Batreiie
The Business q/ Innovation
    Environmental Technology
       Verification Program
       Advanced Monitoring
           Systems Center
   Quality Assurance Project Plan for
           Verification of
 Sediment Ecotoxicity Assessment Ring
             (SEA Ring)

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Verification of the Sediment Ecotoxicity Assessment Ring

                         Draft
                     May 16, 2012

                       Version 1


                     Prepared by
                        Battelle
                   505 King Avenue
              Columbus, OH 43201-2693

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                                         SECTION A

                                PROJECT MANAGEMENT



                             Al VENDOR APPROVAL PAGE



                          ETV Advanced Monitoring Systems Center

                       Quality Assurance Project Plan for Verification of
                          the Sediment Ecotoxicity Assessment Ring

                                            Draft

                                         May  16, 2012


                                         APPROVAL:


                       Name
                       Date
                                            Notice
The U.S. Environmental Protection Agency, through its Office of Research and Development, funded and managed,
or partially funded and collaborated in, the research described herein. It has been subjected to the Agency's peer
and administrative review.  Any opinions expressed in this report are those of the author(s) and do not necessarily
reflect the views of the Agency, therefore, no official  endorsement should be inferred.  Any mention of trade names
or commercial products does not constitute endorsement or recommendation for use.

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                                  A2 CONTENTS


Section                                                                         Page

SECTION A: PROJECT MANAGEMENT	3
Al Vendor Approval Page	3
A2 Contents	4

APPENDICES	5
FIGURES	6
TABLES	6

A3 ACRONYMS AND ABBREVIATIONS	7
A4 DISTRIBUTION LIST	9
A5 VERIFICATION TEST ORGANIZATION	10
    A5.1  Battelle's Test Program Roles and Responsibility	11
    A5.2  Technology Representative	14
    A5.3  EPA	15
    A5.4  Verification Test Stakeholders	16
    A5.5  Reference Laboratories	16
A6 BACKGROUND	18
    A6.1  Technology Need	18
    A6.2  SEA Ring Technology Description	19
A7 VERIFICATION TEST DESCRIPTION AND SCHEDULE	22
    A7.1  Verification Test Description	22
    A7.2  Verification Test Schedule	23
    A7.3  Verification Location	23
A8 QUALITY OBJECTIVES	24
A9 SPECIAL TRAINING/CERTIFICATION	30
A10 DOCUMENTATION AND RECORDS	31

SECTION B: MEASUREMENT AND DATA ACQUISITION	32
Bl EXPERIMENTAL DESIGN	32
    Bl.l  Test Procedures	32
           B 1.1.1  Sediment and Water Sampling	32
           Bl.l.2  Benthic and Aquatic Organism Collection	35
           Bl.l.3  SEA Ring Preparation and Operation	36
    B1.2  Laboratory SEA Ring Test	37
           Bl.2.1  Repeatability (Replicate Variability)	38
           Bl.2.2  Comparability	40
           Bl.2.3  Reproducibility	41
    B1.3  EPA/ASTM Method Laboratory Comparability Tests	41
    B1.4  Operational Factors	43
    B1.5  Supporting Analyses	43
    B1.6  Statistical Analysis	44
B2 SAMPLING METHOD REQUIREMENTS	49
    B2.1  Toxicity Test Breakdown - Collection Test Organisms	49
    B2.2  Collection and Analysis of Tissue Chemical Samples	49

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    B2.3  Collection and Analysis of Water and Sediment Samples	49
B3 SAMPLE HANDLING AND CUSTODY REQUIREMENTS	50
    B3.1  Handling of Aquatic Organisms	50
    B3.2  Sample Custody	50
    B3.3  Sample Receipt	51
B4 ANALYTICAL METHOD REQUIREMENTS	52
    B4.1  Water Analysis	52
    B4.2  Sediment and Tissue Analysis	52
    B4.3  Tissue Lipid Analysis	53
    B4.4  Instrument Calibration Requirements	53
    B4.5  Quality Control	54
B5 Quality Control Requirements	56
    B5.1  Reference Toxicant Test	56
    B5.2  Control Performance	56
    B5.3  Test Conditions Acceptability	56
    B5.4  Comparison to Background Tissue Levels	57
B6 INSTRUMENT/EQUIPMENT TESTING, INSPECTION, AND MAINTENANCE	58
B7 INSTRUMENT CALIBRATION AND FREQUENCY	59
B8 INSPECTION/ACCEPTANCE OF SUPPLIES AND CONSUMABLES	60
B9 NON-DIRECT MEASUREMENTS	61
BIO DATA MANAGEMENT	62

