
“ .1w
GULF
MARINE
TERMINAL
•/TANK.
I. ELEVAIIONS ARE SHO w BASED
THE PLANE CF MEAN LOW WM .
2. PflDROOI APHJC SIm EY DATA P ropu rt
NATCHEZ AND ASSOC.. INC. DATED 11/22/93
FOR SEC11ON$ SEE 1. NO 3 OF 3
PROPOSED MMN1!j j DREDGING
GULF MARINE TERMINAL
NEW HA ,EN HARBOR
(NEW HA iEpi COUNTY), CT.
APPUCA11ON BY
GULF L OMSION
CIJMBERt,AND FARMS. INC.
PrIporid By: HMM Concord, MA
Salle 2 of 3 NO ,a1BER. 1993
4
—I
S -I CMPo
27.1
24.1
15.7
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2.2 Coordination
Early coordination with the NAE Regulatory staff (obtained through the Regulatory
Project Manager) is required to determine the sediment contaminant analyses needed,
and for development and approval of a project Sampling and Analysis Plan (SAP),
including the proper techniques, location and number of samples to be taken (See
Chapter 4).
Prior to any sampling and testing, the applicant must ensure submission to NAE of the
following:
1) A laboratory quality assurance plan (LQAP) providing standard quality
assurance/quality control procedures used by the contractor laboratory (unless
previously submitted) (Note: NAE and EPA are currently programmatically reviewing
LQAPs from laboratories performing testing for this regulatory program. A 24 month
grace period will be allowed for existing labs to submit LQAPs from the effective date of
this document. After that date, any new labs will be required to submit and have
approved LQAPs before any project data can be submitted.); and
2) A project Sampling and Analysis Plan (SAP) providing supplemental project-
specific information on the actual field sampling effort and associated quality assurance
measures (See Chapter 4).
The Federal permitting process (Figure 2) involves a comprehensive evaluative process
and requires a multi-agency review of dredged material suitability decisions, Of prime
importance is Region I, EPA which has the authority to review, approve, or propose
conditions upon permits for open water disposal, and the National Marine Fisheries
Service which reviews project evaluai.ive steps and provides information on endangered
species and Essential Fish Habitat and other biological resources. Early coordination
(application or pre-application) ensures that unnecessary delays do not become a factor
in the review process.
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Figure 2. Generalized Coordination Procedure for Sediment Suitability Determination
‘1
Submits Conducts Sampling;
Application for Submits Results in
Review Proper Format
4 !
r Permits
I Project
Manager
Reviews For
Completeness
and Logs In
Prepares Suitability
Determination; EPA
NMFS, FWS Concur
Testing Need: MAS Concurs
with Applicant’s Sampling and
Analysis Plan or Prepares Sampling
and Analysis Plan
Prepare Public Notice; Completes
Evaluation of Permft Application
I
Permits Project
Manager
iF
MAS, EPA, NMFS and
FWS for Review
‘I ,
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915102
3.0 TIERED TESTING
The tiered approach to testing provides increasing levels of investigative intensity to
generate the information necessary to evaluate the proposed disposal of dredged
material at an open water site. It provides for optimal use of resources by focusing the
least effort on dredging operations where the potential (or lack thereof) for unacceptable
adverse impact is clear, and expending the most effort on operations to determine the
potential for impact. This approach is described in detail in Chapters 4-7 of the 1991
Green Book and Chapters 3-7 of the ITM. These chapters should be read thoroughly in
either manual, depending upon the jurisdiction, to ensure a full understanding of all
tiered testing requirements. A brief description of the tiered testing approach is
presented below and illustrated in Figure 3. Prior to undertaking any testing, applicants
must coordinate with their Corps Project Manager.
3.1. Tier! - Review of Existing Information and Identification of Contaminants of
Concern.
The purpose of Tier I evaluations is to determine if existing information on proposed
dredged material is sufficient to show compliance with regulations and to determine
contaminants of concern. A comprehensive review of existing and readily available
information is required to make this determination, If existing test data are considered
inadequate to evaluate the proposed project, new sediment chemical and biological
testing is required.
3.2. TIer II - Water Column and Potential Bloaccumulation Analyses.
Tier II consists of evaluation of water quality criteria (WQC) compliance using a
numerical based mixing. model (Appendix B, Green Book; Appendix C, ITM) and an
evaluation for potential bioaccumulation using calculations of Theoretical
Bioaccumulatjon Potential (Section 5.2 of the ITM) for non-polar contaminants of
concern. At this time, the TBP is not used for projects subject to MPRSA (Green Book).
3.3. TIer III - Toxicity and Bioaccumulation Testing.
Tier Ill testing is used to provide data that allows an impact assessment of the
contaminants of concern through use of toxicity and bioaccumulation tests with
appropriatejensitive organisms (see Tables 6 and 7 for test organisms). Both water
column toxicity testing and benthic toxicity testing are required. Bloaccumulation testing
is used to determine the potential for uptake of sediment contaminants at the disposal
site by benthic organisms.
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Figure 3. Generalized Tiered Process for Review of Dredging Projects
Does Dredged Material (DM)
I Meet the Testing
j Exclusionary Cnteria (40
I CFR 227.13(b) or 230.60
TIER I
I N Information
° Sufficient to Make a
IL{ Decision?
TIER II
No
‘I,
I
Evaluate Potential Water
J
I
I
L
Column Impact and
Compliance with Water Quality
Criteria
J
t
Evaluate Potential
Bloaccumulation of
Non-polar Contaminants
(Section 404 Projects
Evaluate Water
Column Toxicity
TIER IlI-lV
Does DM
Meet All
T-E
_________ Other
Criteria?
Yes
Yes
Proceed With Remaining
L Regulatory Requirements 1
I
Evaluate Benthic
Ir
‘I,
J
Specify propriate
Alternative or
Management Action
(Section 404 Only)
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915/02
3.4. Tier IV - Long Term Bioassays and Bioaccumulation Tests, Risk Evaluations and
other case-specific testing/evaluations.
Under unusual circumstances, such as when a unique resource or resource area is
involved, it may be necessary to evaluate long-term effects of proposed dredged
material on appropriate sensitive aquatic organisms and human health risks. A risk
assessment, prepared by EPA may be required to interpret bioaccumulation results.
Because of the limited availability of appropriate and widely accepted procedures, each
test is selected to address specific concerns of each disposal operation (Section 7.1
Green Book; and Section 7 of the ITM). In a situation such as described, extremely
close coordination with Region I, EPA and NAE in all aspects of Tier IV testing is
required.
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4.0. SAMPLING METHODOLOGY
The importance of a well-designed sampling program is underscored by the fact that an
evaluation of the potential impacts of a proposed dredging project is only as complete
and reliable as the sampling upon which it is based. The quality of information gathered
through the tiered testing process is impacted by the following sampling related factors:
a) collecting representative samples; b) using appropriate sampling techniques; and c)
protecting or preserving the samples until they are tested. It is the responsibility of the
applicant to ensure that samples taken for a proposed project meet the Quality
AssurancelQuality Control (QA/QC) requirements presented below and discussed in
Chapter 8 of the Green Book (EPA/USACE 1991), Chapter 8 of the Inland Testing
Manual (ITM) (EPA/USACE 1998), and the Quality Assurance manual (EPA/USACE
1995). Failure to meet these requirements or follow any specified procedure
without NAE approval will likely cause rejection of the testing results.
Within 24 months of the effective date of this manual, each Laboratory must have an
approved Laboratory Quality Assurance Plan (LQAP) (see Chapter 2) on file with the
Corps in order for its sampling and test data and analysis to be accepted for permit
applications or for proposed Corps federal dredging and disposal projects. As
mentioned in Section 2, after that date, any new labs will be required to submit and
have approved LQAPs before any project data can be submitted.
4.1 Development of a Project Sampling and Analysis Plan (SAP)
In addition to the LQAP, applicants must have a project Sampling and Analysis Plan
(SAP) (see Chapter 2) which together make up the Quality Assurance Project Plan.
Applicants may submit a proposed sampling plan for approval to NAE, or request a SAP
be prepared by NAE based on submittal of project plans. The plan must be coordinated
with the appropriate state agencies. NAE wilt develop and/or approve the SAP in
coordination with the federal agencies (and state agencies if appropriate). NAE will
provide the approved SAP to the applicant, including the number and location of
samples, the required analytes, target quantitation limits (TQL) (see below) and other
project specific information supplemental to the LQAP. The approved SAP must be
implemented by the applicant. Any changes to the approved SAP must be submitted to
and accepted by NAE and be approved in writing prior to sampling.
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Please note that applicants should not, under any circumstances, undertake field
sampling and analysis without first coordinating with the Corps and receiving an
approved SAP from the Corps.
The following data must be included with the proposed project SAP:
- a brief project description, contract lab name/address and verification that
the LQAP is on file with NAE;
- reference/disposal site location (see below);
- station-specific sampling procedures (including sampling gear and
proposed positioning methodology) and description;
- sample handling/storage procedures; and
- analytical procedures and detection levels (see Chapter 5).
Following approval of the SAP and concurrent with submission of analytical results to
the Permits Project Manager, latitudes and longitudes (using NAD 83) for station
locations must be provided to the Corps by the applicant.
4.2 Sample Collection
1. Sediment samples must be collected according to the approved SAP. In the
instances where vertical grain size homogeneity exists and the project depth is
less than 2 feet, a grab sampler can be used if agreed by the Corps prior to
field sampling. A core sampler should be employed in al other cases to ensure
the samples are representative of materials to dredging depth, including
expected overdepth. To ensure an adequate sample is representative of a
project, NAE must approve the sampling apparatus. The type of equipment used
to collect the samples should be noted as part of the project record. For
example, if coring was used, the type of corer (gravity, vibracore, split spoon,
borings, etc.) and the core liner (polycarbonate or butyrate, etc.) should be added
to the field documentation. Core logs should be provided, along with narratives
describing relative grain sizes, color, odor, strata, core length and depth of
penetration along with other pertinent sediment sampling observations.
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2. In instances where significant (at least 2.0 ft) vertical stratification is evident in
samples, subsampling and testing of each layer may be required to adequately
characterize the materials. If the stratum is less than 2 ft, then the sample may
not be large enough to meet minimum sample requirements for physical and
chemical analyses. A wider diameter core or a grab, for example, will provide for
a larger sample. Information may be necessary for disposal management
decisions, such as the necessity for capping and the availability and volume of
uncontaminated capping materials from within the project site. Decisions as to
the need and type of vertical subsampling must be coordinated with NAE. If
there is no means or time for the field crew to communicate with NAE, the
subsamples should be taken back to the lab and analyzed for grain size, so that
NAE and the applicant can determine if the cores should be vertically
composited. In this case, collection of extra sediment within each stratum is
advised to ensure an adequate sample to run additional analyses if segmentation
is warranted. The goal is to provide the best possible characterization of
the material to avoid overestimating the amount of contaminated material
that may require special and likely more expensive disposal
considerations.
3. In situations where grain size analyses show samples to be comparable and
samples represent a similar project segment(s), compositing of samples could
occur. In all instances where compositing is contemplated and NAE has required
grain size analyses, NAE must review grain size data prior to any compositing,
and will make the final decision on any compositing scheme. Should
compositing be allowed, the individual samples making up the composites must
be archived by the testing laboratory until results of analyses have been
reviewed by NAE.
4. Care should be taken to avoid contamination from sampling gear, grease, ship
winches or cables, airborne dust, vessel engine exhaust, cross contamination
and improper subsampling procedures. Engines should be shut off during
sampling, if possible. If not possible (due to boat traffic, type of workboat,
currents, etc), then the sampling effort should be performed upwind of the
exhaust. It is recommended that core extrusion and sample mixing be performed
in the laboratory. However, if on-board mixing is necessary, this effort must be
performed away from exhaust fumes and any other sources of contamination. In
addition, care must be taken to avoid cross contamination. All core samplers or
other sampling devices must be appropriately decontaminated between samples.
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The applicant must ensure that the workboat has room to store cores vertically
out of the way from contamination and disturbance.
5. A sufficient sediment mass must be collected to meet the objective(s) of the
sampling program. A minimum of approximately 1000 grams of sediment per
sample must be collected for bulk physical and chemical analyses. The mass of
sediment will vary with grain size, density, core depth/diameter and should be
assessed before sampling to ensure adequate mass. It should be noted other
types of analyses require higher masses, e.g., bioaccumulation test which needs
a minimum of 7,500 grams (see Chapter 7, Section 7.2). Material must be
available for analyses and for partitioning of samples to meet archiving
requirements cited in Table 5 of the EPNCorps document entitled QAJQC
Guidance for Sampling and Analysis of Sediments, Water and Tissue for
Dredged Material Evaluations, Chemical Evaluations (EPNUSACE, 1995). The
guidance specified in Methods for Collection, Storage, and Manipulation of
Sediments for Chemical and Toxicological Analyses (EPA 2001 d) shoUld also be
consulted. -
6. The project sampling must be taken at the precise locations required by the NAE
approved SAP. Vessel positioning must be determined, using any number of
techniques including GPSI Loran and surveying equipment. GPS systems need
to be calibrated using known references. In all cases, NAE requires that sample
location latitudes and longitudes be recorded and provided to NAE for each
sampling location for compatibility with the NAE regional database. All data
should be reported in NAD 83 decimal minutes. Locational information for each
sampling point should be recorded in-field on a Station Location Log, Sediment
Sampling Log or similar document and be included as part of the data QA/OC
portion 01 the analytical results. Examples of these types of documents are
included in Appendix A of EPA/USACE (1995).
4.3 Sample Handling, Preservation and Storage
1. The applicant is responsible for ensuring that the sampling, handling and
preservation and storage procedures and the applicable quality assurance/quality
control measures are followed for both sampling and analysis. These
procedures must be adequately described in the approved SAP and the LQAP.
The LQAP must be on file with the Corps in order for test data and analysis to be
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accepted. These Plans are based on the EPNUSACE QA/QC Guidance Manual
dated 1995. The guidance specified in Methods for Collection, Storage, and
Manipulation of Sediments for Chemical and Toxicological Analyses (EPA
2001d) should also be consulted.
2. Samples are subject to chemical, biological and physical changes as soon as
they are collected. It is therefore imperative that, from initiation of collection
activities until samples are analyzed, all applicable Quality Assurance and Quality
Control (QNQC) procedures are followed.
3. Sample preservation should be accomplished onboard the collecting vessel
whenever possible. If final preservation cannot occur onboard, an interim
preservation technique that preserves the sample integrity should be employed.
Onboard refrigeration can be accomplished with coolers and ice while samples
that are to be frozen can be placed in coolers with dry ice. Sediment samples for
biological analysis should not be frozen but preserved at 40 C. No samples
should be allowed to dry. Additional information is given in Chapter 8 of the
Green Book (EPNUSACE 1991) or the QA/QC guidance manual (EPA/USACE,
1995).
4. In general, careful choice of sampling gear and containers should be made for
each group of chemicals analyzed to avoid sample contamination. Prior to
contact with samples, equipment and containers should be cleaned and rinsed.
Specific methodologies and containers are discussed in EPA (2001) and
EPA/USACE (1995). Labels for the containers must be able to withstand
environmental extremes and remain legible.
5. Sample containers should be filled to the top unless the sample is to be frozen, in
which case room for expansion must be allowed. If subsamples are to be taken
from the container, the container is best left about 3/4 full to allow for proper
stirring.
6. Work should start as soon as possible on sediments so as not to exceed the
holding time requirements (as exhibited in EPAJUSACE (1998)). The time
between sample collection and analysis should be minimized to maintain the
integrity of the sample. The longer the sample is stored, the more difficult it
becomes to accurately assess sample results. For example, over time a
relatively low contaminated sample can become increasingly toxic to bioassay
organisms (due to ammonia or other constituents).
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4.4 Sampling of Reference Sediments, Control Sediments and Water
Test procedures are conducted on the control and reference sediments in the same way
as on the dredged material proposed for open water disposal.
A. REFERENCE AND CONTROL SEDIMENTS
1. Sample handling, preservation and storage QAIQC requirements (see
EPA/USACE 1998) are the same for reference and control sediments as those
for the dredged material. Reference samples may be collected with grab
samplers.
2. Reference sampling sites are determined through a Region I EPA and NAE
cooperative program that designates reference locations for each active disposal
site. Current reference sampling sites will be indicated in the approved SAP. The
location of reference sampling sites for each established disposal site are shown
below. Testing laboratories are responsible for collection of control sediments.
3. Rockland 440 7.lt N 68° 58.70’ W
Portland 430 38.6’ N 69° 59.01’ W
Cape Arundel 430 17.9’ N 700 26.02’ W
Massachusetts Bay 42° 22.70’ N 700 30.30’ W
Cape Cod Bay 41° 57.50’ N 700 16.00’ W
New London 41° 16.7’ N 72° 02.0’ W
Cornfield Shoals 41° 15.63’ N 72° 13.32’ W
Central 41° 8.1’ N 72° 50.06’ W
Long Island Sound (LIS)
Western LIS 41° 58.69’ N 73° 29.20’ W
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B. WATER
Should water samples be necessary to prepare sediment elutriate samples, they
should be collected with either a non-contaminating pump or a discrete water
sampler. Samples for preparation of the elutriate should be collected within the
proposed dredging site, at mid-water depth at a location(s) in the vicinity of the
sediment samples, but avoiding any outfalls or other sources of pollution. For
disposal sites < 30 ft depth, one sample mid-depth will be collected. For sites>
30 ft depth, the sample should be a composite of near surface, mid-depth and
near bottom samples (3 ft above the bottom).
2. Control water is analogous to control sediment as it is used for water-column
bioassay control treatments. Control water should be the same water in which
the test organisms were held prior to testing. Field collection of the water (if
collected during the same field sampling as sediment samples), should be done
prior to sediment sampling and at near-bottom depths. The control water may be
clean seawater or adequately aged artificial seawater.
3. Quality Assurance/Quality Control procedures need to be followed for sampling
and analysis.
4.5 Sample Documentation
A complete field record of all procedures must be maintained including station
locations, sample handling, preservation and storage procedures. Any
circumstances potentially affecting the samphng must be noted as they may
prove invaluable in explaining a data anomaly.
2. The following infc,rmation represents the minimum that must be placed on a
sample label.
- Unique identifying code
- Location (station number) and depth
- Analysis or test to be performed
- Preservative and/or storage method
- Date and time of collection
- Special remarks if appropriate
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- Initials or name of person doing the collecting
3. The information on the sample label represents the first step in a sample tracking
(chain of custody) procedure. This procedure for tracking samples from
collection through completion of analyses has to be in place prior to the initiation
of sampling, with appropriate personnel assigned responsibility for the tracking
and sample custody. Example sample labels and Chain of Custody forms are
provided in Appendix A of EPA/USACE (1995).
4. Results of analyses submitted to NAE for evaluation must be made using the
NAE formatted data template available on the Corps NAE website or as a
computer disk. Additionally, the applicant must provide completed QC Summary
Tables (Appendix Il, also available on this website or disk) and hard copy results
of the QC analyses. This format is necessary to facilitate the project review
process and to ensure completeness of the submittal. Project data not submitted
in this format will be considered incomplete and a resubmittal will be required.
The format is provided to the applicant by the Corps when the SAP is approved.
5. As part of the chain-of-custody procedure and insuring an accurate evaluation of
test results, sample designations used to identify sample locations in the field
must be maintained throughout the process from sampling to data presentation.
Records should include field log books, location of samples (latitude, longitude),
positioning technology, sample labels, records of containers, time and conditions
of storage. All sample containers and storage conditions must comply with the
specifications in the Green Book. ITM, the “QA/QC Guidance for Sampling and
Analysis of Sediments, Water and Tissues for Dredged Matenal Evaluations,
Chemical Evaluations” (EPA/USACE 1995) and EPA (2001). Records must be
kept a minimum of 5 years.
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5.0 PHYSICAL AND CHEMICAL ANALYSIS OF
SEDIMENTS
Testing is commonly required to characterize the’physical and chemical properties of
sediments proposed for dredging. The following information supplements Section 9.0 of
the National Ocean Disposal (EPA/Corps 1991) (“the Green Book”) and Inland Testing
(ITM) (EPNUSACE 1998) guidance manuals and the QNQC guidance manual
(EPA/Corps 1995).
5.1. Initial Characterization of Sediment
All individual core samples must be visually inspected prior to their extrusion from the
core liner in preparation for subsampling, homogenization or compositing. Each core
must be described in terms of any discernible sediment strata characterized by changes
in composition, texture, grain size, color and odor (e.g., sulfides, oil).
If no significant strata (< 2 ft thick) are present (as visible in a clear core liner),
sediment in the core liner should be extruded, thoroughly homogenized and samples
taken for physical parameters listed in Tables 1 and 4 (if necessary). If split spoons or
other coring devices are used, the core must be extruded to examine and describe it. A
subsample representative of each core must be archived in case additional or repeat
analyses are required. As discussed in Chapter 4, should any significant strata be
present, the applicant must contact the New England District (NAE) immediately for a
determination of the need for analyses of individual strata prior to any further
manipulation of the core. Archiving the sample in this instance is also required.
Sediment proposed for dredging and reference sediments must be analyzed for grain
size distribution, total organic carbon (TOG) and total solidslpercent moisture (Table 1).