SECTION C: ASSESSMENT AND OVERSIGHT	65
Cl ASSESSMENT AND RESPONSE ACTIONS	65
    Cl.l  Performance Evaluation Audit	65
    C1.2  Technical Systems Audits	66
    C1.3  Data Quality Audits	66
    C1.4  QA/QC Reporting	67
C2 REPORTS to Management	68

SECTION D: DATA VALIDATION AND USABILITY	69
Dl Data Review, Verification, and Validation Requirements	69
D2 Verification and Validation Methods	70
D3 Reconciliation with User Requirements	71

SECTION E: REFERENCES	72
                                  APPENDICES
Appendix A: TEST DATA SHEETS
Appendix B: CONTROL CHARTS
Appendix C: CHAIN OF CUSTODY FORMS
Appendix D: SEA RING MANUAL
Appendix E: LABORATORY SOPs

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                                         FIGURES
Figure 1.  Organizational Chart	12
Figure 2.  Schematic of SEA Ring Technology	20
Figure 3.  Multiple Lines of Evidence Use of SEA Ring Technology	21
Figure 4.  Second Generation SEA Ring Device (left). Field Evaluation in Beach Deployment
          (right)	21
Figure 5.  Overview of Sediment Toxicity and Bioaccumulation Testing Approach with Both
          SEA Ring and Standard Laboratory Tests	34
Figure 6.  Overview of Water Column Toxicity Testing Approach with Both SEA Ring and
          Standard Laboratory Tests	35
Figure 7.  The SEA Ring verification testing will be conducted in 17-gallon HOPE containers
          (Chem-Tainer Industries; left), with concurrent standardized laboratory testing using
          glass beakers such as those shown at right	38
Figure 8.  A Troll 9500 datasonde (In Situ, Inc.) will be used to continuously measure and record
          water quality parameters in one of the SEA Ring exposure chambers associated with
          each treatment type	43


                                          TABLES

Table 1.   Toxicity Test Methodology and QA/QC Requirements for Water Column Toxicity Tests
          Using the Mysid Shrimp Americamysis bahia	25
Table 2.   Toxicity Test Methodology and QA/QC Requirements for Water Column Toxicity Tests
          Using TopsmeltAtherinops qffinis	26
Table 3.   Toxicity Test Methodology and QA/QC Requirements for Solid-Phase Toxicity Tests
          Using the Marine Amphipod Eohaustorius estuarius	27
Table 4.   Toxicity Test Methodology and QA/QC Requirements for Solid-Phase Toxicity and
          Bioaccumulation Tests Using the Marine Polychaete Neanthes arenaceodentata	28
Table 5.   Test Methodology and QA/QC Requirements for 28-Day Bioaccumulation Tests Using
          the Marine Clam Macoma nasuta	29
Table 6.   Summary of Tests and Testing Frequency	39
Table 7.   Test Methods and Equipment	44
Table 8.   Summary of Data Recording Process	64
Table 9.   Summary of Assessment Reports	68

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                            A3 ACRONYMS AND ABBREVIATIONS
%D           percent difference

ADQ         audit of data quality
AMS         Advanced Monitoring Systems
ANOVA      analysis of variance
ASTM        American Society for Testing and Materials

CAB         Cellulose Acetate Butyrate
cc            cubic centimeter
CCV         continuing calibration verification
CETIS        Comprehensive Environmental Toxicity Information System
COC         chain-of-custody
Cu            copper

DO           dissolved oxygen
DQI          data quality indicator

EPA          U.S. Environmental Protection Agency
ERDC        Engineer Research Development Center
ESTCP        Environmental Security Technology Verification Program
ETV          Environmental Technology Verification

GC           gas chromatography

HOPE        high density polyethylene

ICAL         initial calibration
ICP-MS       inductively coupled plasma mass spectrometry
ICV          initial calibration verification

LC50         median lethal concentration
LCS          laboratory control sample
LRB          laboratory record book

MS           Metals Contaminated Sediment

PAH         polycyclic aromatic hydrocarbon
PCB          polychlorinated biphenyl
PE            performance evaluation
ppb           parts per billion
ppm          parts per million
ppt           parts per thousand
PSNS         Puget Sound Naval Shipyard

QA           quality assurance
QAO         quality assurance officer

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QAPP         quality assurance project plan
QC           quality control
QMP         Quality Management Plan

RMO         Records Management Office

SEA Ring     Sediment Ecotoxicity Assessment Ring
SED          surficial sediment
SOP          Standard Operating Procedure
SPAWAR     Space and Naval Warfare
SSC          SPAWAR Systems Center
SWI          sediment water interface

TOC          total organic carbon
TSA          technical systems audit

UHMWPE    Ultra-high molecular weight polyethylene
USAGE       U.S. Army Corps of Engineers

VTC          verification test coordinator

WC          water column

YB           Yaquina Bay, OR

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                                 A4 DISTRIBUTION LIST
Technology Representative
Gunther Rosen
SPAWAR Systems Center Pacific (SSC Pac)
Environmental Sciences and Applied Systems
Code 71751
53475 Strothe Rd., Bldg. Ill
San Diego, CA 92152

EPA
John McKernan, ScD, CIH
U.S. Environmental Protection Agency (EPA)
National Risk Management Research Laboratory
26 W. Martin Luther King Dr.
Cincinnati, OH 45268

Verification  Organization, Battelle
Ramona Darlington, PhD -
AMS Center  Technology Verification Coordinator
Eric Stern - Research Leader/Sediment Management
Rosanna Buhl - Manager/Quality Systems
Amy Dindal - AMS Center Manager
Battelle
505 King Ave.
Columbus, OH 43201
Reference Laboratory
Patricia Tuminello
USAGE ERDC Chemistry Laboratory
3909 Halls Ferry Road
Vicksburg, MS 39180-6199

Dr. Jacob Stanley
USAGE ERDC, Environmental
Laboratory, Risk Assessment Branch
 3909 Halls Ferry Road
Vicksburg, MS 39180-6199

Brandon Swope
SPAWAR SSC Pac Chemistry
Laboratory
53560 Hull Street
San Diego, CA 92152-5001

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                        AS VERIFICATION TEST ORGANIZATION

The verification test will be conducted under the U.S. Environmental Protection Agency (EPA)
Environmental Technology Verification (ETV) Program. It will be performed by Battelle, which is
managing the ETV Advanced Monitoring Systems (AMS) Center through a cooperative agreement with
EPA. The scope of the AMS Center covers verification of monitoring technologies for contaminants and
natural species in air, water, soil and sediments. This verification test will evaluate an in-situ field
sampling technology that determines the toxicity of contaminants in the sediment and water column
(WC), and sediment-water interface on benthic and WC organisms.

The objective of the verification is to test the efficacy and ability of the Sediment Ecotoxicity Assessment
Ring (SEA Ring) to evaluate the toxicity of contaminants in the sediment, at the sediment-water interface,
and WC to organisms that live in those respective environments.  The SEA Ring will improve the
assessment of exposure and response at Department of Defense contaminated sediment and surface water
sites to assist in making accurate and informed management decisions, particularly with respect to
assessment of sediment remedy effectiveness and time-varying exposures. Although the SEA Ring is
used in the field, the verification testing will focus on the ability of the SEA Ring to provide comparable
data (using quantitative and qualitative criteria) to traditional EPA and American  Society for Testing and
Materials (ASTM)-approved laboratory methods under controlled laboratory conditions.  The
performance parameters for this test are repeatability, comparability and reproducibility as well as a
number of operational factors defined in Section B.