In addition, specific gravity, bulk density and Atterberg limits may be required on a case-
by-case basis and are desctibed in Section 5.3.
The grain-size analysis must be conducted according to the methods described in
Plumb (1981) or ASTM (1998 a) and reported as percentages retained by weight in the
following size classes at a minimum:
Gravel
Coarse Sand
Medium Sand
Fine Sand
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Silt/Clay (expressed as “Fines”)
Gravel and sand fractions should be separated using the standard sieve sizes in Table
I (ASTM 1998 a, D 422-63). In addition to reporting the percentages of each size class,
the applicant must graph the cumulative frequency percentages using the Engineering
(ENG) Form 2087 or a similar form (Figure 4). There may be cases where silt and clay
fractions will need to be distinguished. The NAE will provide guidance on whether it is
needed on a case-by-case basis. These will be determined on a case-by-case basis.
Both silt and clay fractions should be quantified by hydrometer (ASTM 1998 a), pipette
or Coulter Counter (Plumb 1981). Further analysis of other size classes may be
required to evaluate suitability for beneficial use or other purposes.
Measure the total solids and calculate percent moisture as described by Plumb
(1981) orAPHA (1995).
Note that the results of the above physical analyses may be used to support compliance
with one or more of the three exclusionary criteria in 40 CFR 227.13(b) for ocean
disposal or support a determination that the material is not a carrier of contaminants
under 40 CFR 230.60 (a) for other aquatic disposal. If physical analyses show that the
dredged material meets one or more of the exclusionary criteria, and if other pertinent,
historical, and site-specific information can support the criteria, the material may be
approved for disposal without further testing.
5.2. Chemical Analysis of Sediment
The chemicals of concern routinely required are listed in Tables 2 and 3. Table I-I in
Appendix I lists additional project-specific contaminants of concern. The routine metals,
PCBs, PAHs and pesticides listed in Tables 2 and 3 were chosen based on their
toxicity, their persistence in the environment, their abihty to bioaccumuiate and their
widespread and consistent occurrence in New England estuarine, marine and
freshwater sediments and organisms.
The target quantitation limits (TQLs) listed in the Tables are performance goals set.
between the lowest, technically feasible quantitation level for routine analytical methods
and available background concentrations at reference areas in the vicinity of the
disposal sites. As a routine data acceptance criteria, the Method Detection Limits
(MDL, see below) for each analyte must be below the listed TQLs, with the caveat that
some sediments with higher % moisture content may have MDLs higher than the TQLs.
MDLs are calculated using the method described in Section 5.4 (C) and must be
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ENG , 2087
FIGURE 4. SEDIMENT GRAIN SIZE GRADATION GRAPH
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. s NOARO SIEVE OPENING IN 1104E 5
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GRAIN SIZE IN MILUMETERS
Sample No.
COBBLES -- GRAVE
0ev Depth
I 0 I n i lE Ia
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Nat w %
LL
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awini No.
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GPO 920-200
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performed at a minimum every six months (see below). Achieving the TQLs is critical to
providing a consistent and accurate quantitation of contaminants of concern and a valid
comparison with known background concentrations in New England estuarine and
marine sediments in reference areas for disposal sites.
As noted in Tables 2 and 3, the specified methods (listed from the Green Book, ITM,
QA/QC manual (EPNUSACE 1995) and the EPA guidelines on clean metals techniques
(EPA 1996a,b,c,d)) are not required, as other acceptable methods are available.
Whichever method is used, it is the applicant’s responsibility to meet the target
quantitation levels and the specified performance standards in Tables Il-I through 11-5,
the attached QC Summary Tables (Appendix II). These performance standards assess
accuracy, as measured by standard reference material, and precision, as measured by
duplicates and matrix spike duplicates, for the contaminant groups listed in Tables 2, 3
and Appendix I. Each applicant must demonstrate that any new lab they choose can
meet these specifications prior to the analysis of any samples by the approval of an
LQAP (see Section 4). Some labs have had difficulties in the past meeting the required
target quantitation levels because of use of inappropriate sample preparation and clean-
up procedures to remove interfering substances typically found in marine sediments
(e.g., sulfides). Appropriate sample preparation, clean-up and analytical methods have
been developed for estuarine/marine sediments by NOM (1993) and EPA research
laboratory at Narragansett, RI (EPA 1993) that have successfully met the TQLs. These
are available from EPA Region I upon request. If the TQLs required auantitation limits
cannot be attained, a detailed explanation must accompany the data providing the
reasons for not attaining the required quantitation limits . Re-analysis may be
necessary.
The concentration and analytical detection limit for each of the following analytes on a
dry weight basis should be reporied as: ppm for metals, ppb for organics, parts per
trillion (pptr) for dioxins/furans and dioxin-like PCBs. Percent solids, used to calculate
dry weight concentrafions, must also be reported. The format for reporting is discussed
in Section 4.5.
As discussed in the Green Book (Section 9.3.2), capillary gas chromatography with
electron capture detection is recommended for analysis of PCBs and pesticides,
whereas GC/MS in the Selected Ion Monitoring (SIM) mode S recommended for the
PAHs and other semi-volatiles to meet the TDLs. Second column confirmation of
pesticides is required. Such confirmation for PCBs IS recommended but not required at
this time. The 18 PCB congeners (listed in Table 3) are those analyzed in the NOAA
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National Status and Trends Program (NOAA 1991). Additional congeners such as the
non-ortho, mono-ortho and di-ortho dioxin-like PCBs (e.g., PCB # 77, 126, 169) may be
required when dioxin is a contaminant of concern.
Total organic carbon (TOC) (Table 3) must be determined on all samples and
subsamples. TOC must be analyzed in duplicate in addition to an SRM or laboratory
control sample (LCS).
The Corps may require analysis of additional contaminants of concern other than those
listed in Tables 2 and 3 as they are identified in the Tier I review. These remaining
pollutants and other potential contaminants of concern and acceptable TQLs are listed
in Appendix I. Required analyses will be documented in the approved SAP.
As a general rule, Gas Chromatography/Mass Spectroscopy (GCIMS) chromatograms
must be scrutinized for the presence of compounds not included on the target analyte
list. These compounds must be tentatively identified and always reported.
5.3. Additional Physical Characterization of Sediment
Additional characterization of the sediments may be required on a case-by-case basis,
for modeling and geotechnical evaluations. These include specific gravity, bulk density
and Atterberg Limits (Table 4). Specific gravity should be measured following APHA
(1995), ASTM (1998 b, D 854-92) or Plumb (1981). Bulk density of sediment should
be determined according to Klute (1986) or DOA (1980). Atterberg Limits may be
required to assess the relative cohesiveness of the sediment. The procedures are
outlined in ASTM (1998 c, D 4318-95)1. The plastic/liquid limits and plasticity index
must be reported on ENG Forms 3838 and 4334 (Appendix I I), respectively, or a
facsimile.
5.4. Quality Control Measures:
The following analytical quality control measures must be followed for the above
referenced methods. They are explained in more detail in Quality Control (QC)
Summary sheets (Tables Il-I through 11-7. Appendix II). Along with reporting the data
generated from the sediment analyses, the applicant’s contractor laboratory is required
to document specified quality control measures in these attached worksheets. All
QNQC for Dioxin/Furan analyses (listed in Appendix I-I) will be documented according
to the methods described in EPA Method 1613.
(a) Physical Analyses: The following QC checks are required for physical analyses
(grain size, total solids) of sediments:
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Sample duplicate
(b) Chemical Analyses: The following QC checks are required for chemical analyses of
sediments:
• Initial calibration
• Calculation of MDLs
• Blind analysis of spiked or performance evaluation material for calibration
verification
• Continuing calibration checks
Analysis of Reference Materials or Laboratory Control Materials
Method blank
• Matrix Spike
- Matrix Spike Duplicate
Analytical replicates
Internal standards
Surrogates
(c) Detection/Quantitation Limits: The detection/quantitation limits used in this manual
are defined as follows:
Method Detection Limit (MDL) is defined as:
A statistical determination based on measured variance that defines the
minimum concentration of a substance that can be detected with 99% confidence
that the analyte concentration is greater than zero. In other words, that the
analyte can be qualitatively detected above signal noise. Quantitative
measurement at the MDL is inaccurate and therefore data reported less than the
Reporting Limit (RL), see below) and greater than or equal to the MDL should be
qualified with a “J” as estimated. Any analytes not detected (below the MDL)
should be reported as one half the MDL and qualified with a “U”. Detection
limits are analyte- and matrix-specific and may also be instrument- and
laboratory-dependent (see beiow).
The procedure described below, based on 40 CFR Part 136, Appendix B, must be
followed to verify the MDL for samples collected for each approved Sampling and
Analysis Plan. This MDL verification must be submitted with the data or performed on a
similar matrix within the previous six months.
Select one representative relatively uncontaminated sample for each matrix and
spike it with the analytes of concern so that the resulting concentration is
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between I and 5 times the TQLs listed in Tables 2, 3, 5, 8 and Table I-I
(Appendix I). Prepare and analyze a total of seven spiked replicates of the
chosen representative sample. Calculate the sample standard deviation (in
concentration units) of the seven measurements for each analyte of concern.
This value must then be multiplied by 3.143 and reported as the MDL.
Target Quantitation limit (TQL):
The TQL is a performance goal set between the lowest, technically feasible
detection limit (i.e., MDL) for routine analytical methods and available
background concentrations at reference areas in the vicinity of the disposal sites.
The goal is to have confidence in measured values in sediment, tissue or water
at concentrations typical of areas near but unaffected by the disposal site or
other pollution sources.
Practical Quantitation Limit (PQL):
The minimum concentration of an analyte or category of analytes in a specific
matrix that can be identified and quantified above the MDL and within specified
limits of precision and bias during routine analytical operating conditions.
Reporting Limit (RL):
The Reporting Limit (RL) is the sample-specific PQL adjusted for sample
processing volumes and factors (such as dilution) which can raise or lower the
PQL. The RL should not be higher than the TQL and is usually 3-5 times higher
than the MDL. However, when sediments of higher % moisture are analyzed, the
RL may be higher than the TQL.
(d) The applicant must submit documentation of all quality control measures performed
during ana ys s of the samples. if any of the corthol limit criteria are exceeded, the
sampling results may not be accepted.
5.5 Data Reporting
All applicants are required to submit physical and chemical bulk sediment data in the
New England District (NAE) format that NAE will use to review its analyses. These
formats are available on the NAE webpage (xxx ) or available on a floppy disk from the
Corps contact listed in Section 1. These data must be supplied both as a hard copy and
on a 3 1/2” floppy disk. The format will be provided by the Corps with the approved
SAP. Non-detects should be reported at one half the MDL. The applicants may submit
their own data summaries and analyses; however, they must also submit the original
data and copies of sampling logs so that the Corps and EPA can conduct independent
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analyses. All submitted data must be clearly presented and traceable to the original
samples and subsamples. No permit will be issued based solely on an applicant’s data
analysis.
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TABLE 1. Parameters used for the physical characterization of sediments.
Parameter Method MeasurelQuantitation limit
Grain Size Plumb 1981;
Distribution ASTM 1998 a
Gravel (>4.75mm) Retained on No. 4 Sieve
Coarse Sand (2.0-4.75mm) Passing through No. 4 and retained on No. 10 Sieve
Medium Sand (0.425-2.0mm) Passing through No. 10 and retained on No. 40 Sieve
Fine Sand (O.O75-0.425mm) Passing through No. 40 and retained on No. 200 Sieve
Sift (O.O05-0.075mm) As determined by Hydrometer, Pipette or Coulter Counter.
Clay (< 0.005mm) As determined by Hydrometer, Pipette or Coulter Counter.
Total Solids! Plumb 1981 1.0% -
Water Content
Total Organic Carbon (TOC) Plumb 1981, 0.1 %
EPA 1992,
Puget Sound Method (PSEP 1986)
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TABLE 2. Metal contaminants of concern, analytical methods and target
quantitation limits (dry weight) routinely analyzed in sediments.
Target
Analytical Quantitation
Metal Method 1 Limit ( pDmj
Arsenic 6010B, 6020, 7060, 7061 0.5
Cadmium GO1OB, 6020, 7130, 7131 0.1
Chromium 6010B, 6020, 7190, 7191 1.0
Copper 6010B, 6020, 7210 1.0
Lead 6010B, 6020, 7420, 7421 1.0
Mercury 7471 0.02
Nickel 6010B, 6020, 7520 1.0
Zinc 6010B, 6020, 7950 1.0
The specified methods are recommendations only. Other acceptable
methodologies capable of meeting the TQLs can be used. Sample preparation
methodology (e.g. extraction and cleanup) and sample size may need to be modified to
achieve the required target quantitat on limits.
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TABLE 3. Organic contaminants of concern, analytical methods and target
quantitation limits (dry weight) routinely analyzed in sediments.
Target
Analytical Quantitation
Chemical Constituent Method 1 Limit 2
TOTAL ORGANIC CARBON (TOC) Plumb 1981, 0.1 %
EPA 1992,
Puget Sound Method
PAHs 8270C-S IM, 20 ppb 2
Acenaphthene
Acenaphthylene
Anthracene
Benzo(a)anthracene
Benzo(a)pyrene
Benzo(b)fluoranthene
Benzo(k)fluoranthene
Benzo(g, h, i)perylene
Chrysene
Dibenzo(a,h)anthracene
Fluoranthene
Fluorene
Indeno(1 ,2,3-c,d)pyrene
Naphthalene
Phenanthrene
Pyrene
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TABLE 3. Organic contaminants of concern, analytical methods and target
quantitation limits (dry weight) routinely analyzed In sediments (continued).
Target
Analytical Quantitation
Chemical Constituent Method’ Limit
PESTICIDES NOAA (1993), 8081B I ppb
Aldrin
cis- and trans-Chiordane
cis- and trans-Nonachlor
Oxychiordane
p,p’-DDT, DDE, ODD
Dieldrin
Endosulfan I and II
End rin
Heptachior
Heptachior epoxide
Hexachlorobenzene
Lindane
Methoxychior
Toxaphene 50ppb
PCB CONGENERS 3 NOM (1993), 8082A I ppb 2
8* 2,4’dICB
18* 2,2’,5 tnCB
28* 2,4,4’ triCB
44* 2,2’,3,5’ tetraCB
49 2,2 ’,4’,5 tetraCB
52* 2,2’,5,5’ tetraCB
66* 2,3’,4,4’ tetraCB
87 2,2’,3,4,5’ pentaCB
101* 2,2’,4,5,5’ pentaCB
105* 2,3,3’,4,4’ pentaCB
118* 2,3’,4,4’,5 pentaCB
128* 2 ,3,3’,4,4’ pentaCB
138* 2,2’,3,4,4’,5’ hexaCB
153* 2,2’,4,4’,5,5’ hexaCB
170* 2,2’,3,3’,4,4’,5 heptaCB
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180* 2,2’,3,4,4’,5,5’ heptaCB
183 2,2’,3,4,4’,5’6 heptaCB
TABLE 3. Organic contaminants of concern, analytical methods and target
quantitation limits (dry weight) routinely analyzed in sediments (continued).
Target
Analytical Quantitation
Chemical Constituent Method 1 Limit
PCB CONGENERS 3 (continued) NOAA (1993), 8082A I ppb 2
184 2,2’,3,4,4’,6,6’ heptaCB
187* 2,2’,3,4’,5,5’,6 heptaCB
195* 2,2’,3,3’,4,4’,5,6 octaCB
206* 2,2’,3,3’,4,4’,5,5’,6 nonaCB
209* 2,2’,3,3’,4,4’, 5,5’,66’ decaCB
The specified methods are recommendations only. Other acceptable
methodologies capable of meeting the TDLs can be used. Sample preparation
methodology (i.e., extraction and cleanup) (EPA 1993; NOAA 1993) and sample
size may need to be modified to achieve the required target quantitation limits.
2 Applies to each analyte listed below unless otherwise noted.
Total PCBs are to be estimated based on the following: Total = 2 X [ sum of 18
NOM summation congeners marked with *] (T.Wade, personal communication).
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TABLE 4. Additional parameters used for the physical characterization of
sediments.
Analytical Measure!
Parameter Method Quantitatinn I irnif
Specific Gravity Plumb 1981 0.01
ASTM 1998 b
APHA 1995
Bulk Density Klute 1986 0.01 g/cm 3
DOA 1980
Atterberg Limits ASTM 1998 c
Liquid Limit
Plastic Limit
Plasticity Index
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6.0 WATER COLUMN EVALUATION
6.1. Tier II. Compliance with Water Quality Criteria/Standards
The discharge of dredged material into the water column and resuspension at an
aquatic disposal site may introduce sediment contaminants into the water column. As
required in 40 CFR 227.6 (c)(1) and 40 CFR 230.10 (b) (1), the discharge must be in
compliance with marine water quality criteria after allowance for mixing for discharges in
federal waters and state water quality standards for discharges in state waters, if
applicable. Based on 40 CFR 227.6, compliance with marine aquatic life water quality
criteria or state water quality standards must be evaluated for every discharge in
federal or state waters. The federal criteria are shown in Table 5. State water quality
requirements for dredged material discharges vary with each state. Each appropriate
state department of environmental protection office, in coordination with NAE, will
assess compliance with applicable state standards using the data described below.
General procedures for these analyses are described in Section 10.1 of the Green Book
unless otherwise noted below.
Step 1: Criteria screen for compliance with EPA Water Quality Criteria
As a first step in evaluating compliance, the applicant may use the dry weight sediment
concentrations of listed contaminants which assumes a total release from the sediments
to the water column as described in Section 10.1.1 of the Green Book and Section 5.1
of the ITM. The model to be used is described below (6.4). As discussed in those
sections, the analysis need only be run for the contaminant of concern that requires the.
greatest dilution for compliance. If the modeled discharge meets the criteria (Table 5),
then no further analysis of water quality criteria (WQC) are needed. If the analysis
shows that the discharge exceeds the criteria, then the standard elutriate test, as
described in Step 2, must be performed.
Step 2: Standard Elutriate Analysis
The dredged-material elutriate preparation is conducted according to the methods
presented in ITM Section 10.1.2.1: (“Standard Elutriate Test”) with the following
modifications (italicized). Samples for the elutriate and the water column toxicity test
(Section 6.2) can be prepared from the same sediment-water mixture. To evaluate
water quality criteria in the liquid phase, the elutriate water must be centrifuged to
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remove particulates in accordance with the guidelines in Section 10.1.2.1 of the ITM.
(The sample is not centrifuged in the case of the water column toxicity test since it
assesses toxicity in the liquid and suspended solid phases (See ITM Section 11.1.4.))
The chemical analysis of the elutriate and dredging site water is discussed in ITM
Section 9.4.2. Chemical Analysis of Water. If “clean” seawater is used to prepare the
elutriate and water column toxicity tests, then, for baseline purposes, the “clean”
seawater must be analyzed for all the chemical parameters measured in elutriate and
dredging site waters.
Table 5 provides the recommended methods and required Target Quantitatiori Limits
(TQLs) for each contaminant of concern. The reference methods in Table 5 should be
consulted when selecting methods for water analysis. So-called “clean” techniques for
sampling (EPA 1995a) and analyses of metals are currently available from EPA and are
listed in Table 5. For extraction and analysis of PCB congeners, the NYDEC method
(NYDEC, 1991) is also recommended. The 18 PCB congeners are listed in Table 3. If
there is doubt about meeting TQLs, the applicant should contact New England District
before any analyses are performed.
Particular note should be taken of the volume of the water samples required to meet the
TQLs for water analysis (Table 5). As a general rule, at least I liter water samples are
necessary for each organic analysis and I liter for metal analyses to provide TQLs that
are below the applicable marine WQC. Larger samples are recommended since there
should be enough left over in case repeat analysis is required. Additional clean-up
steps also may be necessary, especially for the organics. It is important for a valid
mixing evaluation (see Section 6.4 below) that accurate ambient contaminant
concentrations be measured in the field collected ambient disposal site water samples.
To meet the TQLs in Table 5 for the organics in the ambient samples, a larger sample
may be necessary. An example procedure for collecting large field samples can be
found in Appendix Ill.
At a minimum, chemical analysis must be conducted for the inorganic and organic
analytes given in Table 5. Additional contaminants of concern may be requested for
specific projects. Both elutriate (made up of dredging site water and sediments to be
dredged) and disposal site water are to be tested in triplicate. Disposal site water
values are used in the calculation to determine WQC compliance, or, existing data
(provided by the Corps) in the vicinity of the disposal site may be substituted.
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Refer to Green Book and the ITM (Sections 9.4): Chemical Analysis of Water and
Section 10.0 of Guidance for Performing Tier II Evaluations and EPA/USACE (1995) for
general guidance.
6.2. Tier Ill Water-Column Evaluations
Tier Ill water-column tests evaluate the potential for toxicity of the dissolved and
suspended portions of the dredged material that remain in the water column after
discharge of the dredged material. The Tier Ill water-column bioassays are run if the
Tier II evaluations are inconclusive: i.e., there are no WQC for all contaminants of
concern or there is reason to suspect additive or synergistic effects among the
contaminants. The Tier Ill water-column tests involve exposing fish, pelagic
crustaceans and planktonic invertebrate larvae to a dilution series containing dissolved
and suspended components of the proposed dredged material. An overview of the Tier
lii water-column evaluations is presented in the Green Book and the ITM under Section
6.1 in both documents. Technical guidance for performing the tests is provided in the
ITM Section 11.1: Tier Ill Water-Column Toxicity Tests. The NAE will specify to the
applicant which species in Table 6 of this manual will be required for these tests.