The performance of the  SEA Ring will be based on comparison with data obtained from EPA and ASTM
methods for determining the toxicity of contaminated sediment and whole effluents. Both the SEA Ring
exposures and the traditional laboratory exposures will be conducted in the laboratory.  The test methods
will follow those described in standard guidance documents (EPA and USAGE, 1998; ASTM, 2000;
ASTM, 2010).  Over approximately a two-month time period, all exposures will be conducted at the
Navy's Space and Naval Warfare (SPAWAR) Systems Center (SSC) Pacific Bioassay Laboratory,
San Diego,  an Environmental Laboratory Accreditation Program certified laboratory.  An external
laboratory, the  U.S. Army Corps of Engineers (USAGE) Engineer Research Development Center
(ERDC), Vicksburg, MS, will be utilized for verification of sediment and tissue concentrations from
relevant test  samples.  The subject technology is concurrently being evaluated in a project sponsored

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by the Environmental Security Technology Verification Program (ESTCP) Project ER-201130 titled
"Demonstration and Commercialization of the Sediment Ecosystem Assessment Protocol".

The day to day operations of this verification test will be coordinated and supervised by Battelle, with the
participation of the SEA Ring technology representative (SPAWAR).  Battelle will conduct laboratory
testing of the SEA Ring technology at the SPAWAR Systems Center in San Diego, CA.  SPAWAR will
provide the SEA Ring technology for testing and replicating multiple deployments of the technology, and
train Battelle staff on its use. Battelle staff and SPAWAR will operate the technology during verification
testing.

The organization chart in Figure 1 identifies the responsibilities of the organizations and individuals
associated with the verification test.  Roles and responsibilities are defined further below. Quality
assurance (QA) oversight will be provided by the Battelle Quality Manager, and also by the EPA AMS
Center Quality Manager, at EPA's discretion.

A5.1       Battelle's Test Program Roles and Responsibility
Dr. Ramona Darlington is the AMS Center's Verification Test Coordinator (VTC) for this test.  In this
role, Dr. Darlington will have overall responsibility for ensuring that the technical, scheduling, and cost
goals established for the verification test are met.  Specifically, Dr. Darlington will:

       •   Serve as the primary point of contact with SPAWAR;
       •   Prepare the draft quality assurance project plan (QAPP), verification report, and verification
           statement;
       •   Revise the draft QAPP, verification report, and verification statement in response to
           reviewers' comments;
       •   Assemble a team of qualified technical staff to conduct the verification test;
       •   Establish a budget for the verification test and manage staff to ensure the budget is not
           exceeded;
       •   Coordinate with the technology representative for provision of its technology for testing;
       •   Coordinate with SPAWAR personnel for laboratory testing;
       •   Direct the team in performing the verification test in accordance with this QAPP;

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     Battelle
   Management
Battelle AMS Center
 Quality Manager
   Rosanna Buhl
 Quality Assurance
     Officer
   Rosanna Buhl
                                     AMS Center
                                    Stakeholders
  Battelle AMS
 Center Manager
   Amy Dindal
 Verification Test
  Coordinator
Ramona Darlington
                          Verification
                        Testing Leader
                           Eric Stern
           US Navy SPAWAR
             Technology
            Representative
            Gunther Rosen


Battelle Testing
Staff



Reference Laboratories
(SSC Pac Chemistry,
ERDC and SSC Pac
Bioassay Laboratory)
EPA AMS Center
 Project Officer
 John McKernan
                                  EPA AMS Center
                                  Quality Manager
                       Figure 1. Organizational Chart
 Hold a kick-off meeting approximately one week prior to the start of the verification test to

 review the technical, logistical, and administrative critical paths of the verification test.