Technical guidance on conducting water-column bioassays is provided in ITM Section
11.1: ‘T,er Ill: Water Column Toxicity Tests”. Three series of tests are necessary; tests
must be run using a fish (Menidia sp., Cyprinodon variegatus), a crustacean
(Americamysis bahia) and a planktonic larvae (bivalve or echinoderm) (Table 6). The
mysids should be fed as prescribed by EPA (1991b) or ASTM (1998 d,e). Bivalve
larvae and silversides must not be fed (ASTM 1998 d,e,f). Test duration is generally 96
h except planktonic larvae which is 48 h. The procedure for preparing the water column
toxicity test sample is given in Section 11.1.4 of the ITM with the following modifications
(italicized). In cases where the salinity of the dredging site water is detnmenia! to the
health of the test organism (too low), all the toxicity water samples must be prepared
using clean seawater. The necessa,y dilutions may be made using water collected
from clean seawater or aged artificial seawater. Each series should include 100%,
50%, and 10% treatments and a 0% treatment (=100% dilution-water treatment). Clean
seawater in which the organisms were held prior to testing must be run as a control. If
the diluent is the same water the organisms are held in prior to testing, then the control
and 0% treatment are one and the same. There is no reference site water in the water
column toxicity test. Some fine-grained sediments can create turbidity in the test water
even after settling. In this case, the ITM Section 11.1.4 allows mild centrifugation
.until the suspension is clear enough at the first observation time for the organisms to
be visible in the testing chamber.”
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A minimum of five replicates per treatment concentration and a minimum of 10
organisms per replicate are required except for larvae (see next paragraph). As stated
in the Green Book, the applicant should ensure that organisms are not overcrowded in
the test chambers which can stress the organisms and falsely influence the results. The
number of surviving fish and mysids for each replicate must be recorded at 0, 4, 24, 48,
72, and 96 h.
A minimum of five replicates per treatment are also required for the larvae bioassay. A
suspension of fertilized eggs is used in the preparation of the test solutions. The
suspensions containing bivalve larvae should contain 20-30 embryos/mL whereas the
suspensions containing sea urchin larvae should contain 2000 embryos/mL. - Follow
ASTM (1998 g) protocol for the bivalve water-column toxicity test or the procedure in
EPA (1990) in Appendix V for sea urchin larvae. Use a light box or dissecting
microscope to record the number of live animals. Use of an image analyzer as
discussed in this procedure is not required here. For the larval test, centrifugation of a
turbid supernatant is not necessary and should not be performed. The test is
terminated in 48 hours. At this time, the larvae in the 0% treatment should have
reached the appropriate stage of development (straight hinge—D shape for bivalves and
plutel for the sea urchin).
For all test organisms, any sublethal effects such as physical or behavioral anomalies
must also be reported. Daily water quality records must be kept for salinity,
temperature, DO and pH for each test dilution.
6.3 Quality Control Measures
The EPA Region I and NAE require the following QC measures:
(a) Water Chemistry:
For water chemistry in the elutriate test, the analytical methods and TQLs described in
Table 5 and EPA/USACE (1995), are recommended following the appropriate sample
preparation. The analytical quality control measures described in each of the methods
must be followed supplemented with applicable QNQC guidelines described in Section
5.4 (b) and (C) for sediment chemistry. They are explained in more detail in Quality
Control Summary Sheets Tables 11-1-5, (Appendix II of this manual). Along with
reporting the data generated from the chemical analyses, the applicant’s contractor
laboratory is required to document specified quality control measures in these attached
worksheets.
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(b) Water Column Toxicity tests: The EPA Region I and NAE require the following QC
measures:
• All bioassays must be performed under the conditions specified in each of the
test species sheets in Appendix V in either natural seawater or a synthetic
seawater adjusted to salinity appropriate for the test species and disposal site
(generally 25-30 & c).
• The survival rate requirements in the Control treatments must be achieved.
Failure to meet the applicable requirements below will likely invalidate the testing
procedures and require retesting of the control and test samples.
Control mortality requirements: 10% mean
Control abnormality requirements:
<30% for oyster and mussel larvae, or
. 40% for clam larvae)
(c) The applicant must submit documentation of all quality control measures performed
during analysis of the samples. If any of the control limit criteria are exceeded, the data
may not be accepted.
6.4. NUMERICAL MODELS FOR INITIAL-MIXING EVALUATIONS
This section explains descnbes how the Corps of Engineers uses numerical models to
evaluate testing results from water column bloassays. The Corps and EPA will run the
numerical models and make the evalu t ons; applicants or their agents do not need to
run the models.
In general, initial-mixing evaluations for compliance with water quality criteria and
toxicity will be performed by NAE as part of their assessment of each project. The
following information supplements the national guidance in the ITM Appendix C:
Evaluation of Mixing (EPNACE 1998) and Appendix B (EPA/USACE 1991).
Numerical models are components of the Tiers II and Ill water-column evaluations. The
model used, STFATE, is contained in the Automated Dredging and Disposal
Alternatives Management System (ADDAMS) from the ITM (updated software and is
not referenced in the 1991 Green Book). However, this updated model is available for
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unrestricted distribution from the U.S. Army Corps of Engineers Environmental
Research and Development Laboratory (formerly the Waterways Experiment Station)
web page (http://www.wes.army.mil/el/elmodels/jndexhtml)
The appropriate model is wn only for the contaminant of concern that requires the
greatest dilution. If the contaminant requiring the greatest dilution is shown to meet the
LPC, all of the other contaminants that require less dilution will also meet the LPC.
The STFATE initial-mixing model can be run on IBM®-compatible personal computers.
STFATE computes the movement of dredged material from an instantaneous dump and
from a hopper dredge that falls as a hemispherical cloud. To properly apply this model,
the total time required for the dredged material to leave the disposal vessel should not
be greater than the time required for the material to reach the bottom. The model
applies to both split-hull barge and hopper disposal.
This model accounts for the physical processes that determine the short-term fate of
dredged material in the water column as it is disposed at open-water sites. The models
assume that the dredged material behaves as dense liquid, and simulate the movement
of the disposed material as it falls through the water column and spreads over the
bottom. They do not account for resuspension or other long-term post-disposal
phenomena on the water-column or benthic environment.
Input data for the models are grouped into the following general areas:
Description of the disposal operation
Description of the disposal site
Description of the dredged materials
Model coefficients
Controls for input, execution, and output
tIM Appendix C: Evaluation of Mixing, Table C-2 lists each model’s necessary input
parameters and their corresponding units. Applicants must provide the following
parameters: volume in barge, vessel course and speed, barge length and width, and
post-disposal draft of barge. . Additional descriptions and guidance for selection of
values for many of the model parameters are provided in the Appendix C text and
directly on-line in ADDAMS.
For discharge in federal waters, the results of the toxicity test will be used to determine
compliance with the Limiting Permissible Concentration (LPC). The results of the water-
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column tests are used to calculate the median lethal concentration (LC 50 ). The LPC for
the dredged material is I % of the LC 50 . If the numerical mixing model predicts that the
concentration of dredged material in the water column will not exceed 1% of the LC 50
concentration either outside the disposal site or within the disposal site 4 hours after
discharge of the dredged material, the proposed discharge of dredged material meets
the water-column LPC. If either of these criteria are not met, the dredged material does
not meet the water-column LPC. For compliance of discharges in state waters general
guidelines are explained in Section 11.1.6 and Appendix C of the ITM. Here, the state
environmental regulatory agency needs to be consulted to determine the mixing
requirements for compliance with the water quality criteria in that state. Such mixing
guidelines can vary with each state.
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TABLE 5. REQUIRED CONTAMINANTS, RECOMMENDED METHODS, TARGET QUANTITATION LIMITS AND
FEDERAL WATER QUALITY CRITERIA USED IN WATER QUALITY CRITERIA COMPLIANCE DETERMINATION
RECOMMENDED TARGET FEDERAL WATER
QUALITY
CONTAMINANT TEST METHOD 2 QUANTITATION LIMIT(upIlj CRITERION ( ugIL )
Metals 1
Arsenic 200.9, 1632 1 69
Cadmium 200.9, 1637 1 42
Chromium (VI) 218.6, 1636 1 1100
Copper 200.9, 1639, 1640 0.6 4.8
Lead 200.9, 1639, 1640 1 210
Mercury 245.7, 1631, 04 1.8
Nickel 200.9, 1639, 1640 1 74
Selenium 200.9, 1639 1 290
Silver 200.9 0.5 1.9
Zinc 200.9, 1639 1 90
Pesticides. 3510B, 8081 B 4
Aldrin 0.26 1.3
Chlordane 0.02 0.09
Chloropyrifos 0002 0.011
Die ldrin 0.14 0.71
4,4’DDT 0.D3 0.13
a-Endosulfan 0 007 0.034
b-Endosulfan 0.007 0.034
Endrin 0.007 0.037
Heptahlor 0.01 0.053
Heptachior
Epox lde 0.01 0.053
Lindane 0.26 1.3
Toxaphene 0.04 0.21
Industrial Chemicals
PCBs 5 NYDEC, 3510B, 8082A 0006 0.03
Pentachlorophenol 3510B, 8270C 2.60 13
TABLE 5. REQUIRED CONTAMINANTS, RECOMMENDED METHODS, TARGET QUANTITATION LIMITS AND
FEDERAL WATER QUALITY CRITERIA USED IN WATER QUALITY CRITERIA COMPLIANCE DETERMINATION
(continued)
Determined as “total recoverable metals”.
2 Except for chromium and mercury, samples can be digested by Method 200.2 (EPA, 1991) and extracted by
chelatjon/e,m fJon as described under “Metals-14” S 9.2 (EPA, 1979, revised 1983), prior to analysis by Method
200.9. EPA Clean metal techniques (1600 series) are described in EPA (1995 abc) and EPA (1996 a,b,c,d).
Bloom and Crecelius (1983) method for determining mercury concentrations.
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Pesticides and PCBs can be extracted from the water by Methods 35106 and analyzed by Method 8081A (EPA
1986); PCB congener analysis by NYDEC (1991) are also recommended
Total PCBs will be estimated based on the summation of these congeners and using the equation total PCBs = 2 X
[ sum of 18 congeners] (1. Wade, personal communication).
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TABLE 6. ORGANISMS REQUIRED FOR THE WATER COLUMN BIOASSAY’
Group Organism Scientific Name Test Duratinn
Fish: 96h
Silverside Menidia sp.
Sheepshead minnow Cyprinodon variegatus
I I Mysid shrimp Amencamysis bahia 96h
Ill Planktonic larvae: 48h
Blue mussel Mytilus edu!is
American oyster Crassostrea virginica
Hard clam Mercenaria mercenaria
Coot clam Mulinia laterafis
Sea urchin Athacia pqnctulata
1 One type of organism must be tested from each group
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7.0 BENTHIC EFFECTS EVALUATION
The benthic effects evaluation involves whole sediment toxicity and bioaccumulation
testing. The general procedures for Tier Ill toxicity tests are described in Sections 11.2
of the ITM (EPA/ACE 1998) and Green Book (EPA/USACE 1991) and freshwater
testing manual (EPA 2000). Tier lii bioaccumulation tests are described in Section 12.1
of the ITM and the freshwater manual (EPA 2000).
7.1 Tier Ill - Whole Sediment Toxicity Tests
The purpose of the sediment toxicity tests is to determine whether the sum of the
sediment contaminants in combination with the physical characteristics will elicit a toxic
response to exposed organisms after the material is deposited into the marine
environment.
For marine and estuarine disposal, two test species of those listed in the Toxicity
section of Table 7 are required —one of the three marine amphipod species (depending
on salinity and grain size) and the mysid shrimp. Currently only one species is required
for freshwater disposal. Species-specific test conditions are listed in Appendix V and
are detailed in EPA (1994a) for estuarine/marine amphipods, EPA (1991b) for the mysid
and Sections 11 and 12 of the freshwater testing manual (EPA 2000) for the freshwater
amphipod and midge fly larva.
General guidance for the collection, handling and storage of sediments for biological
testing are described in Section 4 of this manual and Section 8 of the Green Book.
Section 8 of the EPA amphipod test manual (EPA 1 994a) must be consulted for specific
guidance related to the amphipod sediment toxicity tests. The Corps will specify any
compositing of sediment samples that will be allowed in consultation with federal/state
regulatory agencies.
Specific guidance on procedures for setting up, performing and breaking down the test
is provided in EPA (1 994a) for the amphipod species, and EPA (1991b) for the mysid
species. All sediments tested must be press-sieved with a 1 or 2 mm sieve to remove
unwanted debris and predators before being added to the test chambers. All data
should be reported on the forms supplied in EPA (1994a, Appendix A, Figures A1-A5)
or a close facsimile. In addition to the parameters on the forms, all observations on
mortality, the formation of tubes or burrows, amphipod emergence from sediment, and
any physical or behavioral abnormalities must be recorded.
Bulk sediment chemistry, for the project specific contaminants of concern, Total Organic
Carbon (TOC) and grain size analyses may be required by NAE on subsamples of the
sediments that are biologically tested. Subsamples of the dredged material, reference
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and control sediments used in the test must be archived for possible future bulk analysis
lithe Corps or EPA requires them to be analyzed.
As a general rule, the applicant is required to seek approval from the Corps and EPA on
project specific procedures for any sediments requiring treatment for ammonia toxicity.
Amphipods and mysids are generally sensitive to sediment ammonia. Excessive
ammonia concentrations may cause mortalities in these species. Because ammonia
toxicity can generally change with ephemeral environmental conditions such as
temperature, salinity, oxidation state and pH, excessive ammonia concentrations can
confound the mortality endpoint of interest to the dredging regulatory program which
focuses on more persistent toxics. To account for this potential false positive, the EPA
and Corps have devised methods to reduce ammonia toxicity before any test begins
(Sections 11.4.5-11.4.5.3 of the EPA amphipod manual (EPA 1994a), as amended by
the “Errata” sheet for pages 80-82 of that document). Therefore, to avoid toxicity from
ammonia, the applicant must insure that the sediment porewater total ammonia and un
ionized ammonia concentrations are below 20 mg/L and 0.4 mg/L, respectively before
any amphipod is added to a test chamber. Collect porewater for ammonia and pH
determinations at test initiation before the test organisms are added to exposure
chambers. This will require setting up dummy chambers for porewater collection.
Recommended procedures to collect porewater are described in Appendix VII. After
treatment, the pore water concentrations must be maintained below the above values
for 24 hours before the animals are added to the test chamber. Total and unionized
ammonia levels must be monitored in the pore-water on days 1, 5 and 10 during the
test. These measurements should be made in at least one chamber (“dummy”
chambers for porewater collection) or using peepers (see Section 6.2.1 of EPA 2001d)
for each homogenized sediment treatment level (control, reference, dredge site) tested.
All samples require triplIcate analysis.
For the mysid, Americamysfs ( Mysidopsis) bahia, the applicant must follow the
guidance in the June 14, 1994 memo to Mario Del Vicario from Elizabeth Southerland
(Appendix VIII). Here, the concern is unionized ammonia in the overlying water (1 cm
above the sediments). The applicant must insure that the water concentrations are
below 0.6 mg/L at pH of 7.9-8.0 or 0.3 at pH of 7.5 before any animals are added to the
test chambers. In this case overlying water is monitored each day.
As indicated in the Green Book and ITM, all control survivorship must be at least 90%
for the test to be valid. The reader is referred to other QAIQC requirements in Section
7.3 of this manual.
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7.2 Tier Ill - Bioaccumulation Testing
Bioaccumulation tests provide a measure of exposure of deposit-feeding marine
animals to bioavailable sediment contaminants. In this case, representatives of a
bivalve and a polychaete worm species are exposed for a 28 day period to dredging
site, reference and control sediments. To clarify recommendations in the Green Book,
the 28 day exposure test is required for organic contaminants of concern as well as for
metals. General technical guidance is provided in Section 12.1 of the ITM (EPA/ACE
1998), Section 13 of the freshwater methods manual (EPA 2000) for freshwater
disposal and Lee et a!. (1989), as cited in the former documents.
The two required species for marine/estuarine disposal are listed in the Bioaccumulation
section of Table 7— the sandworm, Nereis virens, and the bivalve Macoma nasuta.
Each species must be exposed in separate aquaria because of the predatory behavior
of Nerds. It should be noted that use of another set of aquaria will require a
proportionally greater amount of sediments to be collected and processed. For
freshwater disposal, the oligochaete, Lumbriculus variegatus is used.
All aquaria must have a sediment depth of at least 5 cm. At least 20 specimens of each
species are required in each test chamber, although more may be necessary to conduct
the prescribed tissue analyses at the end of the test exposure. It is the applicant’s
responsibility to insure that the laboratory provides enough animal tissue (size/number)
to run subsequent chemical analyses. Generally, it is desirable to produce 50 g (wet
weight) for each replicate and species. The number of animals and the size of the
aquarium will vary with the size of individual animals acquired for the test. For the
species in Table 7, tissue/sediment loading should not exceed I g tissue (wet weight
minus shell) to 50 g sediment (wet weight) (Lee, EPA Newport Lab, personal
communication). If dioxin/furan levels are required, then a separate set of aquaria may
be required to provide adequate tissue for analyses to achieve the required TQLs.
Those constituents generally requiring analysis are listed in Table 8, but may include
other contaminants as determined by the Tier I review and/or chemical testing of the
sediments. The final decision on which project-specific contaminants are required is
made by the Corps in consultation with other Federal/state regulatory agencies.
Recommended tissue extraction and analytical methods are provided in NOAA (1993),
EPNUSACE (1995) or EPA (1993). The applicant must insure the contracted
laboratory can reasonably achieve the required TDLs listed in Table 8 and Appendix I, if
applicable. The sample preparation methods for animal tissue described in EPA
(1993) and EPNUSACE (1995) are highly recommended. As mentioned above, 50
grams of tissue (wet) per replicate is recommended (or enough to obtain acceptable
TQLs). In addition to the contaminants, the lipids of each clam and worm tissue
replicate should be analyzed using a modified Bligh and Dyer (1959) method developed
by the U.S. EPA Narragansett Laboratory, (EPA 1995 d)(see Appendix IX). A copy of
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this method is included as Appendix IX of this document. Percent water, solids and lipid
must be reported for each species and replicate.
All appropriate QA/QC measures listed in Sections 9 and 12 of the ITM and
EPA/USACE (1995) must be followed. Tissues of organisms randomly selected prior to
initiation of bioaccumulation testing (pre-test analyses) must be analyzed and reported
for all contaminants analyzed in the exposed organisms. A subsample of these pre-test
samples of tissue of each species must be archived as the applicant may be required to
analyze this tissue at a later date for specified contaminants.
As with toxicity tests, daily records must be kept of salinity, temperature, DO, pH, flow
rate, obvious mortalities and any sublethal effects. Failure of organisms to burrow into
the sediment or any other physical or behavioral abnormalities must also be recorded.
All bivalves (whether pre or post-test) must be depurated for 24 hours in “clean
seawater ’ prior to freezing. Polychaetes will not depurate in seawater alone and
therefore require a 24 hour depuration with “clean sand.”
7.3 Quality Control Measures
(a) The EPA Region I and NAE require the following biology QC measures:
All marine/estuarine bioassays must be performed under the conditions specified in
each of the test species sheets in Appendix V in either natural seawater or a synthetic
seawater adjusted to salinity appropriate for the test species and disposal site (generally
25-30 WOO) Adherence with the applicable test acceptability requirements in EPA
(1994a) must be documented for Ampe/isca abdita, Eohaustorius estuarius and
Leptocheirus plumulosis and in EPA (2000) for Hyalella azteca. Likewise, the QA
procedures cited in the ITM and EPA (2000) must be followed and documented for
bioaccumulat ion testing.
Bulk physical and chemical testing may be required for each sediment sample tested for
biological analyses to insure the testing was performed on representative samples.
This will be determined on a case by case basis.
The survival rate requirements in the Control treatments must be achieved. Failure to
meet the applicable requirements below will likely invalidate the testing procedures and
require retesting of the control, reference, and test samples.
Sediment toxicity control mortality requirements: 10% mean (amphipods control
mortality 10% mean and no individual chamber 20% mortality)
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Where control mortality >10% for sediment bioaccumulation samples, determine
whether the following conditions exist: a) adequate replicates to obtain statistical power
b) stressed organisms C) contaminated control sediment d) contamination of test system
e) quality control problems f) adequate tissue for chemical analyses
(b) For tissue chemistry in the bioaccumulation testing, the applicant should follow
similar QA/QC guidelines as described in Section 5.4 (b) and (C) for sediment chemistry.
The analytical quality control measures in the above described methods must be
followed supplemented with the guidelines described in Section 5.4 (b) and (C). The
measures are explained in more detail in Quality Control Summary Sheet (Tables Il-I
through 11-8, (Appendix II). Along with reporting the data generated from the chemical
analyses, the applicant’s contractor laboratory is required to document specified quality
control measures in these attached worksheets. All QNQC for Dioxin/Furan analyses
(listed in Appendix I-I) will be documented according to the methods described in EPA
Method 1613.
(C) The applicant must submit documentation of all quality control measures performed
during analysis of the samples. If any of the control limit criteria are exceeded, the data
may not be accepted.
7.4 Statistical Analysis
Toxicity and bloaccumulation data should be analyzed as indicated in Appendix D of the
“Evaluation of Dredged Material Proposed for Discharge in Waters of the U.S —Testing
Manual” (EPNACE 1998) (summarized in Table 9). As discussed in Appendix D, these
methods are described in many popular general statistics texts such as Winer (1971),
Steel and Torrie (1980), Sokal and Rohlf (1981), Dixon and Massey (1983), Zar (1984)
and Snedecor and Cochrane (1989). In addition, Conover (1980) is recommended for
nonparametric tests. Most of these tests are included in commercially available
statistics software packages. Relative to detection levels, alt undetected analytes must
be reported in the data as one half the method detection level (MDL) as defined in
section 5.4.
7.5 Data Reporting
All applicants are required to submit toxicity and bioaccumulation data in the New
England District (NAE) format. The format will be provided by the Corps with the
approved SAP. The appropriate QC Summary sheets described above must also be
submitted with the data. The applicants may submit their own data summaries and
analyses; however, they must also submit the original data and copies of sampling logs
so that the Corps and EPA can conduct independent analyses. All submitted data must
be clearly presented and traceable to the original samples and subsamples. No permit
will be issued based solely on an applicant’s data analysis.
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TABLE 7. ORGANISMS REQUIRED FOR THE WHOLE SEDIMENT TOXICITY AND BIOACCUMULATION TESTS 1
Test
GrouDJTaxa Habitat Scientific Name Duration
TOXICITY lOd
I Amphipod 1 Marine/Estuadne and fine grain Ampelisca abdita
Estuarine Leptocheipjs plum ulosus
Marine/Estuarine nd coarse grain Eohaustor,us estuarius
Freshwater Hyalella azteca
II Non-Amphipods
Mysid Marine/Estuarine Americamysis bahia
Midge larva Freshwater Chironomus fentans
BIOACCUMULATION 28 d
I Bivalve Marine/Estuanne Macoma nasuta
II Polychaete worm Marine/Estuarine Nereis virens
Oligochaete 2 Freshwater Lumbriculus variegatus
‘One species from this grouping is required depending upon disposal site Conditions
2 Only one bioaccumulat ion test species is available and required for freshwater tests
7-6
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TABLE 8. TISSUE CHEMICAL CONSTITUENTS AND TARGET QUANTITATION LIMITS
ROUTINELY USED FOR BIOACCUMULATION EVALUATIONS OF PROPOSED
DREDGED MATERIAL.
Target
Analytical Quantitation
Limit
Chemical Constituent (wet weight)
TOTAL LIPIDS EPA (1995c) 0.1%
TOTAL WATER CONTENT EPA (1986, 1987) 0.1%
METALS ppm 2
Arsenic 200.8, 7061 0.5
Cadmium 200.8, 7131A 0.1
Chromium 200.8, 7191 1.0
Copper 200.8,7211 1.0
Lead 200.8, 7421 1.0
Mercury 7471 0.02
Nickel 200.8, 6010A 1.0
Zinc 200.8, 7950 1.0
ORGANICS
Pesticides 8081B 2 I ppb
Aidrin
cis- and trans-Chiordane
cis- and trans-Nonachior
Oxychiordane
p,p’-DDT, DDE, DDD
Dieldrin
Endosulfan I and II
Endrin
Heptachlor
Heptachior epoxide Hexachlorobenzene
Lindane
Methoxychlor
Toxaphene 50 ppb
7-7
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9/5102
TABLE 8. CHEMICAL CONSTITUENTS AND QUANTITATION LIMITS ROUTINELY
USED FOR BIOACCUMULATION EVALUATIONS OF PROPOSED DREDGED
MATERIAL (continued).
Target
Analytical Quàntitation
Limit
Chemical Constituent (wet weight)
PCB Congeners 3 8082A 2 0.5
ppb 2
8 2,4’dICB
18 2,2,5 triCB
28 2,4,4 triCB
44 2,2’,3,5’tetraCB
52 2,2’,5,5’ tetraCB
66 2,3’,4,4’tetraCB
101 2,2’,4,5,5’ pentaCO
105 2,3,3’,4,4’ pentaCB
118 2,3’,4,4’,5 pentaCB
128 2’,3,3’,4,4’ pentaCB
138 2,2’,3,4,4’,5’ hexaCB
153 2,2’,4,4’,5,5’ hexaCB
170 2,2 ’,3,3’4,4’,5 heptaCB
180 2,2’,3,4,4’,5,5’ heptaCB
187 2,2’,3,4’,5,5’,6 heptaCB
195 2,2’,3,3’,4,4’,5,6 octaCB
206 2,2’,3,3’,4,4’,5,5’,6 nonaCB
209 2 2 ,3 ,3 ’,4 ,4’ ,5,5’,6,6’ dec&CB
PAHs 1625C, 8270C, 8100, 2 Oppb 2
NOM (1 993)2
Acenaphthene
Acenaphthylene
Anthracene
Benzo(a)anthracene
Benzo(a)pyrene
Benzo(b)fluoranthene
Benzo(k)fluoranthene
Benzo(g, h ,i)perylene
Chrysene
Dibenzo(a, h)anthracene
Fluoranthene
7-8
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TABLE 8. CHEMICAL CONSTITUENTS AND QUANTITATION LIMITS ROUTINELY
USED FOR BIOACCUMULATION EVALUATIONS OF PROPOSED DREDGED
MATERIAL (continued).
Target
Analytical Quantitation
Limit
Chemical Constituent Method 1 (wet weight)
PAHs (continued) 1625C, 8270C, 8100, 20 ppb 2
NOAA (1993)2
Fluorene
lndeno(1 ,2,3-cd)pyrene
Naphthalene
Phenanthrene
Pyrene
1 The specified methods are recommendations only. Other acceptable
methodologies capable of meeting the TQLs may be used. Sample preparation
methodology (e.g. extraction and cleanup) and sample size may need to be
modified to achieve the required target quantitation limits.
2 Applies to each analyte listed below unless otherwise noted.
Total PCBs are to be estimated based on the following: Total = 2 X [ sum of 18
NOAA summation congeners] (T. Wade, personal communication).
7-9
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TABLE 9. RECOMMENDED STATISTICAL METHODS FOR BIOLOGICAL TESTING 1
Statistic Method
Normality Shapiro-Wilk’s Test;
Kolmogorov-Smirnov (K-S) Test
Normality tests found in SYSTAT or
SPSS
Equality of Variances Bartlett’s Test (should not be used to test
equality of ranks)
Levene’s Test
Fm Test
Cochran’s Test
Parametric Fisher’s Least Significant Difference
(LSD) (if raw or transformed are normally
distributed) in conjunction with analysis of
variance (ANOVA).
Nonparametric LSD on rankits (= van der Waerden’s
Test in Conover (1980)) (if the data
converted to rankits are found to be
normally distributed); or Conover T-Test
(Conover 1980) (if the variances of the
ranks are not significantly different); or
One tailed T-Test for unequal variances
for each pair of treatments (if the ranks
are significantly unequal).
1 Summarized from Appendix D (EPA/ACE 1998)
7-10
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9/5/02
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ASTM. 1998 g. Standard Guide for Conducting Acute Toxicity Tests with Echinoid larvae
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8-1
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9/5/02
Bligh, E.G. and W.J. Dyer. 1959. A rapid method of total lipid extraction and purification.
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Marine sediments: Water column and Interstitial Toxic Effects. Environmental Toxicology
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Burgess, R.M. 1995. US EPA Environmental Research Lab, Narragansett, RI. Telephone
conversation with D. Tomey.
Conover, W.J. 1980. Practical Nonparametric Statistics. 2nd Ed. John Wiley & Sons, New
York, NY 493 pp.
Dixon, W.J. and F.J. Massey. 1983. Introduction to Statistical Analysis. 4th Ed. MaGraw-
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DOA. 1980. Laboratory Soils Testing. Engineering and Design, Engineer Manual EMI 110-
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Laboratory, Cincinnati, OH.
EPA. 1983. Interim Guidelines and Specifications for Preparing Quality Assurance Project
Plans. EPA-600/4-83-004. Prepared by Environmental Protection Agency, Office of Re-
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EPA. 1984. Guidance for the Preparation of Combined/Work Quality Assurance Project
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Regulations and Standards (OWRS) QA-1, Washington, DC.
EPA. 1986. Test Methods for Evaluating Solid Waste. Environmental Protection Agency,
Office of Solid Waste and Emergency Response, Washington, DC.
EPA. 1987. Quality Assurance/Quality Control (QNQC) for 301(h) Monitoring Program:
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9/5/02
Guidance on Field and Laboratory Methods. EPA 430/9-86-004. NTIS Number PB 87-
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Protection by Tetra Tech, Inc., Bellevue, WA.
EPA. 1989. Preparing Perfect Project Plans; A Pocket Guide for the Preparation of Quality
Assurance Project Plans. EPN600/9-89/087. Prepared by Risk Reduction Engineering
Laboratory, Cincinnati, OH. October 1989. 62 pp.
EPA. 1990. ERL-N Standard Operating Procedure Conducting the Sea Urchin Larval
Development Test. ERL-N SOP 1.03.007 Revision 0, December 1990. u.s.
Environmental Protection Agency, Environmental Research Laboratory, Narragansett, RI
l5pp.
EPA. 1991a. Methods for the Determination of Metals in Environmental Samples. EPA-
600/4-91-010. Environmental Services Division, Monitoring Management Branch.
EPA 1991 b. Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters
to Freshwater and Marine Organisms, 4th ed. EPA600/4-90/027. Office of Research and
Development, Washington, D.C. 20460.
EPA. 1992. Determination of Total Organic Carbon in Sediment. Environmental
Protection Agency Region II. Environmental Services Division, Monitoring Management
Branch, Edison, NJ.
EPA. 1993. Recommended Analytical Techniques and Quality Assurance/Quality Control
Guidelines for the Measurement of Organic and Inorganic Analytes in Marine Sediments
and Tissue Samples. Draft, Prepared by US EPA Environmental Research Laboratory,
Narragansett, RI. 83 pp.
EPA. I 994a. Methods for Assessing the Toxicity of Sediment-Associated Contaminants
with Estuarine and Marine Amphipods. U.S. Environmental Protection Agency. Office of
Research and Development. Washington D.C. EPN600/R-94/025.
EPA. I 994b. Short-term Methods for Measuring Chronic Toxicity of Effluents and surface
waters to Marine and Estuarine Organisms. Second Ed. U.S. Environmental Protection
Agency. Office of Research and Development, Cincinnati, OH. 341 pp. EPN600/4-91-
8-3
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9/5/02
003.
EPA. 1994 c. EPA Requirements for Quality Assurance Project Plans. Current Draft
Version: August 1994. u.s. Environmental Protection Agency, QualityAssurance Division,
Washington D.C. EPA QNR-5.
EPA. 1995 a. Method 1669: Sampling Ambient Water for Trace Metals at EPA Water
Quality Criteria Levels. U.S. Environmental Protection Agency, Washington D.C. EPA
821 -R-95-034
EPA. 1995 b. Method 1632: Determination of Inorganic Arsenic Trace Elements in Water
by Hydride Generation Flame Atomic Absorption. U.S. Environmental Protection Agency,
Washington D.C. April 1995, Draft EPA 821-R-96-028
EPA. 1995 c. Method 1636: Determination of Hexavalent Chromium by Ion
Chromatography. U.S. Environmental Protection Agency, Washington D.C. April 1995,
Draft EPA 821-R-96-029
EPA. 1995 d. AED Laboratory Operation Procedure Measurement of Total Lipids using
Modification Bligh-Dyer Method. Dated March 15, 1995. U.S. Environmental Protection
Agency. Atlantic Ecology Division. Narragansett, RI
EPA. 1996 a. Method 1631: Mercury in Water by Oxidation, Purge and Trap, and Cold
Vapor Atom Fluorescence Spectrometry. U.S. Environmental Protection Agency,
Washington D.C. January 1996, Draft EPA 821-R-96-001
EPA. 1996 b. Method 1637: Determination of Trace Elements in Ambient Waters by
Chelation Preconcentration with Graphite Furnace Atomic Absorption. U.S. Environmental
Protection Agency, Washington D.C. January 1996, Draft EPA 821-R-96-004
EPA. 1996 c. Method 1639: Determination of Trace Elements in Ambient Waters by
Stabilized Temperature Graphite Furnace Atomic Absorption. U.S. Environmental
Protection Agency. Washington D.C. January 1996, Draft EPA 821-R-96-006
EPA. 1996 d. Method 1640: Determination of Trace Elements in Ambient Waters by On-
line Chelation Preconcentration and Inductively coupled Plasma-Mass Spectrometry. U.S.
Environmental Protection Agency, Washington D.C. January 1996, Draft EPA 821-R-96-
007
EPA. 1998. EPA Guidance for Quality Assurance Project Plans, Final: February 1998.
U.S. Environmental Protection Agency, Quality Assurance Division, Office of Research &
Development, Washington, D.C. EPA QA/G-5.
8-4
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9/5/02
EPA. 2000. Methods for Measuring the Toxicity and Bloaccumulation of Sediment-
associated Contaminants with Freshwater Invertebrates. Second Edition, dated March
2000. U.S. Environmental Protection Agency, Office of Research and Development,
Washington D.C. EPA/600/R-99/-064.
EPA. 2001 a. Appendix A, Method 608 -- Organochiorine Pesticides and PCBs. 625. Title
40 Code of Federal Regulations, Part 136.
EPA. 2001 b. Appendix A, Method 625 -- Base/Neutrals and Acids. Title 40 Code of
Federal Regulations. Part 136.
EPA. 2001 c. Appendix B to Part 136 — Definition and Procedure for the Determination
of the Method Detection Limit. Revision 1.11. Title 40 Code of Federal Regulations, Part
136.
EPA. 2001 d. Methods for Collection, Storage, and Manipulation of Sediments for
Chemical and Toxicological Analyses: Technical Manual (EPA-823-B -01- 002) October
2001. EPA, Standards and Health Protection Division (4305), Office of Science and
Technology, Washington, DC.
EPA. 2001 e. EPA Requirements for QualityAssurance Project Plans, Final: March 2001.
U.S. Environmental Protection Agency, Quality Staff, Office of Environmental Information,
Washington, D.C. EPA QA/R-5.
EPA/USACE. 1977. Ecological Evaluation of Proposed Discharge of Dredged Material
into Ocean Waters, Implementation Manual for Section 103 of Public Law 92-532 (Marine
Protection, Research, and Sanctuaries Act of 1972). Environmental Protection Agen-
cy/Corps of Engineers Technical Committee on Criteria for Dredged and Fill Material,
Environmental Effects Laboratory, U.S. Army Engineer Waterways Experiment Station,
Vicksburg, MS. 2nd printing 1978.
EPA Region 1/USACE New England Division. 1989. Guidance for Performing Tests on’
Dredged Material To Be Disposed of in Open Waters. Environmental Protection Agency,
Region I, Boston, MA/U.S. Army Corps of Engineers, New England Division, Waltham, MA.
32 pp.
EPA/USACE. 1991. Evaluation of Dredged Material for Ocean Disposal (Testing Manual).
Environmental Protection Agency/U.S. Army Corps of Engineers. U.S. Army Engineer
Waters Experiment Station, Vicksburg, MS.
EPNUSACE. 1995. QA/QC Guidance for Sampling and Analysis of Sediments, Water
8-5
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9/5/02
and Tissue for Dredged Material Evaluations Chemical Evaluations. Environmental
Protection Agency/U.S. Army Corps of Engineers. U.S. Environmental Protection Agency,
Office of Water, Washington D.C. EPA 823-B-95-001
EPNUSACE. 1998. Evaluation of Dredged Material for Pmposed forDischarge in Waters
of the U.S. - Testing Manual, Inland Testing Manual. U.S. Environmental Protection
Agency, Office of Water, Washington D.C.
Hampson, B.L. 1977. The Analysis of Ammonia in Polluted Sea Water. Water Research
11:305-308
Klute, A. (ed) 1986. Methods of Soil Analysis Part I. Physical and Mineralogical Methods
2nd ed. American Society of Agronomy, pp 363-375
Lee, H. Ill, B.L. Boese, J. Peltier, M. Sinsor, D.T. Sprecht and R.C. Randall. 1989.
Guidance Manual: Bedded Bioaccumulation Tests. ERL-N Contribution No. NI 11, EPA
600/X-89/302. Newport, OR.
Lee, H. III. 1995. US EPA Environmental Research Lab, Newport, OR. Telephone
conversation with 0. Tomey.
McFarland, V.A. and J.U. Clarke. 1989. Environmental occurrence, abundance, and
potential toxicity of polychlorinated biphenyl congeners: Considerations for a congener -
specific analysis. Environ. Health Perspect. 81: 225-239.
Merks, A.G.A. 1975. Determination of Ammonia in Sea Water with an Ion-Selective
Electrode. Netherlands J. Sea Res. 9: 371-375.
NOM. 1991. Second Summary of Data on Chemical Contaminants in Sediments from the
National Status and Trends Program. NOAA Technical Memo. NOS OMA 59. U.s. Dept.
Commerce, NOAA National Ocean Service, Rockville, MD.
NOM. 1993. Standard Analytical Procedures of the NOAA National Analytical Facility.
NOAA Tech. Mem. NMFS F/NWC-92, 1986-89. National Status and Trends Program,
National Oceanic and Atmospheric Administration, NOAA N/0MA32, 11400 Rockvil le Pike,
Rockville, MD 20852. 3rd ed.
NYDEC. 1991. Analytical Method forthe Determination of PCB Congeners by Fused Silica
Capillary Column Gas Chromatography with Electron Capture Detector. NYSDEC #91-11.
Available from Larry Bailey, New York State Department of Environmental Conservation,
8-6
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9/5/02
50 Wolf Road, Albany, NY 12233, Phone 518-457-7471.
Plumb, R.H., Jr. 1981. Procedure for Handling and Chemical Analysis of Sediment and
Water Samples. Tech. Rep. EPA/CE-81-1. Prepared by Great Lakes Laboratory, State
University College at Buffalo, Buffalo, WY, for the Environmental Protection Agency/U.S.
Army Corps of Engineers Technical Committee on Criteria for Dredged and Fill Material.
U.S. Army Engineer Waterways Experiment Station, Vicksburg, MS.
Pruell, Richard. 1995. US EPA Environmental Research Lab, Narragansett, RI. Telephone
conversation with D. Tomey.
Puget Sound Estuary Program (PSEP). 1986. Total Organic Carbon (TOC). Pages 23-26
in Recommended Pmtoco!s for Measuring Conventional Sediment Variables in Puget
Sound. Prepared for U.S. Environmental Protection Agency, Region 10, Seattle, WA by
Tetra Tech, Inc. Bellevue WA.
Rice, C.D., F.A. Espourteille, and R.J.Hugget. 1987. Analysis of Tributyltin in Estuarine
Sediments and OysterTissue, Crassostrea virginica. Appl. Organomet. Chem. 1:541-544.
SchwartzT.R., D.E.Tillit, K.P. Feltz and P.H. Peterman. 1993. Determination of Mono-and
Non-o,o’-Chlorine Substituted polychlorinated Biphenyls in Aroclors and Environmental
Samples, Chemosphere 26(8):1443-1 460.
Seriano J.L., A M. El-Husseini and T.L. Wade. 1991. Isoletion of Planar Polychicrinated
Biphenyls by Carbon Column Chromatography, Chemosphere 23(7): 915-924.
Snedecor, G.W. and G.C.Cochrane. 1989. Statistical Methods. 8th Ed. The Iowa State
University Press, Ames, IA 507 pp.
Sokal, R.R. and F.J. Rohlf. 1981. Biometry. 2nd Ed. W.H. Freeman and Company, San
Francisco, CA 859 pp.
Steel, R.G.D. and J.H. Tome. 1980. Principles and procedures of Statistics. 2nd Ed.
McGraw-Hill Company, New York, NY. 633 pp.
Ulher, AD. and G.S. Durrel. 1989. Measurement of Butyltin Species in Sediments by n-
pentyl Derivation with Gas Chromatography/Flame Photometric Detection (GCIFPD)
Battelle Ocean Sciences Project N-0519-6100, Duxbury, MA.
Wade, T. 1996. Geochemical and Environmental Research Group, Texas A & M
8-7
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9/5/02
University, College Station, TX. Telephone conversation with D. Tomey.
Winer, B.J. 1971. Statistical Principles in Experimental Design. 2nd Ed. MaGraw-Hill Book
Company, New York, NY 907 pp.
Zar. J.H. 1984. Biostatistical Analysis. 2nd Ed. Prentice-Hall, Inc., Englewood Cliffs, NJ
717 pp.