 Responsibility for each aspect of the verification test will be established by the VTC;

 Ensure that all quality procedures specified in this EPA Quality Level III QAPP and in the

 AMS Center Quality Management Plan (Battelle, 2011) are followed;

 Ensure that confidentiality of sensitive technology information is maintained;

 Assist SPAWAR as needed during verification testing;

 Become familiar with the operation of the technology through instruction by SPAWAR;

 Prepare a deviation report for any departure from the QAPP during the verification, obtain the

 requisite  EPA approvals, and distribute the approved report as specified in the AMS Center

 Quality Management Plan (QMP);

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       •   Respond to any challenges raised in assessment reports, audits, or from test staff
           observations, and institute corrective actions as necessary; and
       •   Coordinate distribution of the final QAPP, verification reports, and verification statements.

Ms. Amy Dindal is Battelle's Manager for the AMS Center.  As such, Ms. Dindal will oversee the various
stages of verification testing.  Ms. Dindal will:
       •   Review the draft and final QAPP;
       •   Attend the verification test kick-off meeting;
       •   Review the draft and final verification report and verification statement;
       •   Ensure that necessary Battelle resources, including staff and facilities, are committed to the
           verification test;
       •   Maintain communication with EPA's technical and quality managers; and
       •   Issue a stop work order if Battelle or EPA QA staff discovers adverse or non-consistent
           findings that are derived from technology failure or physical deployment conditions that will
           compromise test results.
Technical staff from Battelle, including Mr. Eric Stern, will support Dr. Darlington in planning and
conducting the verification test.  The responsibilities of the technical staff will be to:
       •   Assist Dr. Darlington (VTC) in preparing the QAPP;
       •   Review the draft and final QAPP;
       •   Attend the verification test kick-off meeting;
       •   Ensure that confidentiality  of sensitive vendor information is maintained;
       •   Support Dr. Darlington in responding to issues raised in assessment reports and audits;
           and
       •   Review the draft and final  verification reports and verification statements.

Ms. Rosanna Buhl is Battelle's QA Manager for the AMS  Center. Ms. Buhl will:
       •   Review the draft and final QAPP;
       •   Delegate to other Battelle quality staff any Quality Assurance Officer (QAO) responsibilities
           assigned below as needed to meet project schedules;
       •   Review and approve QAPPs, QAPP amendments, deviations and audit reports;
       •   Work with the VTC and Battelle's AMS Center Manager to resolve data quality concerns and
           disputes; and

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        •   Recommend a stop work order if audits indicate that data quality or safety is being
           compromised.

Ms. Buhl will also be the QAO for this test.  In this capacity she will:
        •   Attend the verification test kick-off meeting and lead the discussion of the QA elements of
           the meeting checklist;
        •   Prior to the start of verification testing, verify the presence of applicable training records,
           including any training on test equipment/technologies;
        •   Conduct a technical systems audit (TSA) at least once during the verification test;
        •   Conduct audits to verify data quality;
        •   Prepare and distribute an audit report for each audit;
        •   Verify that audit responses for each audit finding and observation are appropriate and that
           corrective action has been implemented effectively;
        •   Communicate to the VTC and/or technical staff the need for immediate corrective action if an
           audit identifies QAPP deviations or practices that threaten data quality;
        •   Provide a summary of the QA/quality control (QC) activities and results for the verification
           reports;
        •   Review the draft and final verification report and verification statement; and
        •   Communicate data quality concerns to the VTC.

A5.2       Technology Representative
The technology representative is US Navy SPAWAR. Mr. Gunther Rosen is the Navy's representative
and point of contact.  The technology was developed  and patented by  SPAWAR and the  University of
Michigan. A commercial technology vendor, Zebra-Tech, Ltd., is supporting SPAWAR in an effort
(funded by ESTCP) towards commercialization and standardization of the hardware and approach,
respectively. As part of the ESTCP project technology transition goals, the verified prototype of the
technology will ultimately be made commercially available through Zebra-Tech, or another vendor,
depending on who pursues licensing rights. The  responsibilities of the technology representative are:
        •   Review and provide comments on the draft QAPP;
        •   Accept (by signature) the final QAPP prior to test initiation;
        •   Participate in the kick-off meeting for the verification test;

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       •   Provide two SEA Ring technologies to carry out comparative analysis during the verification
           test;
       •   Supply instructions on the use of the technology, and written consent for test staff to carry out
           verification testing; and
       •   Review and provide comments on the draft verification report and verification statement for
           their respective technology.