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9. APPENDICES
I. Additional Priority Pollutants of Concern and Target Quantitation Limits
II. Quality Control Summary Sheets
Ill. Forms for Atterberg Limits
IV. Procedures for Collection of Large Volume Water Samples
V. Sea Urchin Larval Toxicity Test Procedure
VI. Species-Specific Testing Condtions
VII. Pore Water Collection Procedure
VIII. Procedures forAddressing Ammonia Presence in Mysidopsis Sediment
Toxicity Tests (Elizabeth Southerland Memo to Mario P. Del Vicarlo,
dated June 14, 1994)
IX. AED Laboratory Operating Procedure, Measurement of Total Lipids
using Modified Bligh-Dyer Method
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APPENDIX I
Additional Priority Pollutants of Concern and Target Quantitation Limits
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9/5/02
APPENDIX I.
TABLE I-I. Additional chemical constituents 1 , EPA analytical methods and target quantutation limits used for the chemical examination of proposed
dredged material and tissue for bioaccumulation testing.
Sediment Tissue
Analytical Target Target
Chemical Constituent Method Quantitation Limit(dry wt) Quantitation Limit (wet wfl
METALS (ppm) (ppm)
Antimony 7040, 7041 2.5 0.1
Beryllium 7090, 7091 2.5 0.1
Selenium 7740, 7741 1.0 02
Silver 7760 0.2 0 1
Thallium 7840 0.2 0 1
MISCELANEOUS (ppm) (ppm)
Cyanide 9010, 9012 2.0 1.0
Acid Volatile Sulfides Allen et al. (1991) 0.01 umol/g N/A
Organotins Uhier & Duffel (1989) 10 ppb 10 ppb
Rice et al. (1987)
DlOXlNS/DIBENzoFu N 5 8290. 1613 (pptr)
2,3,7.8-TCDD/.TCDF 1 0.5
1.2,3,7,8-PeCDD/-PeCDF 5 0.5
2,3,4,7,8-PeCDF 5 5
1,2,3,4,7,8-HxCDD/ -H COF 5 5
1,2.3,6,7,8-HXCDD/-HxCDF 5 5
1.2,3,7,8,9-HXCDD/-HxCDF 5 5
2.3,4.6,7,8-HxCDF 5 5
1.2.3 ,4.6.7,8-HpCDD/-HpCDF 5 5
1.23.4,7 ,8,9-HpCDF 5 5
OCDD/OCDF 10 10
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9/5/02
TABLE I -i. Additional chemical constituents 1 , EPA analytical methods and quantitation limits used for the chemical examination of proposed
dredged material (continued).
Sediment Tissue
Analytical Target Target
Chemical Constituent Method Quantitation Limit(drv wt) Quantitation Limit (wet wt )
(ppb) (ppb)
WHO PCB CONGENERS 16682 0.252 0.52
PCB -77
PCB-81
PCB-105
PCB-114
PCB-118
PCB - 123
PCB - 126
PCB - 156
PCB - 157
PCB - 167
PCB-169
PCB - 189
BASE/NEUTRALS (ppb) (ppb)
Aromatic Hydrocarbons 82702 202 202
Biphenyl
Benzo(e)pyrene
2 -6-Dimethylnaphtha lene
I -Methyiphenanthrene
I -Methylnaphthalene
2-Methylnaphtha lene
Perylene
(ppb) (ppb)
Phthalates 1625C, 3540, 82502 502 202
Dimethylphtha lata
Diethy lphthalate
Di-n-butylphtha lata
Butyl benzyl phthalate
Bis(2-ethylhexyl) phthalate
Di-octyl phthalate
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TABLE I-I. Additional chemical constituent&, EPA analytical methods and detection limits used for the chemical examination of proposed
dredged material (continued).
Chemical constituents on this optional list would be stipulated by the Corps of Engineers in cooperation with other Federal resource agencies.
Any additional chemicals can be found in EPNUSACE (1995) or other EPA standard guidance
2 Includes all compounds listed.
Includes all compounds listed unless otherwise noted
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APPENDIX II
QUALITY CONTROL SUMMARY SHEETS
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Table li-I: Completeness Checklist
Quality Assurance/Quality Control Questions
Yes/No? Comments?
1 Was the report signed by the responsible applicant approved representative?
2. Were the methods for sampling, chemical and biological testing descnbed in the
Sampling and Analysis Plan (SAP) and the Laboratory QA Plan (LQAP) followed?
3. If not, were deviations documented?
4. Was the SAP approved by the New England District?
5. Did the applicant use a laboratory with a LQAP on file at the New England
District?
6. Did the samples adequately represent the physical/chemical variability in the
dredging area?
7. Were the correct stations sampled (include the precision of the navigation method
used)?
8. Were the preservation and storage requirements in Chapter 8 of the EPNCorps
QNQC Manual (EPNUSACE 1995) and EPA (2001d) followed?
9. Were the samples properly labeled?
10. Were all the requested data included?
11. Were the target quantitation limits (TOLs) met?
12. Were the chain-of-custody forms properly processed
13 Were the method blanks run and were the concentration below the acceptance
criteria?
14. Was the MDL study performed on each matrix (with this data submission) or
within the last 6 months?
15 Were the SRM/CRM analyses within acceptance criteria?
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116 Were the matrix spike/matrix spike duplicates run at the required frequency and
was the percent recovery/RPD within the acceptance criteria?
17. Were the duplicate samples analyzed and were thc RPDs within the required
acceptance criteria?
18. For each analytical fraction of organic compounds, were recoveries for the
internal standard within the acceptance criteria?
19. Were surrogate recoveries within the required acceptance criteria?
20. Were corrective action forms provided for all data?
21. Were all the species-specific test conditions in Appondix V met?
22. dVere the test-specific age requirements met for each test species?
23. Was the bulk physicaL/chemical testing performed on the sediments/composites
that were biologically tested?
.
24. Were the mortality acceptance criteria met for the water column and sediment
toxicity tests?
-
25. Were the test performance requirements in Table 11
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9/5/02
Table 11-2: QualIty Control Summary To be completed for Sediment and Tissue Matrices
Quality Control (QC)
Element
Acceptance Criteria*
Criteria
Met?
Yes/No
List results outside
criteria
(Cross-reference results
table in data report)
Location of
Results
(Retained at Lab
or in Data
Initial Calibration
Must be performed prior to the
analysis of any QC sample or field
sample.(.c20 % RSD for each
compound)
Package)
Retained at Lab
Calculation of Method
Detection Limits (MDLs)
For each matrix, analyzed once per 6
month period (or with each group of
field samples if MDL data have not
been submitted in previous 6
months), - See Section 5.4 for MDL
procedure
In Data Package
Calibration Verification (Second
Source)
Once, after initial calibration (80 -
120% recovery of each compound)
Retained at Lab
Continuing Calibration
At the beginning of every 12 hour
shift(j 15% D)
Retained at Lab
Standard Reference Materials
Within the limits provided by vendor
In Data Package
Method Blank
No target analytes> TDL
Matrix Spike/Matrix Spike
Duplicate (MSIMSD)
One set (MS/MSD) per group of field
samples. Must contain all target
analytes. (Recovery Limits 50-
120%; RPD <30%)
In Data Package
In Data Package
•
Parameter: Polyaromatic Hydrocarbons (PAH) and other base-neutrals
Method Reference
Number: 8270C
-------
9/5/02
Quality Control (QC)
Element
Acceptance Criteria*
Criteria
Met?
Yes/No
List results outside
criteria
(Cross-reference results
table in data report)
Location of
Results
(Retained at Lab
or in Data
Analytical Replicates
Analyze one sample ri duplicate
(water in triplicate) for each group of
field samples (% RSD <30)
Package)
In Data Package
Surrogate Recoveries
Calculate % recovery. (30 - 150%
Rec.)
In Data Package
Internal Standard Areas
Wthjri 50 -200% of internal
standards in continuing calibration
check
In Data Package
* The Quality Control Acceptan Cntena are general guidelines. If alternate criteria are used, they must be documented
in this table.
-------
9/5/02
Table 11-3: Quality Control Summary
To be completed for Sediment, Tissue and Water Matrices
Parameter: Pesticides
Method Reference Number: 8081 B
— Quality Control (QC)
Element
Acceptance Criteria*
Criteria
Met?
YeslNo
List results outside
criteria
(Cross-reference results
table in data report)
Location of
Results
(Retained at Lab
or in Data
Initial Calibration
Must be performed prior to the
analysis of any QC sample or field
sample.(<20 % RSD for each
compound)
Package)
Retained at Lab
Calculation of Method
Detection Limits (MDLs)
For each matiix, analyzed once per 6
month period (or with each group of
field samples if MDL data have not
been submitted in previous 6
months), - See Section 5.4 for MDL
procedure
In Data Package
Calibration Verification (Second
Source)
Once, after initial calibration. (80 -
120% recovery of each compound)
Retained at Lab
Continuing Calibration
Every 20 injections (± 15 % D)
Retained at Lab
Standard Reference Materials
Within the limits provided by vendor
In Data Package
Method Blank
No target analytes> TOL
In Data Package
-------
9/5/02
Quality Control (QC)
Element
Acceptance Criteria*
Criteria
Met?
Yes/No
List results outside
criteria
(Cross-reference results
table in data report)
Location of
Results
(Retained at Lab
or in Data
Matrix Spike/Matijx Spike
Duplicate (MS/MSD)
One set (MSIMSD) per group of field
samples. Must contain all target
anaiytes. (Recovery Limits 50-120%;
RPD <30%)
Package)
In Data Package
Analytical Replicates
Analyze one sample in duplicate
(water In triplicate) for each group of
field samples (% RSD < 30)
In Data Package
Surrogate Recovenes
Calculate % recovery. (30 - 150%
Rec.)
In Data Package
* The Quality Control Acceptance Critena are general guidelines, If alternate criteria are used, they must be documented
in this table.
-------
9/5/02
Table 11-4: Quality Control Summary To be completed for Sediment, Tissue and Water Matrices
Parameter: Polvchorjnated Biphenvis (PCB congeners)
Method Reference Number:8082A
Quality Control (QC)
Element
Acceptance Criteria*
Criteria
Met?
YeslNo)
List results outside
criteria
(Cross-reference
results table in data
Location of
Results
(Retained at Lab
or in Data
Initial Calibration
Must be performed prior to the
analysis of any OC sample or field
sample.(<20 % RSD for each
compound)
report)
Package) —
Retained at Lab
Calculation of Method
Detection Limits (MDLs)
-
For each matrix, analyzed once per 6
month period (or with each group of
field samples if MDL data have not
been submitted in previous 6
months), - See Section 5.4 for MDL
procedure
In Data Package
—
Calibration Verification (Second
Source)
Once, after initial ca’ibration. (80 -
120% recovery of each compound)
Retained at Lab
Continuing Calibration
Every 20 injections (± 15 % D)
Retained at Lab
Standard Reference Materials
Wthin the limits prov:ded by vendor
In Data Package
Method Blank
No target arialytes > TDL
In Data Package
Matrix Spike/Matrix Spike
Duplicate (MS/MSD)
One set (MS/MSD) per group of field
samples. Must contain all target
analytes. (Recovery Limits 50-120%,
RPD <30%)
In Data Package
-------
9/5/02
Quality Control (QC)
Element
Acceptance Criteria*
Criteria
Met?
YesiNo)
List results outside
criteria
(Cross-reference
results table in data
report)
Location of
Results
(Retained at Lab
or in Data
Package)
Analytical Replicates
Analyze one sample in duplicate
(water in triplicate) for each group of
field samples (% RSD <30)
In Data Package
Surrogate Recoveries
Calculate % recovery. (30 - 150%
Rec.)
In Data Package
* The Quality Control Acceptance Criteria are general guidelines. If alternate criteria are used, they must be documented
in this table.
-------
9/5/02
Table 11-5: QualIty Control Summary To be completed for Sediments, Tissue and Water Matrices
Parameter: Metals
Method Reference Numbers: Various Reference Numbers
Quality Control (QC)
Element
Acceptance Criteria*
Criteria
Met?
Yes/No
List results outside
criteria
(Cross-reference results
table in data report)
Location of
Results
(Retained at Lab
or in Data
Linear Range Determination for
IcP
Performed Quarterly
Package)
Retained at Lab
Initial Calibrabon for AA, Hg
Performed Daily (Correlation
Coefficient O.995)
Retained at Lab
Calculation of Method
Detection Limits (MDLs)
For each matrix, analyzed once per 6
month period (or with each group of
field samples if MDL data have not
been submitted in previous 6
months), - See Section 5.4 for MDL
procedure
In Data Package
Initial Calibration Verification!
Continuing Calibration
Venfication
Hg: 80 -120% recovery
Other metals: 90- 110% recovery
Retained at Lab
Initial Calibration Blank!
Continuing Calibration Blank
No target analytes> Instrument
Detection Limit (IDL)
Retained at Lab
Standard Reference Materials
Within the limits provided by vendor
In Data Package
Method Blank
No target analytes> TDL
In Data Package
-------
9/5/02
Quality Control (QC)
Element
Acceptance Criteria*
Criteria
Met?
Yes/No
List results outside
criteria
(Cross-reference results
table in data report)
Location of
Results
(Retained at Lab
or in Data
Sample Spike! Sample
Duplicate
One set per group of field samples.
Must contain all target analytes.
Recovery Limits (75-125%: RPD <
20% or < 35%)
Package)
In Data Package
Analytical Replicates
Analyze one sample in duplicate
(water in triplicate) for each group of
field samples (% RSD <30)
In Data Package
* The Quality Control Acceptance Criteria are general guidelines. If alternate criteria are used, they must be documented
in this table.
-------
9/5/02
Table 11-6: Quality Control Summary To be completed for Sediment, Tissue and Water Matrices
Parameter: Other Organic Chemicals not listed
Method Reference Number
Quality Control (QC)
Element
Acceptance Criteria*
Criteria
Met?
Yes/No
List results outside
criteria
(Cross-reference results
table In data report)
Location of
Results
(Retained at Lab
or in Data
Initial Calibration
Must be performed orior to the
analysis of any QC sample or field
sample.(c20 % RSD for each
compound)
Package)
Retained at Lab
Calculation of Method
Detection Limits (MDLs)
For each matrix, analyzed once per 6
month period (or with each group of
field samples if MDL data have not
been submitted in previous 6
months), - See Sect;on 5.4 for MDL
procedure
In Data Package
Calibration Verification (Second
Source)
Once, after initial calibration (80 -
120% recovery of each compound)
Retained at Lab
Continuing Calibration
At the beginning of every 12 hour
shift ( ± 15 % D)
‘
Retained at Lab
Standard Reference Materials
Within the limits provided by vendor
In Data Package
Method Blank
No target analytes> TDL
In Data Package
-------
9/5/02
Quality Control (QC)
Element
Acceptance Cnteria*
Criteria
Met?
Yes/No
•
List results outside
criteria
(Cross-reference results
table in data report)
Location of
Results
(Retained at Lab
or In Data
Matrix Spike/Matrix Spike
Duplicate (MS/MSD)
One set (MS/MSD) per group of field
samples. Must contain all target
analytes. (Recovery Limits 50-
120%; RPD <30%)
•
Package)
In Data Package
Analytical Replicates
Analyze one sample in duplicate
(water in triplicate) for each group of
field samples (% RSD < 30)
In Data Package
Surrogate Recoveries
Calculate % recovery. (30 - 150%
Rec.)
In Data Package
Internal Standard Areas
Within 50-200% of internal
standards in continuing calibration
check
In Data Package
* The Quality Control Acceptance Criteria are general guidelines. If alternate criteria are used, they must be documented
in this table.
-------
9/5/02
Table 11-7: Quality Control Summary To be completed for Sediments only
Quality Control (QC)
Element
Acceptance Criteria*
Criteria
Met?
YeslNo
List results outside
criteria
(Cross-reference
results table in data
Location of
Results
(Retained at Lab or
in Data Package)
Analytical Replicates
Analyze one sample in duplicate for
each group of field samples (%
RPD <25%)
report)
In Data Package
Total Organic Carbon -
Standard Reference
Materials
Wthin the limits provided by vendor
In Data Package
Total Organic Carbon
Analytical Replicates
Analyze one sample in duplicate for
each group of field samples (%
RSD <30)
In Data Package
Parameter: Sediment Grain Size and Total Organic Carbon Analyses
Method Reference
Numbers:
-------
9/5/02
Table 11-8: Quality Control Summary To be completed for Biological Testing only
Parameter: Toxicity Testing _________________________ Method Reference Numbers:
Quality Control (QC)
Element
Acceptance Criteria*
Criteria
Met?
Yes/No
List results outside
criteria
(Cross-reference
results table In data
Location of
Results
(Retained at Lab
or in Data
Test condition requirements for
each species:
Temperature, Salinity, pH, D.O.,
NH 4 + (Total, Un-ionized)
Test conditions w:thin the
requirements specified for each
species
report)
Package)
In Data Package
Test species age
Age/health within guidelines for
each species (Appendix V)
In Data Package
Bulk physicalIchemi l analyses
(If required by the Sampling plan)
Required? If so, performed? Yes or
No
In Data Package
Water column toxicity test:
In Data Package
Control mortality
10% mean
Control abnormality
c 30% musseI/oy ter; 40% clam
larvae
Sediment toxicity test:
-_____________
In Data Package
Control mortality
10% mean (no chamber> 20%)
Compliance with applicable See EPA (1994a) Section 9; Table
test acceptability requirements 11.3
inTable 11.3 (EPA 1994a)
* The Quality Control Acceptance Criteria are general guidelines. If alternate criteria are used, they must be documented
in this table.
-------
9/5/02
APPENDIX III
FORMS FOR ATTERBERG LIMITS
-------
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-------
9/5/02
APPENDIX IV
PROCEDURES FOR COLLECTION OF LARGE VOLUME WATER
SAMPLES
-------
AED LABORATORY OPERATING PROCEDURE AED LOP 2.02.001
OPERATION OF HIGH VOLUME WATER SAMPLER (formerly 2.01 .002)
FOR EXTRACTION OF NON-IONIC ORGANIC ANALYTES REVISION 1
March 1996
PAGE 1 OF 5
POINT OF CONTACT:
Richard McKinney, Chemistry Group
Atlantic Ecology Division
US Environmental Protection Agency
27 Tarzwell Dr.
Narragansett, RI 02882
1.0 OBJECTIVES
The objective of this document is to describe the recommended field use of the high
volume water sampling apparatus. This apparatus concentrates particulate material on
a glass fiber filter and extracts dissolved non-ionic organic contaminants polychiorinated
biphenyls and chlorinated pesticides on polyurethane foam plugs from a large (10-20 L)
water sample. Also included in this LOP is information that may be useful in trouble
shooting problems encountered.
2.0 MATERIALS AND EQUIPMENT
- High volume pump
- Stainless steel coated hoses
- Filter housing
- Foam plug housings (loaded with extracted plugs)
- Generator
- Pre-combusted Type A/B glass fiber filters 293 mm
- Acetone rinsed stainless steel cans with tops
- TWO 18 L containers with DI water
- Labeling tape
- Lab marker
- Lab notebook
- Gloves (field gloves and plastic lab gloves)
- Large ziplock bags
-------
AED LABORATORY OPERATING PROCEDURE AED LOP 2.02.001
OPERATION OF HIGH VOLUME WATER SAMPLER (formerly 2.01.002)
FOR EXTRACTION OF NON-IONIC ORGANIC ANALYTES REVISION 1
March 1996
PAGE 2 OF 5
- Teflon tape
- Duct tape
- Cooler with ice
- Forceps
- Spatulas
- Filter housing wrench
— Crescent wrenches 1 1/4” (2), 11/16”, 1”, 7/8”
- Two large adjustable wrenches
- One hammer
3.0 PROCEDURE
3.1 Preparation
3.1.1. If the pump, hoses, filter housing, and foam plug housings have not been
recently used, they should be cleaned well with Alconox and tap water. Ifpossible, the
pump should be set up in the lab and tap water circulated through it. Any parts of the
apparatus that can be should be thoroughly rinsed with DI water prior to use.
Note: The stainless steel covering the hoses is frayed in some places. It is
advisable to wear work gloves whenever manipulating them to avoid cutting your hands.
3.1.2. Filters should be individually wrapped in clean aluminum foil and
combusted in a 450°C oven, for 6 hours. After the filters have been combusted it is
extremely important that they not be bent, twisted or disturbed in any way. They should
be taken out of the oven and immediately placed in a covered container in which they
can remain until it is time for them to be used. There should be one filter for each
sample, one for each field blank and at least three extra.
3.1.3. Filter containers (stainless steel cans with tops) should be washed, rinsed
with DI water and cleaned with acetone.
-------
AED LABORATORY OPERATING PROCEDURE AED LOP 2.02.001
OPERATION OF HIGH VOLUME WATER SAMPLER (formerly 2.01.002)
FOR EXTRACTION OF NON-IONIC ORGANIC ANALYTES REVISION 1
March 1996
PAGE 3 OF 5
3.1.4. The procedure for the preparation for the foam plugs is included in the LOP
for the ai a1ysis of dissolved organics using foam plugs (AED LOP 2.03.0 18). The
housings should be wrapped in clean aluminum foil for transport to the field.
3.2 Field Use
3.2.1. The pump will float when placed in the water however, a safety line should
be tied from it to the boat.