A5.3       EPA
EPA's responsibilities in the AMS Center are based on the requirements stated in the Environmental
Technology Verification Program Quality Management Plan (EPA, 2008). The roles of specific EPA
staff are as follows.

The EPA's AMS Center Quality Manager will:
       •   Review the draft QAPP;
       •   Perform one external TSA during the verification test, at EPA's discretion;
       •   Notify the EPA AMS Center Project Officer of the need for a stop work order if the external
           audit indicates that data quality is being compromised;
       •   Prepare and distribute an assessment report summarizing results of any external audits; and
       •   Review draft verification report and verification statement.

Dr. John McKernan is EPA's Project Officer for the AMS Center.  Dr. McKernan will:
       •   Review the draft QAPP;
       •   Approve the final QAPP;
       •   Review and approve deviations to the approved final QAPP;
       •   Appoint a delegate to review and approve deviations to the approved  final QAPP in
           his absence, in order that testing progress will not be delayed;
       •   Review the first day  of data from the verification test and provide immediate
           comments if concerns are identified;
       •   Review the draft verification report and verification statement;
       •   Oversee the EPA review process for the QAPP, verification report, and verification
           statement; and

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       •   Coordinate the submission of verification reports and verification statements for final EPA
           approval.

A5.4       Verification Test Stakeholders
This QAPP and the verification report and verification statement based on testing described in this
document will be reviewed by experts in the fields related to aquatic sediment toxicity and
bioaccumulation (bioassay) sampling and testing. The following experts have been providing input to
this QAPP and have agreed to provide a peer review.
       •   Marc Greenberg, PhD - EPA Environmental Response Team, Edison, NJ
       •   Guilherme Lotufo, PhD - ERDC, Vicksburg, MS
       •   Damn Greenstein - Southern California Coastal Water Research Project, Costa Mesa, CA

 The responsibilities of verification test stakeholders and/or peer reviewers include:
       •   Participate in technical panel discussions (when available) to provide input to the test design;
       •   Review and provide input to the draft QAPP; and
       •   Review and provide input to the verification report/verification statement.

In addition, this technology category was reviewed with the broader AMS Center Stakeholder
Committees during the regular stakeholder teleconferences. Toxicity testing has been a long-standing
priority area for the AMS Center, with verifications and/or protocols completed in the areas of drinking
water, wastewater, and soil toxicity. This sediment toxicity technology verification was discussed with
the EPA Project Officer in May 2011.

A5.5       Reference Laboratories
Two reference laboratories will be utilized for verification of test exposures and/or bioaccumulated
concentrations of selected contaminant classes. The responsibilities of the reference laboratories for this
verification test include:
       •   Acknowledging receipt of samples and completing the chain-of-custody (COC) forms for the
           samples;
       •   Analyzing all samples for copper (Cu) (SPAWAR) or polychlorinated biphenyl (PCB)
           congeners (ERDC);
       •   Providing analysis results and supporting laboratory documentation within 30 days of receipt

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           of samples; and
       •   Providing documentation as requested (such as Standard Operating Procedures [SOPs]) for an
           independent TSA of laboratory procedures.
The SSC Pacific Chemistry Laboratory will analyze seawater samples to verify control and spiked
samples for Cu. The SSC Pacific Laboratory technical point of contact for Cu measurements will be
Brandon Swope. He is responsible for providing SOPs and appropriate QA reporting for the verification
test.  In lieu of participating in the performance evaluation (PE) audit, the SSC Laboratory will provide
results from its two most recent Cu PE samples to the Battelle Quality Manager. SOPs will be obtained
and reviewed from the external laboratory.