3.2.2. Pass the intake hose through the water filling it completely with water. This
is necessary to prime the pump. Attach the intake hose to the pump.
3.2.3. Attach the outflow hose to the pump.
3.2.4. Start the generator and start the pump. There should be a strong flow of
water out of the outflow hose.
3.2.5. Once the pump is primed, it may be turned off as long as the operators are
careful not to allow air to enter the device. At this time, open the filter housing and very
carefully place one filter on the screened platform. Hand tighten the screws and then
completely tighten them with the filter housing wrench.
3.2.6. Attach the hose from the bottom of the filter housing to the top of the foam
plug housing.
3.2.7. To take a seawater sample, place the end of the intake hose in the water
making sure not to introduce any air into the system. Start the pump for 5 seconds. Stop
the pump. Attach the hose from the outflow of the pump to the top of the filter housing.
Open the air bleed valve on the top of the filter housing. Start the pump. Shut the air
bleed valve once the air stops coming out (approximately 5 seconds). There should be
a trickle of water coming out of the foam plug. A second hose may be attached to the
outflow of the foam plug housing and the end placed in the empty 1 8L DI water
container. This will make it possible to measure the volume of water sampled.
-------
AED LABORATORY OPERATING PROCEDURE AED LOP 2.02.001
OPERATION OF HIGH VOLUME WATER SAMPLER (formerly 2.01.002)
FOR EXTRACTION OF NON-IONIC ORGANIC ANALYTES REVISION I
March 1996
PAGE 4 OF 5
3.2.8. Pump 18 liters or other amount of water through the apparatus. Turn off
the pump. If the apparatus has not been used recently or was last used in a contaminated
area it would be advisable to take another field blank before sampling the seawater.
3.2.9. Open the air bleed valve on the filter housing. Unscrew the housing top and
carefully remove the top. Examine the filter to see if it is intact. If it is, use the spatulas
to fold the filter and place it in the stainless steel can. Label the can.
3.2.10. Replace the ends of the foam plug housing. Label the housing and wrap
it in aluminum foil. Place the filter and foam plug on ice in the cooler.
4.0 QA/QC
The primary concern at the point of collection of samples for further analysis is to verify
that the system is free from initial contamination and that no cross contamination occurs
between sample locations. This is accomplished by the collection of field blanks as
necessary.
4.1 Field Blanks
4.1.1. To take the field blank, place the end of the intake hose in the DI water
container making sure not to introduce any air into the system. Start the pump for 5
seconds. Stop the pump. Attach the hose from the outflow of the pump to the top of the
filter housing. Open the air bleed valve on the top of the filter housing. Start the pump.
Shut the air bleed valve once the air stops coming out (approximately 5 seconds). There
should be a trickle of water coming out of the foam plug.
4.1.2. Pump as much of the 18 liters of DI water as you can through the apparatus
without getting any air in the system. This should take approximately 10-15 minutes.
Turn off the pump. If the apparatus has not been used recently or was last used in a
contaminated area it would be advisable to take another field blank before sampling the
seawater. Place the intake hose in the second 18 liters of DI water before changing the
-------
AED LABORATORY OPERATING PROCEDURE AED LOP 2.02.001
OPERATION OF HIGH VOLUME WATER SAMPLER (formerly 2.01 .002)
FOR EXTRACTION OF NON-IONIC ORGANIC ANALYTES REVISION 1
March 1996
PAGE 5 OF 5
foam plug and the filter. If not taking a second field blank, the intake hose may be place
back into the seawater.
4.1.3. Open the air bleed valve on the filter housing. Unscrew the housing top and
carefully remove the top. Examine the filter to see if it is intact. If it is, use the spatulas
to fold the filter and place it in the stainless steel can. Label the can.
4.1.4. Replace the ends of the foam plug housing. Label the housing and wrap it
in aluminum foil. Place the filter and foam plug on ice in the cooler.
5.0 TROUBLE SHOOTING
5.1. Pump is on, no water flow - The pump has not been primed properly. Purge
the intake hose of air and reattach. Hold the outflow hose and the foam plug lower in the
boat.
5.2. The filter housing leaks - Wipe standing water off of the top of the housing.
Use the filter wrench to tighten the screws.
5.3. Leaks occur at hosing attachments - Use teflon tape to wrap the male
connectors prior to use.
5.4. Filters break - Experience has shown the breaking filters usually are the result
of rough handling. Place the next filter on and make sure to shield the housing and filter
from the wind while putting the filter on.
6.0 REFERENCES
None.
-------
9/5/02
APPENDIX V
SEA URCHIN LARVAL TOXICITY TEST PROCEDURE
-------
AED LOP 1.03.007
REVISION 1
November, 1996
PAGE! 0F8
POINT OF CONTACT:
Anne Kuhn-Hines
Diane Nacci
Atlantic Ecology Division
US Environmental Protection Agency
27 Tarzwell Dr.
Narragansett, RI 02882
1. OBJECTIVES
The purpose of the sea urchin larval development test is to detennine the effects of effluents
and water samples on survival, growth, and development of larvae of the sea urchin,
Arbacia punctulata.
2. MATERIALS AND EQUIPMENT
- Facilities for holding and acclimating test organisms.
- Laboratory sea urchin culture unit -- See culturing LOP. To test effluent or receiving water
toxicity, sufficient eggs and sperm must be available.
- Environmental chamber or equivalent facility with temperature control (2O±1 C) for controlling
temperature during exposure.
- Water purification system -- Millipore Super-Q, Deionized water (DI) or equivalent. - Balance
- - Analytical, capable of accurately weighing to 0.0001 g.
- Reference weights, Class S -- for checking performance of balance.
- Air pump -- for supplying air.
- Air lines, and air stones -- for aerating water containing adults.
- Vacuum suction device -- for washing eggs.
- pH and DO meters -- for routine physical and chemical measurements. Unless the test is being
conducted to specifically measure the effect of one of the parameters, portable,
field-grade instruments are acceptable.
- Transformer, 10-12 Volt, with steel electrodes -- for stimulating release of eggs and sperm.
- Centrifuge, bench-top, slant-head, variable speed -- for washing eggs.
- Fume hood -- to protect the analyst from formaldehyde fumes.
- Dissecting microscope -- for counting diluted egg stock.
-------
AED LABORATORY OPERATING PROCEDURE AED LOP 1.03.007
CONDUCTING THE SEA URCHIN LARVAL REVISION 1
DEVELOPMENT TEST November, 1996
- Compound microscope -- for examining and counting sperm cells and fertilized eggs.
- Compound microscope with CCD digital camera and low powered objectives (2-lOx
magnification) -- for use with image analyzer (quantification of growth endpoint).
- Cambridge Instruments Quantimet 520 image analyzer with IBM PC/AT (or equivalent) and
video display-- for quantification of growth endpoint.
- Sedgwick-Rafter counting chamber — for counting egg stock and final examination of larvae.
- Hemacytometer, Neubauer -- for counting sperm.
- Count register, 2-place -- for recording sperm and egg counts.
- Refractometer -- for determining salinity.
-Thermometers, glass or electronic, laboratory grade -- for measuring water temperatures.
-Thermometers, bulb-thermograph or electronic-chart type -- for continuously recording
temperature.
- Ice bucket, covered -- for maintaining live sperm.
- Centrifuge tubes, conical, 15 mL -- for washing eggs.
- Cylindrical glass vessel, 8-cm diameter -- for maintaining dispersed egg suspension.
- Beakers -- at least six Class A, borosilicate glass or non-toxic plasticware, 1000 mL for
making test solutions.
- Glass dishes, flat bottomed, 20-cm diameter -- for holding adult urchins during gamete
collection.
- Wash bottles -- for deionized water, for rinsing small glassware and instrument electrodes and
probes.
- Volumetric flasks and graduated cylinders -- Class A, borosilicate glass or non-toxic plastic
labware, 10-1000 mL for making test solutions.
- Syringes, 1-mL, and 10-mL, with 18 gauge, blunt-tipped needles (tips cut off) -- for collecting
sperm and eggs.
- Pipets, volumetric -- Class A, 1-100 mL.
- Pipets, automatic -- adjustable, 1-100 mL.
- Pipets, serological -- 1-10 mL, graduated.
- Pipet bulbs and fillers -- PROPIPETR, or equivalent.
- Tape, colored -- for labelling tubes.
- Markers, water-proof-- for marking containers, etc.
- Sea Urchins (approximately 12 of each sex).
- Scintillation vials, 20 mL, disposable -- to prepare test concentrations.
- Parafilm -- to cover tubes and vessels containing test materials.
- Gloves, lab coat, disposable -- for personal protection from contamination.
- Safety glasses.
- Data sheets (one set per test) -- for data recording (Figure 1).
-------
AED LABORATORY OPERATING PROCEDURE AED LOP 1.03.007
CONDUCTING THE SEA URCHIN LARVAL REVISION 1
DEVELOPMENT TEST November, 1996
- Acetic acid, 10%, reagent grade, in sea water -- for preparing killed sperm dilutions.
- Fomialin, 10% in seawater -- for preserving eggs.
- pH buffers 4, 7, and 10 (or as per instructions of instrument manufacturer) for standards and
calibration check.
- Reagent water -- defined as distilled or deionized water that does not contain substances which
are toxic to the test organisms.
- Effluent, surface water, and dilution water.
- Saline test and dilution water -- The salinity of the test water must be 30%o. The salinity
should vary by no more than ± 2%o among the replicates.
3. PROCEDURE
A. Test Solutions
1. Samples are used directly as collected when sample salinity is between 28 and 32
parts per thousand. If samples do not require salinity adjustment natural seawater is
used in all washing and diluting steps. Local uncontaminated water may be used as
an additional control.
2. If salinity adjustment is required, prepare 3 L of control water at 30%o using
hypersaline brine (see Brine LOP). This water is used in all washing and diluting
steps and as control water in the test. Natural sea water and uncontaminated local
waters may be used as additional controls.
3. Effluent/receiving water samples are adjusted to salinity of 30 0/00.
4. The selection of the effluent test concentrations should be based on the objectives of
the study. A dilution factor of 0.5 is used with this procedure, starting with a high
concentration of 70% effluent (for freshwater effluents). If the effluent is known or
suspected to be highly toxic, a lower range of effluent concentrations should be used.
5. Three replicates are prepared for each test concentration, using 10 mL of solution in
disposable liquid scintillation vials. A 50% (0.5) concentration series can be
preparedby serially diluting test concentrations with control water.
6. All test samples are equilibrated at 20± 1°C before addition of sperm.
-------
AED LABORATORY OPERATING PROCEDURE AED LOP 1.03.007
CONDUCTING THE SEA URCHIN LARVAL REVISION 1
DEVELOPMENT TEST November, 1996
B. Collection and Preparation of Gametes for the Test
Select four females and place in shallow bowls, barely covering the shell with
seawater. Stimulate the release of eggs by touching the test with electrodes from the
transformer. Collect about 3 mL of eggs from each female using a syringe with a
blunted needle. Remove the needle from the syringe before adding the eggs to a 15
mL conical centrifuge tube. Pool the eggs. The egg stock may be held at room
temperature for several hours before use. Note: The egg suspension maybe prepared
during the 1-h sperm exposure.
2. Select four males and place in shallow bowls, barely covering the animals with
seawater. Stimulate the release of sperm by touching the shell with steel electrodes
connected to a 12 V transformer (about 30 seconds each time). Collect the sperm
(about 0.25 mL) from each male, using a 1 mL disposable syringe fitted with an
18-gauge, blunt-tipped needle. Maintain the syringe containing pooled sperm sample
on ice. The sperm must be used in a toxicity test within 1 h of collection.
3. Using control water, dilute the pooled sperm sample to a concentration of about 5 X
l0 sperm/mL (SPM). Estimate the sperm concentration as described below:
a. Make a sperm dilutions of 1:50, 1:100, 1:200, and 1:400, using 30%o
seawater, as follows:
1. Add 400 uL of collected sperm to 20 mL of sea water in Vial A. Mix
by gentle pipetting using a 5-mL pipetter.
2. Add 10 mL of sperm suspension from Vial A to 10 mL of seawater in
Vial B. Mix by gentle pipetting using a 5-mL pipetter.
3. Add 10 mL of sperm suspension from Vial B to 10 mL of seawater in
Vial C. Mix by gentle pipetting using a 5-mL pipetter.
4. Add 10 mL of sperm suspension from Vial C to 10 mL of seawater in
Vial D. Mix by gentle pipetting using a 5-mL pipetter.
-------
AED LABORATORY OPERATING PROCEDURE AED LOP 1.03.007
CONDUCTING THE SEA URCHIN LARVAL REVISION 1
DEVELOPMENT TEST November, 1996
5. Discard 10 mL from Vial D. (The volume of all suspensions is 10
mL).
b. Make a 1:2000 killed sperm suspension and determine the SPM.
1. Add 10 mL 10% acetic acid in seawater to Vial C. Cap Vial C and
mix by inversion.
2. Add 1 mL of killed sperm from Vial C to 4 mL of seawater in Vial E.
Mix by gentle pipetting with a 5-mL pipetter.
3. Add sperm from Vial B to both sides of the Neubauer hemacytometer.
Let the sperm settle 15 mm.
4. Count the number of sperm in the central 400 squares on both sides of
the hemacytometer using a compound microscope (400X). Average
the counts from the two sides.
5. SPM in Vial E = i0 4 x average count.
c. Calculate the SPM in all other suspensions using the SPM in Vial E above:
SPM in Vial A = 40 x SPM in Vial B
SPMinVia1B = 2OxSPMinVialE
SPMinVia ID = 5xSPMinVia IE
SPM in original sperm sample = 2000 x SPM in Vial B
d. Dilute the sperm suspension with a concentration greater than 5 x 1 SPM to
5x 10 SPM.
Actual SPMJ(5 x 10 ) = dilution factor (DF)
[ (DF) x 10] - 10 = mL of seawater to add to vial.
4. Wash the pooled eggs three times using control .water with gentle centrifugation
(SOOxg or the lowest possible setting) for 3 mm using a tabletop centrifuge). If the
wash water becomes red, the eggs have lysed and must be discarded.
-------
AED LABORATORY OPERATING PROCEDURE AED LOP 1.03.007
CONDUCTING THE SEA URCHIN LARVAL REVISION I
DEVELOPMENT TEST November, 1996
a. Dilute the egg stock, using control water, to about 2000 ± 200 eggs/mL.
1. Remove the final wash water from the eggs and transfer the washed
eggs (by refilling the centrifuge tube with control water and
repeatedly inverting to resuspend the eggs) to a beaker containing a
small amount (about 50 mL) of control water. Add control water to
bring the eggs to a volume of 200 mL (“egg stock”).
2. Mix the egg stock using gentle aeration. Cut the point from a pipet
tip and transfer I mL of eggs from the egg stock to a vial containing 9
mL of control water. (This vial contains an egg suspension diluted
1:10 from egg stock).
3. Mix the contents of the vial using gentle pipetting. Cut the point
from a pipet tip and transfer I mL of eggs from the vial to a
Sedgwick-Rafter counting chamber. Count all eggs in the chamber
using a dissecting microscope at lOX (“egg count”).
4. Calculate the concentration of eggs in the stock. Eggs/mi, = lOx (egg
count). Dilute the egg stock to 2000 eggs mL by the formula below.
b. If the egg count is equal to or greater than 200:
(egg count) - 200 = volume (mL) of control water to add
to egg stock
c. If the egg count is less than 200, allow the eggs to settle and remove enough
control water to concentrate the eggs to greater than 200, repeat the count,
and dilute the egg stock as above. 100 mL of egg stock are required to
perform this test.
d. Transfer 1 mL of the diluted egg stock to a vial containing 9 mL of control
water. Mix well, then transfer 1 mL from the vial to a Sedgwick-Rafter
counting chamber. Count all eggs using a dissecting microscope. Confirm
that the final egg count = 200/mi, ± 20.
-------
AED LABORATORY OPERATING PROCEDURE AED LOP 1.03.007
CONDUCTING THE SEA URCHIN LARVAL REVISION 1
DEVELOPMENT TEST November, 1996
5. Mix the egg stock well, subsample 100 mL, and place the subsample in a clean
beaker. Add 10 mL of the proper sperm dilution to the beaker and mix well. This
will result in a egg:sperm ratio of 1:2500, which should allow acceptable egg
fertilization 1 hr after sperm addition.
C. Start of the Test
1. Mix the diluted embryo suspension (2000 embryos/mL), using gentle aeration. Add
1 mL of diluted egg suspension to each test vial using a wide mouth pipet tip.
Incubate covered for 48 hours 20± 1°C.
D. Termination of the Test
1. Terminate the test and preserve the samples by adding 2 mL of 10% formalin in
seawater to each vial.
2. Vials may be evaluated immediately or capped and stored for as long as one week
before being evaluated.
3. Each vial is thoroughly mixed and a 1 mL aliquot added to a Sedgwick-Rafter
counting chamber for microscopic observation and image analysis. The total number
of larvae and of appropriately developed larvae (pluteii) are counted to determine
survival and development for each treatment. Fifty larvae per replicate are also
observed using the image analysis system and measured for maximum length, total
area, and shape (a function relating observed shape to that of a circle).
4. QA/QC
A. STATISTICAL ANALYSIS AND DATA USAGE
1. Tabulate and summarize the data.
2. An estimate of the effluent concentration which would cause a 50% toxic effect
(EC5O) for each parameter is calculated using Trimmed Spearman-Karber analysis
(Hamilton, Russo, and Thurston, 1977). One-way analysis of variance (ANOVA)
followed by Dunnetts Procedure (Dunnett, 1955) is used to compare single
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AED LABORATORY OPERATING PROCEDURE AED LOP 1.03.007
CONDUCTING THE SEA URCHIN LARVAL REVISION 1
DEVELOPMENT TEST November, 1996
treatments to the control in order to estimate no effect and least effect concentrations
(NOEC and LOEC values).
3. Data are used along with other toxicity tests in assessing the toxicity of an effluent or
receiving water.
5. TROUBLE SHOOTING
1. Toxic substances may be introduced by contaminants in dilution water, glassware,
sample hardware, and testing equipment.
6. REFERENCES
Dunnett, C.W. 1955. A multiple comparisons procedure for comparing several treatments with a
control. JASA 50:1096-1101.
Hamilton, M.A., R.C. Russo, and R.V. Thurston. 1977. Trimmed Spearman-Karber method for
estimating median lethal concentrations in toxicity bioassays. Environ. Sci. Technol.
1 1(7):714-719.
US EPA. 1988. Short-Tenn Methods for Estimating the Chronic Toxicity of Effluents and
Receiving Waters to Marine and Estuarine Organisms. Weber, C.I., et al (eds). EPA Office
of Research and Development EPA-600/4-87/028 (May 1988).
-------
9/5/02
APPENDIX VI
SPECIES-SPECIFIC TESTING CONDITIONS
-------
E-l
SUM1 ’1ARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR MYSID
SHRIMP, Mysidopsis bahia, M. bigelowi, M. almyra, Neomysis americana, Hobnesimysis costasa, ACUTE
TOXICITY WATER COLUMN TESTS
1. Test type:
2. Test duration:
3. Temperature:
4. Salinity:
5. Light quality:
6. Light intensity:
7. Photoperiod.
8. Test chamber size:
9. Test solution volume:
10. Renewal of test solutions:
11. Age of test organisms
12. No. organisms per test chamber:
13. No. replicate chambers per concentration:
14. No. organisms per concentration:
15. Feeding regime
16. Test chamber cleaning:
17. Test solution aeration.
18. Dilution water:
19. Test concentrations:
20. Dilution series:
21. Endpoint:
Static Non-renewal
96 h
20±1°C. or 25±1°C for
Mysidopsis bahia
Mysidopsis bigelowi
Mysidopsis almyra
20±1°C for
NeomysLc americana
12±1°C for
Hol,nesiniysis costata
25-30 %o ±10% except for Holmesimysis costata which
is to be 32-34 %o ±10%
Ambient Laboratory
10-20 uE/m 2 /s (50-100 ft-c)
16USD
250 mL minimum
200 ml. minimum
None
I -5d; 2 4 hrangeinage
10 minimum
5 minimum
50 minimum
Anemia nauplii are made available while holding pnor
to the test; feed 0.2 mL of concentrated suspension of
Anemia naupili 24 h old, daily (approximately 100
nauplii per mysid)
None
If needed to maintain DO> 40% for:
Mysidopsis bahia
Mysidopsis bigelowi
Mysidopsis almyra
Neomysis americana
and DO> 60% saturation for:
Hobnesinzysis costata
(<100 bubbles/mm.)
Natural seawater or modified GP2, Forty Fathoms® or
equivalent, artificial seawater prepared with Millipore
MILLI-Q® or equivalent or deionized water
Three concentrations for site sediment, and control
water
100%, 50%, 10%
Survival
-------
E-2
22. Sampling and sample holding requirements: <8 wk (sediment); elutriates are to be used within 24 h
- of preparation
23. Sample volume required: i L per site
24. Test acceptability criterion: 90% survival in controls
REFERENCE:
USEPA. 1991. Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and
Marine Organisms, 4th Ed. EPAJ600/4-90/027.
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E-7
SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA, FOR
SHEEPSIEEAD MINNOW, Cyprinodon variegatus, INLAND SILVERSIDE, Menidia be, yllina,
ATLANTIC SILVERSIDE, M. menidia, TIDEWATER SILVERSIDE, M. peninsulae, ACUTE
TOXICITY WATER COLUMN TESTS
1. Test type:
2. Test duration:
3. Temperature:
4. Salinity:
5. Light quality:
6. Light intensity:
7. Photoperiod
8. Test chamber size:
9. Test solution volume:
10. Renewal of test solutions:
11. Age of test organisms:
12. No. organisms per test chamber:
13. No. replicate chambers per concentration.
14. No. organisms per concentration:
15. Feeding regime:
16. Test chamber cleaning.
17. Test solution aeration:
18. Dilution water
19. Test concentrations:
20. Dilution series:
21. Endpoint:
22. Sampling and sample holding requirements:
23. Sample volume required:
24. Test acceptability criterion:
REFERENCE:
Static Non-renewal
96 h
20 or 25±1°C
Sheepshead minnow: 5-30 %o ± 10%
Silversides: 5-32 %o ± 10%
Ambient Laboratory
10-20 uE/m 2 /s (50-100 ft-c)
161J8D
250 mL minimum
200 mL minimum
None
Sheepshead minnow: I - 14 d; 24-h range in age
Silversides: 9 - 14 d; 24-h range in age
10 minimum
5 minimum
50 minimum
Anemia nauplii are made available while holding prior
to the test; add 0.2 mL Anemia nauplii concentrate at
48h
None
If needed to maintain DO> 40% saturation
(<100 bubbles/nun.)
Natural seawater or modified GP2, Forty Fathoms® or
equivalent, artificial seawater prepared with Mifipore
MILLI-Q® or equivalent or deionized water
Three concentrations for site sediment, and control
water
100%. 50%, 10%
Survival
<8 wk (sediment); elutriates are to be used within 24 h
of preparation
4 L per site
90% survival in controls
USEPA. 1991. Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and
Marine Organisms, 4th Ed. EPA/600/4-90/027.
-------
E-9
REFERENCE
Adapted in part from the Menielia sp. protocol published in:
USEPA. 1991. Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and
Marine Organisms, 4th Ed. EPA/60014-90-027.
and from EPA in-house expertise, ERL-Narragansen, RI.
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E- 13
SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR OYSTER,
Crassostrea vfrginica, AND MUSSEL, Mytilus edulLc, ACUTE TOXICITY WATER COLUMN TESTS
1. Test type:
2. Test duration:
3. Temperature:
4. Salinity:
5. Light quality:
6. Light intensity:
7. Phocopenod:
8. Test chamber size:’
9. Test solution volume:’
10. Renewal of test solutions:
11. Age of test organisms:
12. No. organisms per test chamber:
13. No. replicate chambers per concentration:
14. No. organisms per concentration.
15. Feeding regime:
16. Test chamber cleaning:
17. Test solution aeration.
18. Dilution water:’
19. Test concentrations:
20. Dilution series:
21. Endpoint
22. SampLing and sample
23. Sample volume required
24. Test acceptability’ criterion:
* - Protocol dependent
REFERENCE:
Static Non-renewal
48 h
25±10 C for Crassosirea virginica
16±1° C for Mytilus edulis
18-32± 1 %
Ambient Laboratory
10-20 uFJm 2 /s (SO-i 00 ft-c)
16L18D
1L
500 mL
None
Larvae less than 4 h old
7,500 - 15,000
5 minimum
22500 - 45,000
None
None
None
Natural seawater or modified GP2, Forty
Fathoms®, artificial seawater prepared with
Millipore MILLI-Q® or equivalent or deionized
water
Three concentrations for site sediment, and control
water
None
Shell development to hinged, D -shaped
prodissoconch I larva
<8 wk (sediment); elutriates are to be used within
24 h of preparation
1 L per site
70% or greater survival and 70% shell
development in controls
ASTM. 1989. E 724-89. Standard guide for conducting static acute toxicity tests starting with embryos of four
species of saltwater bivalve molluscs. Annual Book of ASTM Standards, Vol. 11.04. American Society for
Testing and Materials, Philadelphia, PA.
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E-14
SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR SEA
URCHINS, Strongylocesarosus sp., Lytechinus pktus, AND SAND DOLLAR, Dendrarter sp., ACUTE
TOXICITY WATER COLUMN TESTS
1. Test type: Static Non-renewal
2. Test duration: 48 h
3. Temperature: 12°C
4. Salinity: 30-32 %o
5. Light quality: Ambient Laboratory
6. Light intensity: 10-20 uEIm 2 /s (50-100 ft-c)
7. Photoperiod: Not essential
8. Test chamber size: 20 mL minimum
9. Test solution volume: 10 mL minimum
10. Renewal of test solutions: None
ii. Age of test organisms: 1 h embryos
12. No. organisms per test chamber: 2000
13. No. replicate chambers per concentration: 3 minimum
14. No. organisms per concentration: 6000 minimum
15. Feeding regime: None
16. Test chamber cleaning: None
17. Test solution aeration: None
18. Dilution water: Natural seawater or modified GP2, Forty Fathoms® or
equivalent, artificial seawater prepared using Millipore
MILLI-Q® or equivalent or deionized water and 3x
brine to maintain constant salinity across tests
19. Test concentrations: Three concentrations for site sediment, and control
water
20. Dilution series: 100%, 50%, 10%
21. Endpoint: Survival, Embryo Development
22. Sampling and sample holding requirements: <8 wk (sediment); elutriates are to be used within 24 h
of preparation
23. Sample volume required: I L per site
24. Test acceptability criterion: 70% survival and 70% normal embryo development
in controls
REFERENCE:
USEPA. 1990. Conducting the Sea Urchin Larval Development Test. ERL-Narragansect Standard Operating
Procedure 1.03.007.
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E-17
SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR THE
AMPHIPOD, Ampelisca abdita, ACUTE TOXICITY SEDIMENT TESTS
1. Test type:
2. Test duration:
3. Temperature:
4. Salinity:
5. Light quality:
6. Light intensity:
7. Photopenod:
8. Test chamber size:
9. Test solution volume:
10. Sediment depth:
11. Renewal of test solutions:
12. Age of test organisms:
13. No. of organisms per test chamber:
14. No. replicate chambers per sediment:
15. No. organisms per sediment:
16. Feeding regime:
17. Test chamber cleaning:
18. Test solution aeration:
19. Dilution water:
20. Test concentrations:
21. Dilution series:
22. Endpoint
23. Sampling and sample holding requirements:
24. Sample volume required:
25. Test acceptability criterion:
REFERENCE:
Static Non.renewal*
lOd
20°C
20 to 35 %o
Ambient Laboratory
10-20 uEIm 2 /s (50-100 ft-c)
Continuous Light
IL
Vol to 950 mL
4 cm minimum
None ”
Immature amphipods, or mature females only
20
5
100 to 150
None
None
Trickle-flow (< 100 bubbleslmin.)
Natural seawater or modified GP2, Forty Fathoms® or
equivalent, artificial seawater prepared using Millipore
MILLI-Q® or e uivaicnt or deionized water
Site sediment, a reference sediment and a control
sedmient
N/A
Survival
<8 wk
2L
90% survival in controls
ASTM. 1994. E1367-92. Standard guide for conducting 10-day static sediment toxicity tests with marine and
estuaiine amphipods. Annual Book of ASTM Standards, Vol. 11 04. American Society for Testing and
Materials, Philadelphia, PA.
* Static renewal, intermittent flow or continuous flow tests may be used where it is necessary to maintain
water quality parameters, e.g., dissolved oxygen (DO) and where ammonia is a water quality parameter of
concern (cf. Section 11.2.2). For static renewal tests the overlying dilution water should be changed every 48
h at a minimum .
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E-18
SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR THE
AMPHIPOD, Leptocheinss pluinulosus, ACUTE TOXICITY SEDIMENT TESTS
1. Test type: Static Non renewal*
2. Test duration: 10 d
3. Temperature: 20-25°C
4. Salinity: 20 % (range 2 - 32 % )
5. Light quality: Ambient Laboratory
6. Light intensity: 10-20 uE/m 2 Is (50-100 ft-c)
7. Photopetiod: 16USD
8. Test chamber size: 1 L
9. Test solution volume Vol. to 950 mL
10. Sediment depth: 2 cm minimum
11. Renewal of test solutions: None
12. Age of test organisms: Mature 3 - 5 mm mixed sexes
13. No. of organisms per test chamber: 20
14. No. replicate chambers per sediment: 5
15. No. organisms per sediment: 100
16. Feeding regime: None
17. Test chamber cleaning: None
18. Test solution aeration: Trickle-flow (< 100 bubbles/mm.)
19. Dilution water: Natural seawater or modified 0P2, Forty Fathoms® or
equivalent, artificial seawater prepared with Millipore
MILLI-Q® or equivalent or deionized water
20. Test cOncentrations: N/A
21. Dilution series: N/A
22. Endpoint: Survival
23. Sampling and sample holding requirements: <8 wk
24. Sample volume required: 2 L
25. Test acceptability criterion: 90% survival in controls
REFERENCE:
ASTM. 1994. El367-92. Standard guide for conducting 10-day static sediment toxicity tests with marine and
estuarine amphipods. Annual Book of ASTM Standards, Vol. 11.04. American Society for Testing and
Materials, Philadelphia, PA.
Schiekat, C.E., B.E. McGee and E. Reinharz. 1992. Testing sediment toxicity in Chesapeake Bay using the
amphipod Leptocheirus plumulosus: an evaluation. Environ. Toxicol. Chem. 11: 225-236.
* Static renewal, intennittent flow or continuous flow tests may be used where it i; necessary to maintain
water quality parameters, e.g., dissolved oxygen (DO). For static renewal tests the overiy di1utiu i tttei
should be changed every 48 h at a minimum.
-------
SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR TilE
AMPHIPOD, Eohaustorius esluarius, ACUTE TOXICITY SEDIMENT TESTS
1. Test type: Static Non-renewals
2. Test duration: 10 d
3. Temperature: 15±3°C
4. Salinity. 2 to 28 %o
5. Light quality: Ambient Laboratory
6. Light intensity: 10-20 uE/m 2 /s (50-100 ft-c)
7. Photoperiod Continuous.Light
8. Test chamber size: I L
9. Test solution volume: Vol. to 950 mL
10. Sediment depth: 2 cm minimum
11. Renewal of test solutions: None*
12. Age of test organisms: Mature amphipods,3 -5 mm, mixed sexes
13. No. of organisms per test chamber: 20
14. No. replicate chambers per sediment. 5
15. No. organisms per sediment: 100
16. Feeding regime: None
17. Test chamber cleaning None
18. Test solution aeration: Tnckle-flow (< 100 bubbles/nun.)
19. Dilution water: Natural seawater or modified GP2, Forty Fathoms® or
equivalent, artificial seawater prepared using Millipore
MILLI-Q® oz equivalent or deionized water
20. Test concentrations: Site sediment, a reference sediment and a control
sediment
21. Dilution series: N/A
22. Endpoint: Survival
23. Sampling and sample holding requirements: <8 wk
24. Sample volume required: 2 L
25. Test acceptability criterion: 90% survival in controls
REFERENCE:
ASTM. 1994. E1367-92. Standard guide for conducting 10-day static sediment toxicity tests with marine and
estuarine amphipods. Annual Book of ASTM Standards, Vol 11.04. American Society for Testing and
Materials, Philadelphia, PA.
Static renewal, intermittent flow or continuous flow tests may be used where it is necessary to maintain
water quality parameters, e.g., dissolved oxygen (DO) and where ammonia is a water quality parameter of
concern (cf. Section 11.2.2). For static renewal tests the overlying dilution water should h a banged every 48
h at a minimum.
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E-25
SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR THE
FRESHWATER AMPHIPOD, Hyalella azteca, ACUTE TOXICITY SEDIMENT TESTS
1. Test type:
2. Test duration:
3. Temperature:
4. Salinity
5. Light quality.
6. Light intensity:
7. Photoperiod:
8. Test chamber size:
9. Test solution volume:
10. Sediment depth:
11. Renewal of test solutions:
12. Age of test organisms:
13. No. organisms per test chamber:
14. No. replicate chambers per sediment:
15. No. organisms per sediment:
16. Feeding regime:
17. Test chamber cleaning:
18. Test solution aeration:
19. Dilution water:
Dilution series
Endpoint
Sampling and sample holding requirements:
Sample volume required:
Test acceptability criterion:
Static Non renewal*
lOd
20 - 25OC
0-15 %o
Ambient Laboratory
19-20 uEfm 2 /s (50-100 ft-c)
16L18D
300 mL minimum
Variable, depending on test type
2 cm minimum
None*
7- 14d
10 minimum
5 minimum
20. Test concentrations:
50 minimum
Variable (None, Tetrafin, YCr”, rabbit chow,
maple leaves)
None
Tnckje-flow (<100 bubbles/mm.)
Moderately hard synthetic water prepared using
Millipore MILLI-Q® or equivalent deionized
water and reagent grade chemicals or 20% DMW,
receiving water, or synthetic water modified to
reflect receiving water hardness
Site sediment, a reference sediment and a control
sediment
N/A
Survival
<8 wk
2L
80% survival in controls
21.
22.
23.
24.
25.
* Slurry of Yeast, Cereal flakes, Trout chow
-------
E-26
REFERENCES:
ASTM. 1994. Method E1383-94. Standard guide for conducting sediment toxicity tests with freshwater
invertebrates. Annual Book of ASTM Standards, Vol. 11.04. American Society for Testing and Materials,
Philadelphia, PA.
USEPA. 1994. Methods for measuring the toxicity and bioaccumulation of sediment-associat j contaminants
with freshwater invertebrates. EPA 6001R-94/024 U.S. Environmental Protection Agency. Duluth, MN.
* Static renewal, intermittent flow or continuous flow tests may be used where it is necessary to maintain
water quality parameters, e.g., dissolved oxygen (DO) and where ammonia is a water quality parameter of
concern (ef. Section 11.2.2). For static renewal tests the overlying dilution water should be changed every 48
h at a minimum.
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E-3 I
SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR MYSID
SHRIMP, Mysidopsi.r bahia, M. bigelow4 M. almyra, Neomysis americana, Holmesimysj.s costala, ACUTE
TOXICITY SEDIMENT TESTS
I. Test type:
2. Test duration:
3. Temperature:
4. Salinity:
5. Light quality:
6. Light intensity:
7. Photopenod:
8. Test chamber size:
9. Test solution volumeS
10. Sediment depth:
11. Renewal of test solutions:
12. Age of test organisms:
13. No. organisms per test chamber:
14. No. replicate chambers per concentration:
15. No. organisms per concentration.
16. Feeding regime:
17. Test chamber cleaning:
18. Test solution aeration:
19. Dilution water:
20. Test concentrations:
Static Non renewa1*
lOd
20±1°C: or 25±1°C for
Mysidopsis bahia
Mysidopsis bigelowi
Mysidopsis almyra
20±1°C for
Neoi’nysis americana
12±1°C for
Holmesimysis costata
25-30 %o ±10% except for Holmesimysis costata which
is to be 32-34 %o ±10%
Ambient Laboratory
10-20 uEfm 2 /s (50-100 ft-c)
16L/8D
250 mL (minimum)
200 mL (minimum)
2 cm minimum
None*
I - 5 d; 24 h range in age
10 minimum
5 minimum
50 mirumum
Anemia nauplii are made available while holding prior
to, but not during, the test; feed 0.2 mL of concentrated
Suspension of Artemia nanplii 24 h old, daily
(approximately 100 nauplii per mysid)
None
If needed to maintain DO> 40% saturation for:
Mysidopsis boizia
Mysidopsis bigelowi
Mysidopsis almyra
Neomysis americana
and DO> 60% saturation for:
Hobnesimysis costota
(<100 bubbles/mm.)
Natural seawater or modified GP2, Forty Fathoms® or
equivalent, artificial seawater prepared with Millipore
MILLI-Q® or equivalent or deionized water
Site sediment, a reference sediment and a control
sediment
N/A
21. Dilution series:
-------
E-32
22. Endpoint: Survival
23. Sampling and sample holding requirements: <8 wk
24. Sample volume required: 1 L
25. Test acceptability criterion: 90% survival in controls
REFERENCE:
Modified from:
USEPA. 1991. Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and
Marine Organisms, 4Th Ed. EPAI600/4-901027.
* Static renewal, intermittent flow or continuous flow tests may be used where it is necessary to maintain
water quality parameters, e.g., dissolved oxygen (DO) and where ammonia is a water quality parameter of
concern (cf. Section 11.2.2). For static renewal tests the overlying dilution water should be changed every 48
h at a minimum.
-------
E-33
SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR MIDGFS,
Osirononsu, Seniwu AND C sjpw*&c ACUTE TOXICITY SEDIMENT TESTS
1. Test type:
2. Test duration:
3. Temperature:
4. Salinhy
5. LIght quality:
6. LIght intensity:
7. Photopedod:
& Test cbamber size:
9. Test solution volume:
Sediment deptlL
Renewal of test solutions:
Age of test organisms:
No. organisms per test chamber:
No. replicate chambers per concentration:
No. organisms per concentration:
Feeding regime:
Test chamber cleaning:
Test solution aeration:
Dilution water
Test concentrations:
Dilution series:
Endpoint:
Sampling and sample holding requirements:
Sample volume required:
Test aceeptability criterion:
Slursy of Yeast, Cereal flakes, Trout chow.
Static Non .renewal°
10 d
20 or 25°C
0%o
Ambient Laboratosy
10-20 ua n 2 , (50-100 ft.c)
16Li D
300 n iL minimum
100 niL sediment minimum; overlying water variable
depending on test type
2 cm minimum
None°
1st - 2nd luster
10 minimum
5 minimum
50 minimum
Variable (None, Tetramin, YCF°)
None
Trickle-flow (< 100 bubbles/mm.)
Variable
Site sediment, a reference sediment and a control
sediment
N/A
Survival
<6 wk
4L
a 70% suMval in controls
Ingersoll, CO. and MX. Nelson. 1990. Testing sediment toxicity with Hyalella azreca (Amphipoda) and
rfpa ius (Dlptera). pp. 93-109. W.G. Landis and W.H. van der Scballe, eds., Aquatic
ToxicoIo , and Risk Assessment: Thirteenth Volume. ASTM Si? 109b. American Sodety for
Testing and Materials, Philadelphia, PA.
ASTh1. 1991. New standard guide for conducting solid-phase sediment toxicity tests with freshwater
Invertebrates. ASTM Draft Document E1383. American Society for Testing and Materials,
Philadelphia, PA.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
-------
E-34
• Static renewal, Intermittent flow or ntinuous flow tests may be used where ft Is necessamy to
maintain water quality parameters, e.g , dissolved ozygen (DO). For static renewal tests the overlying
dilution water should be changed ovemy 48 b at a minimum.
-------
E-46
SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR THE
POLYCHAETE, Nereis virens, SEDIMENT BIOACCUMUL 1flON TESTS
I. Test type:
2. Test duration:
3. Temperature:
4. Salinity:
5. Light quality
6. Light intensity:
7. Photoperiod
8. Test chamber size:
Test solution volume:
Sediment depth:
Renewal of test solutions:
Age of test organisms:
No. organisms per test chamber:
No. replicate chambers per sediment:
No. organisms per sediment:
Feeding regime:
Test chamber cleaning:
Test solution aeration:
Dilution water:
20. Test concentrations:
Dilution series:
Endpoint:
Sampling and sample holding requirements:
Sample volume required:
Test acceptability criterion:
REFERENCE:
Flow-through or Static Renewal
28d
10 to 20°C
20%
Ambient Laboratory
10-20 uEIrn 2 /s (50-100 ft-c)
16L/8D, l4L/IOD, 1211121)
1 L (beaker) or large chamber with multiple worms
composited into a single replicate (e.g.. 20 worms in 20
gallon aquarium)
> 750 mL/worm
4 cm
Row-through = 5-10 vol/d; Static Renewal = 3x/weel t
adult (3 - 15g)
One per IL beaker, 20 per 20 gallon aquanum
5-8 (depending on desired statistical power)
5-8 (assumes values to be determined on individuals)
None
As needed
Moderate, as needed
Natural seawater or modified GP. Forty Fathomr® or
equivalent, artificial seawater prepared with Millipore
MILLI-Q® or equivalent or deionized water
Site sediment, a reference sediment and control
sediment
N/A
Bioaccumulatjon
<8 wk
200 mL per worm
Adequate mass of organisms at test completion for
detection of target analyte(s)
Lee II, H., B. Boese, J. Pelletier, M. Winsor, D. Specht and R. Randall. 1989. Guidance Manual: Bedded
Sediment Bioaccumujatjon Tests. EPAJ600/x-89/302. U.S. Environmental Protection Agency. 232 pp.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
21.
22.
23.
24.
25.