The USAGE, ERDC Environmental Chemistry Lab, in Vicksburg, MS, will analyze sediment and tissue
samples from the technology representative for PCB congener measurements.  Ms. Patricia Tuminello
will be the point of contact at ERDC. She is responsible for providing SOPs and appropriate QA
reporting for the verification test.  The ERDC laboratory will participate in a PE audit (see Section C1.1)
since the laboratory is not accredited.

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                                      A6  BACKGROUND
A6.1       Technology Need
The ETV Program's AMS Center conducts third-party performance testing of commercially available
technologies that detect or monitor natural species or contaminants in air, water, soil, and sediment.  The
purpose of ETV is to provide objective and quality assured performance data on environmental
technologies so that users, developers, regulators, and consultants can make informed decisions about
purchasing and applying these technologies.  Stakeholder committees of buyers and users of such
technologies recommend technology categories, and technologies within those categories become
priorities for testing. Among the technology categories recommended for testing are toxicity testing
technologies, including sediment and aqueous toxicity for assessment of environmental quality in marine,
freshwater and estuarine systems.

Traditionally, the bioavailability and toxicity of contaminated sediments or water samples are assessed on
grab or composite samples collected in the field and tested in a laboratory. In the laboratory, test
organisms are added to site sediment or water samples in beakers and exposed under controlled
conditions (e.g., temperature, pH, salinity, photoperiod, feeding regime, aeration) for a specified time
period (e.g., EPA, 1994a; EPA, 2000; ASTM, 2000; ASTM, 2010). This laboratory-based method of
assessing sediment quality, although widely used and well established, does not necessarily represent the
true in-situ exposure and effects to organisms in the field. This is especially true when the source of
contamination is ephemeral, meaning exposure varies over time and with ambient conditions.  Another
challenge with laboratory testing is that sediment sample manipulation removes the natural vertical
contaminant stratification, which in turn alters the exposure to test organisms.  Such manipulation may
also result in alteration of the contaminant bioavailability through processes including degradation,
volatilization, and redox changes. Sediment samples removed from the field undergo physical and
chemical changes which change the bioavailability and toxicity of the contaminants and may lead to
misleading results in the laboratory and subsequent difficulty in program decision making.

In addition, laboratory tests may overestimate toxicity from sediment-associated contaminants due to
buildup of contaminant concentrations in the overlying water as toxicants desorb from the sediment into
the WC. In aqueous exposures, laboratory tests may also misrepresent actual exposure in the field when
static exposures are used as a means of assessing the potential for adverse effects of a time-varying

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stressor (e.g., stormwater runoff, combined sewer overflow, etc.).  The limitations of standard laboratory
toxicity testing and chemical analyses lead to potentially inappropriate and costly management decisions.

A6.2       SEA Ring Technology Description
The SEA Ring (U.S. Patent No. 8,011,239) is an integrated, versatile, field tested, toxicity and
bioavailability assessment device.  Figures 2 and 3 show top and side views of the patented, first
generation version of the SEA Ring technology. The second generation model (Figure 4) will be the
version used in this ETV verification.  The second generation system is the commercialized version of the
prototype, which was designed to be more user-friendly, more autonomous, and more rigorous to
withstand environmental conditions over exposure time. The unit consists of 10  cylindrical chambers
fixed to a circular ultra-high molecular weight polyethylene (UHMWPE) platform. The top end of each
chamber is fitted with an integrated, multifunctional cap. The cap includes both  overlying water intake
and outlet ports, and an organism delivery port (opening for an optional modified plastic 30 cubic
centimeter [cc] syringe). The intake port connects to a peristaltic pump that is housed in the center of the
device and powered by rechargeable batteries stored in a separate housing underneath the pump. The
pump is programmable to provide chamber water volume exchange at a rate (range ~6 to >25 turnovers
per day) desired for the  site- or project-specific preferences.