-------
E-49
SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR THE
OLIGOCHAETE, Lumbriculus variegatus, SEDIMENT BIOACCUMULATION TESTS
1. Test type: Static Non renewalt or Overlying Water Renewal
2. Test duration: 28 d
3. Temperature: 20 - 25°C
4. Salinity: 0 %o
5. Light quality: Ambient Laboratory
6. Light intensity: 10-20 uE/m 2 /s (50-100 ft-c)
7. Pbotoperiod: 16L18D
8. Test chamber size: 4 L minimum
9. Test solution volume: 1 L
10. Sediment depth: 3 cm
11 Renewal of test solutions; Variable
12. Age of test organisms: Mixed Age Adults
13. No. organisms per test chamber: 5 g (—500-1000) (Minimum)
14. No. replicate chambers per sediment: 4 minimum
15. No. organisms per sediment: N/A
16. Feeding regime: None
17. Test chamber cleaning: None
18. Test solution aeration: If needed to maintain DO> 40% saturation
(<100 bubbles/mm.)
19. Dilution water: Moderately hard synthetic water prepared using
Millipore MILLI-Q® or equivalent, deionized water
and reagent grade chemicals or 20% DMW, receiving
water, or synthetic water modified to reflect receiving
water hardness
20. Test concentrations: Site sediment, a reference sediment and a control
sediment
21. Dilution series: N/A
22. Endpoint: Bioaccumu lation
23. Sampling and sample holding requixements: <6 wk
24. Sample volume required: 4 L
25. Test acceptability criterion: Adequate mass of organisms at test completion for
detection of target analyte(s)
-------
E-50
REFERENCES:
Ankley, G.T.. R.A. Hake, D.A. Benoit, E.N. Leonard, C.W. West, G.L. Phipps, YR. Mattson and L.A.
Anderson. 1993. Development and evaluation of test methods for benthic invertebrates and sediments:
effects of flow rate and feeding on water quality and exposure conditions. Arch. Environ. Contain. Toxicoi
25:12-19.
Phipps, G.L., G.T. Ankiey, D.A. Benoit and V.R. Mattson. 1993. Use of the aquatic oigochaete Lumbriculus
variegazus for assessing the toxicity and bioaccumulation of sediment-associated contaminants. Environ
Toxicol. Chem. 12:269-279.
* Static renewal, intermittent flow or continuous flow tests may be used where it is necessary to maintain
water quality parameters, e.g., dissolved oxygen (DO) and where ammonia is a water quality parameter of
concern (cf. Section 11.2.2).
-------
E-5 I
SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR THE
MACOMA CLAM, Macoma nasula, SEDIMENT BIOACCUMULATION TESTS
I. Test type:
2. Test duration:
3. Temperature:
4. Salinity:
5. Light quality:
6. Light intensity:
7. Photoperiod:
8. Test chamber size:
9. Test solution volume:
Sediment depth:
Renewal of test solutions:
Age of test organisms-
No. organisms per test chamber:
No. replicate chambers per sediment.:
No. organisms per sediment:
Feeding regime:
Test chamber cleaning
Test solution aeratiow
Dilution water
20. Test concentrations:
21. Dilution series:
22. Endpoint:
23. Sampling and sample holding requirements:
24. Sample volume required:
25. Test acceptability criterion:
Flow-through or Static Renewal
28 d
12- 16°C
25%o
Ambient Laboratory
10-20 uE/m 2 /s (50-100 ft-c)
12L112D, 16L/8D, 1OLI14D
2SOmL - I L (beaker)
> 750 mLfbeaker (e.g., len 250 mL beakers in 8L
aquarium)
50 g wet wt sediment per g wet flesh (without shell)
Flow-through = 5-10 volld; Static Renewal = 3 x./wk
2-4yr,28-45 znmshell length
One (1) per beaker maximum
5 - 8 (depending on desired statistical power)
5 - 8 (assumes values to be determined on individuals)
None
As needed
Moderate, as needed
Natural seawater or modified GP2, Forty Fathoms® or
equivalent, artificial seawater prepared with Millipore
MIW-Q® or equivalent or deionized water
Site sediment, a reference sediment and a control
sediment
10.
11.
12.
13.
14
15.
16.
17.
18.
19.
N/A
Bioaccumulation
<8wk
8L
Adequate mass of organisms at test completion for
detection of target analyte(s)
-------
E-52
REFERENCES:
Lee II, H., B. Boese, J. Pelletier, M Winsor, D. Specht, and R. Randall. 1989. Guidance Manual: Bedded
Sediment Bioaccumulatjon Tests. EPA/600/x-89/302. 232 pp.
Ferraro, S., H. Lee II, R. Ozretich, and D. Specht. 1990. Predicting bioaccumulation potential: A test of a
fugacity-based model. Arch. Environ. Contamin. Toxicol. 19:386-394.
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9/5/02
I
APPENDIX VII.
PORE WATER COLLECTION PROCEDURE
-------
9/5/0 2
APPENDIX VII
Collection of Porewater:
Total and unionized ammonia must be analyzed on the sediment interstitial water using the
ion-selective electrode method (Merks 1975) following the manufacturer’s instructions or
the colorimetric method as described in (Bower and HoIm-Hansen (1980). Interstitial water
should be extracted by centrifuge using the method described in Burgess et al. (1993).
Here, 200 ml of sediment are placed in a 250 ml Teflon centrifuge tube and centrifuged at
4°C for 3 h at 4,000 rpm (2520 G). Burgess (personal communication) indicated that, in
most cases, I h may be adequate. In general, about 20 ml of interstitial water would be
needed.
Analysis of Ammonia:
Total Ammonia may be analyzed using the ammonia probe method (Merks 1975), or the
colorimetric method (Bower & HoIm-Hansen 1980). Acceptable detection limits are 0.1
mgIL. Unionized Ammonia can be calculated using the dissociation model of Whiffield
(1972) as programmed by Hampson (1977). All samples require triplicate analysis.
-------
9/5/02
APPENDIX VIII
PROCEDURES FOR ADDRESSING AMMONIA PRESENCE IN
MYSIDOPSIS SEDIMENT TOXICITY TESTS (ELIZABETH
SOUTHERLAND MEMO TO MARIO P. DEL VICARIO, DATED JUNE 14,
1994)
-------
flt3il4/94 17:51 ‘Th02 26fl 9830 0ST-S. SD
1 UNITED STATES ENVIRONMENTAL PROTECTiON AGENCY
_____ WASHINGTON, D.C. 20460
JJN 1 4 1994
MORA )UM
STJBJECT: Recommendations for •Conductixiq Sediment Toxicity
Test with Mysidopsis bahia when amiionia may be
Present at Toxic Levels
YROM: Elizabeth Southerland, Acting Director • J
Standards and applied Science Division (4305)
Office of Science •and Technology
TO: Mario P. Del Vicario, Chief
Marine and Wetlands Protection Branch.
U.S. EPA Region 2
The purpose of this memorandum is to provide guidance to
U.S. EPA Region 2 on conducting the mysid texi-day solid phase
seljment toxicity test to evaluate dredged material for open
water disposal. This guidance is provided in response to a
letter mailed to Region 2 on April 22, 1994 from Monte Greges,
U.S. . rmy Corps of Engineers, New York District, requesting
guida nce on running the mysid test when ainnonia is present at
potentially toxic concentrations.
The Office of Science and Technology held a. conference call.
on May 2.6, 1994 with. EPA and U.S. i rmy Corps of Engineers
scientists and our consultants to develop an acceptable protocol
for running the mysid test when ammonia may be present at toxic
levels. The following protocol was recommended by conference
call, participants who are identified below as recipients of this
memorand u m.
1. The Corps of Engineers and EPA issued joint guidance on
December 21, 1993 offering recommendations, based on the
• best available information, for reducing ammonia levels in
test systems used for acute amphipod sediment bioassay’s.
When running mysid tests, it is recommended that the
procedure described in the December 21. memorandum be used
with modifications pertaining specifically to Mvsidonsis
bahi .
2 • The Corps of Engineers/EPA December 21 guidance memorandum
states that at certain open-water dredged material disposal
sites (e.g. dispersive situations and situations with well—
oxygenated overlying ter, ammonia and hydrogen sulfide
-------
( )6!14194 17:52 ‘202 260 9830 OST—S4SD
may not be con1- mthants of concern If chenical evidence of
ammonia is present at toxicologically important levels (i.e.
a unon a concentrations exceeding the species—specific
acceptability ranges), and ammonia is not a cont ninant of
concern, the laboratory analyst running the mysid ten-day
sediment toxicity test should reduce ammonia in the in the
test system overlyin water to the appropriate acceptable
level before adding the test organism.
3. or 4ysidopsis bahia , the species-specific acceptable level
for unionized ammonia concentration in the test system
overlying water (i.e sublethal water column concentration
for a ten-day sediment test) is 0.6 ing/L in tests run at
26±160, 31± g/1 g salinity, and pE of 7.9—8.0 using one day
old organisms . At a test pif of 7.5, the acceptable
concentration of iznion-ized ammonia is 03 mg/L. These
acceptability levels were derived on the basis of acute
toxicity tests conducted with a nmonia by D.C. Miller, S.
Poucher, J.A. Cardin, and. 1). ffansen at EPA’s Environmental
Research Laboratory, &arragansett, Rhode Island.
4. If unionized ammonia levels in the test system overlying
water exceed the acceptability level for vsidop js bahip
(0.6 mg/L at p 7.9—8.0 or 0.3 m g I I , at pif 7.5) the system
should be flushed at a rate of two volume replacements per
day until it reaches a concentration of unionized ammonia at
or below the acceptability level. Overlying water should be
aerated during flushing, and the analyst should measure the
overlying water ammonia concentration each day until the
acceptable concentration is reachea. Overlying water should
be sampled approximately 1 cm. above the sediment surface.
5. kfter adding the test organisnis to the systei i, the aflalyst
should ensure that ammonia colicentrat ions remain within an
accaptable range by conducting the toxicity test with
continuous flow or volume replacement not to exceed two
voli cs per day. It is recommended that overlying water
concentration of ammonia be measured again at the end of the
test.
6 • Accurate measurement of sample pH is crucial in the
calculation of the unionized ammonia fraction. EP A’s
Narragansett laboratory recommends the use of specific
equipment and procedures for determining pH of seawater (see
Attachment 1)
We are sending this memorandum concurrently to EPA Region 2
and the conference call participants who recommended guidance. We
ask that conference call participants provide any comments or
modifications of the recommended procedure to Tom Armitage of my
staff by 3une 24, 1994. We will notify Region 2 if any changes
in the guidance are required.
-------
06/14/94 17:53 ‘ ‘202 260 9830 OST—SASD 0O4
Attacbment
• cc: Bob Engler (COE WES)
Torn Dillon (COE WES)
David Moore (coE WES)
Monte Greg es (COE NY D1s :ict)
Gary Ankley (EPA ORD)
Don Miller (EPA ORD)
Norm Rubenstein (EPA O D)
Rick Swartz (EPA ORD
Tom Chase (EPA OWOW)
Alex Lechich (EPA Region 2)
Joel. O’Conner (EPA Region 2)
Dave Toney (EPA Region 1)
John Scott (SAIC)
-------
96/14194 17 53 e202 200 9830 OST-SASD oo5
1
Use of criteria far developing water quality-based peit limits and
for dasigniog waste treab nt facilities requires the selection of an
appropriate wasteload allocation ncdel. Dynamic w de1s are preferred fot the
application qf these criteria (U.S. A 1985b). Limited data or other
considerations might make their use practical, in which case one should
rely on a steady—state de1 (U.S. 1986).
Water quality standards for a ia developed Erca these criteria should
specify use f j n 1 nitoring methods which are coeparable to the
analytical methods iip1oyed to generate the toxicity data base. Total
‘ 4an y be measured using. an automated idopbei o1 blue method, such as
described by Techuicon Industrial Systems (1973) or U.S. E P A (1979) method.
350.1. Tin-ionized ani.oiiia concentrations should be calculated using the
dissociation del of *itfield (1974) as progran d by ffa s i (1977J -. mis
progr was used to calculate ui st of the un-ionized values for saltwater
______ listed in Table 1 and 2 of this document. 1 ccurate measur nt of
sai 1e pE is crucial in the calculation of the im-tonized rtn 4 a fracti .
following equ4is nt end procedures were used by EZIt in tbn iw 4 a
toxicity st” es to e ’)’ance the precision of measurenents In salt water.
The pE meter reported two decisel p3.eoes. A Ross electrode with ceramic
junction used due to its rapid response time; an automatic temperature
compensation probe provided temperature correction. Note that the
respousiveness of a new electrcxle may be enhanced by holding it in sea water
for several days prior to use. p-.. . National Buresu of Standards buffer
solutions for r Hhration preferred for their stability were (1) potassium
28
-------
14 94 09:23 ‘ 202 260 9330 l)ST.S SD
STai ,
____ UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
•. IQo’
ENV RONM ” E ’FA 1 U O A1Z RY
L W L Iv
,, .GANSEr HCOE ISLJ.NO
May 20, 1994
Subjecr Mysid No Effect NH 3 Concen ation for Lethality and pH Issues for Sediment
Toxicity Test Protocols
From: Don C. !vliller
Research Aquatic Biologist, ERL,N
To: Tom Arniitage
Office of Science and Technology (4305)
The following nfo ation is provided in response to the May 16, 1994 can xence call on
sediment toxicity testing where high concentrations of ammonia are present. No mysid tests
are dix ct1y applicable to estimate a 10 day no lethal effect coccen ation for NH .However,
da for other expostue periods are available.
1.Webelievcthat 0.6m 3 /Linthewatercoluumshouldbesublethalforl0day
sediment tests with one day old My3idopsi. bahia at 26 l° C, 31±1 g/lcg salinity and apE of
7.9-8.0. At a test pH of 7i. the sublctha.I concenn ation should be approxin ately 0.3 nig
NH,
The 0.6 mg/L value is supported by:
a. foiw day acute results for Test 16 per J. Cardin 8115186 memo, atached. Test 16
pertains to the present question as it was conducted at the above conditions. The LC5O
is 1.7 tug NH4.. The 7% mortality observed in the 0.95 ing/L tme r piobably is
not significant and may be a no effect concenuarion for a four day test - For 10 day
sediment tests,ihe lower nea ent concentration (038 mg/L) may be required because
the 10 day continued cxposntc may result in m ia1ity at lower concenrinrinnc
b. a 32 day chronic value, 0.232 .mg N H 1 /L , which xepz ents a lower bound no effect
couccnuadon (Miller or al., 1990, attached). This value is based on a 4n anr effect
on survival at 0.331 mg/L at the same test conditions as above This lower protection
• concennuzion zcflcct the eaxcr sensitivity of mysids after mat axion and young
die dpOUch. Since sdonotappeatunnlthy 12 tO 14(at
25°), the lower cbi nic value should not be applied to 10 day se iini nt ,
assuming one day old pnhn I are used.
Therecoromended o.3mgNH 3 /LatpH7iissupporredbyacutetcstsatpH8.Oandl.O
(Figure 2B, MUlct, Ct aL). These results suggest mysid acute sensitivity to vmnnia may
increase as iwth as two-fold at pH 7.5, teladve to pH 8.0, hence requiring the 50% reducdon
in the concentration expected to be sublethal.
2. ALso I portant but not specifii aUy stated in the subject protocol, axe the pincaittioris
-------
flf Ii 4 i)9 23 ‘ 2O2 26i) 53fl
: j nfl
neccssai-v to accumtely measure pH in seawater. .A cura :e caicularion of NH, cor centrations
in the test waLer requires acctwaxe pH measurement. However, measuring pH in ea water is
not straight forward, as indicated iii Miller et al. (See discussion. first para ’aph). Enclosed is
a recore nded from the implementanon section of the EPA saltwater criteria for
r c’nia. We suggest this issue be highlighed in the pro ocoI.
3. Should addidonal studies be desired to better describe the NH no effect concenu-4tion for
inysids, we recommend: (a) flow through testing, using a pH con oiler, or at a niinirnum, 24
h monito ng of pH dining day one, and (b) the tests be conducted for the range of pH
conditions expected in sedini t test ing. The variance shown in the atrneh paper (I gure 2B)
for static tests is due to pH drift in tests which were nor monitored over eight. In contrast,
Figure 2A shows good agreement may be achieved with flow through testi where there vj
24 h monitoring of pH dining day one.
‘mthn nts: Cardin memo
ller et aL paper
NH 3 c±eria implementation
without ap -hments . N. Jawoiski
CL Peach
-------
9/5/02
APPENDIX IX.
AED LABORATORY OPERATING PROCEDURE, MEASUREMENT OF
TOTAL LIPIDS USING MODIFIED BLIGH-DYER METHOD
-------
AED LABORATORY OPERATING PROCEDURE AED LOP 2.03.02 1
MEASUREMENT OF TOTAL LIPIDS USING Revision 0
MODIFIED BLIGH-DYER METHOD. March 15, 1995
Page 1 of3
POINT OF CONTACT:
Environmental Chemistry Group
Atlantic Ecology Division
U.S. Environmental Protection Agency
27 Tarzwell Drive
Narragansett, RI 02882
1. OBJECTIVE
This document defines a procedure based on a modification of the method reported by Bligh
and Dyer (1959). This procedure is used to analyze marine tissues for total lipid content.
2. MATERIALS
Solvents
Methanol - Baxter Pesticide Grade
Chloroform - Baxter Pesticide Grade (ethanol free)
Deionized water
Glassware
TurboVap tubes, 25m1 scintillation vials, and 50m1 centrifuge tubes muffled at 450 degrees F for 6
hours.
Equipment
Mayer N-Evap Analytical Evaporator
Zymark TurboVap Evaporator
Sorvall RC2-V Centrifuge
Kinematica Homogenizer with 12mm tip.
-------
AED LABORATORY OPERATING PROCEDURE AED LOP 2.03.021
MEASUREMENT OF TOTAL LIPIDS USING Revision 0
MODIFIED BLIGH-DYER METHOD. March 15, 1995
Page 2 of 3
3. ANALYTICAL PROCEDURE
All Trophic Transfer samples were stored at -20 degrees c immediately after collection and thawed
just prior to analysis. Solvent ratios in the following procedure are expressed in the order:
chloroform/methanol/water.
3.1) For lobster muscle, place 1 Og wet homogenized tissue in a 50m1 centrifuge tube. For lobster
hepatopancreas and Nereis tissue place 5g wet homogenized tissue in a tared 25m1 scintillation vial.
3.2) Calculate the amount of water that is in the sample by using the formula:{ grams wet x (1 -
dry/wet ratio)] = (ml)water. The (ml)water is used to calculate the appropriate amounts of
chloroform and methanol to add to the centrifuge tube to obtain a solvent volume ratio of 1/2/0.8.
Thus, to calculate the amount of chloroform needed for 4m1 of water in the sample, multiply 4m1 x
1.25 = Sm! chloroform and 2 x chloroform = lOmi methanol. The ratio of
chloroformlmethanollwater in the centrifuge tube or vial is now 5/10/4 or 1/2/0.8 . Add the
appropriate amounts of chloroform and methanol to the centrifuge tube and blend with a 12mm
polytron tip for 60 seconds.
3.3) Add an additional volume of chloroform to the centrifuge tube/vial that is equal to the amount
used in step 2. Blend for 30 seconds.(Solvent volume ratio 1/1/0.4)
3.4) Add an additional volume of water to the centrifuge tube/vial that is equal to the amount
calculated in step 2. Blend for 30 seconds. (Solvent volume ratio 1/1/0.9)
3.5) Cap the tube/vial and centrifuge for 10 minutes. Draw off the chloroform and dispense it into a
turbovap tube for muscle tissue or a 25m1 scintillation vial for hepatopancreas and Nereis tissue.
3.6) Rinse all transfer tools with small portions of chloroform, collecting the washes in the centrifuge
tube or scintillation vial.
3.7) Add an additional volume of chloroform equal to 2 times the amount used in step 2 to the
remaining tissue in the centrifuge tube or vial. Blend for 30 seconds. (Solvent volume ratio 1/1/0.9)
3.8) Cap the tube/vial and centrifuge for 10 minutes. Draw off the chloroform and transfer to the
turbovap tube. Rinse transfer tools with small portions of chloroform into the tube or vial.
3.9) Repeat steps 7 and 8 except shake manually instead of using the Polytron.
3.10) If the extract is cloudy or contains an emulsion, pass it through a layer of sodium sulfate and
collect. Repeat as needed to clarify extract. Rinse apparatus with small portions of chloroform.
-------
AED LABORATORY OPERATING PROCEDURE AED LOP 2.03.021
MEASUREMENT OF TOTAL LIPIDS USING Revision 0
MODIFIED BLLGH-DYER METHOD. March 15, 1995
Page 3 of 3
3.11) For muscle tissue, volume reduce the extract under a nitrogen stream in the turbovap tube to
imI then transfer to a 25m1 scintillation vial and blow to dryness under nitrogen in an N-Evap
evaporator. For hepatopancreas and Nereis tissue extracts (which are already in a 25m1 scintillation
vial) reduce to dryness in the N-Evap evaporator.
3.12) Place the uncapped scintillation vial in an oven at 100 degrees c for 1 hour then allow the vial
to cool in a desiccator for 1 5mm and weigh.
3.13) Calculate the weight percent of total lipid in the sample using the formula: ((g)lipid / (g)dry
sample weight) * 100 = percent lipid.
4. REFERENCES
Bligh, E.G. and W.J. Dyer. 1959. Canadian Journal of Biochemistry and Physiology, 37(8),
pp. 2-8.
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