The SEA Ring was designed to evaluate toxicity in the WC, sediment water interface (SWI), and/or
surficial sediment (SED; Figure 3). The SED chambers are open on the bottom,  are 10 inches in length,
2.75 inches in diameter, and extend 5 inches below the base of the system. Small sediment dwelling
organisms can be introduced into the SED chambers through the organism delivery port built into the cap
with a modified 30 cc plastic syringe.  The syringe is plugged with a silicone stopper inside the test
chamber to retain the organisms until desired release. For larger organisms a 1A inch stainless  steel mesh
is integrated into the bottom opening of the exposure chamber, allowing organisms to be preloaded prior
to deployment. The WC and SWI chambers are 5 inches in length, 2.75 inches in diameter, and have a
closed bottom. The bottom consists of a solid plastic polyethylene cap or mesh insert for water quality
chambers. Organisms for the WC and SWI tests can be loaded in the laboratory  or in the field
immediately prior to deployment. In the center of the circular platform there is a custom-built peristaltic
pump and battery. These components are fully encased and water tight. The intake to the test chambers
is located on top of the cap. Each inlet is directly connected to the pump through individual tubes that
pass over the pump roller.  As the pump  rotor turns, compressing and releasing pressure on the tubing,
ambient water from the surrounding area is circulated through each chamber. A  water quality sensor or

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passive sampler can also be attached to one of the chambers (Figure 3). Water quality sensors are used to

measure a variety of physical parameters including pH, temperature, depth, salinity, conductivity, and

dissolved oxygen (DO) from inside the exposure chambers.
                                                              ~--39
                         Figure 2. Schematic of SEA Ring Technology
                                (U.S. Patent Number 7,758,813)

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Overlying
Water



*. v
v
,—




Sediment
Water
Interface

1
0


9 ฐ .
n 9 ฐ
>
*




s ire
4
" Surface Passive
Sediment Sampler

1


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1-
            Figure 3. Multiple Lines of Evidence Use of SEA Ring Technology
Figure 4. Second Generation SEA Ring Device (left). Field Evaluation in Beach Deployment
                                        (right)

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                 A7 VERIFICATION TEST DESCRIPTION AND SCHEDULE

The purpose of the test is to generate performance data on an innovative in-situ field technology for
assessing contaminated sediment and WC toxicity and bioaccumulation potential using indigenous
organisms. The ease of use and comparability of the technology to EPA and ASTM methods will be
evaluated utilizing multiple species, varied sediment types, and chemicals often identified as
contaminants of concern (e.g., metals such as Cu and organics such as PCBs) in the near-shore aquatic
environment. The data generated from this verification test are intended to provide technology users with
information on its performance in controlled laboratory settings prior to its use in the field.

A7.1       Verification Test Description
The purpose of this QAPP is to specify procedures for verification testing of the SEA Ring to assess
contaminated sediment and WC toxicity to aquatic and benthic organisms. The primary evaluation will
assess survival, growth, and bioaccumulation of contaminants in aquatic and benthic organisms exposed
in the SEA Ring compared to responses achieved in the laboratory using standard ASTM and EPA
methods. In performing the verification test, Battelle will follow the technical and QA procedures
specified in this QAPP, and will comply with the data quality requirements in the AMS Center QMP
(Battelle, 2011).

The SEA Ring tests will be evaluated on the following performance parameters, described in detail in
Section B:
       •   Repeatability;
       •   Comparability;
       •   Reproducibility; and
       •   Operational factors (qualitative assessment).
Operational parameters including ease of use, training and sustainability (sampling time, waste produced,
and the amount of protective equipment required by the individual operating the technology) will also be
evaluated by Battelle staff. More details on the test design are provided in Section B.I.3.

Testing will be conducted in the laboratory over a two-month period by Battelle staff with support from
the technology representative. SEA Ring and concurrent bench-top tests  following the EPA and ASTM
methods will be set up and evaluated in the SSC Pacific Bioassay Laboratory. With the exception of PCB

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congener analyses in sediment and tissue by USAGE ERDC Chemistry Laboratory, all analyses will be
performed at the SSC Pacific Bioassay Laboratory.

Subsequent to verification testing, Battelle will prepare one Verification Report for the laboratory
evaluations.  The report will describe the SEA Ring performance on assessing sediment and WC toxicity
to aquatic and benthic organisms.

QA procedures include a TSA and two audits of data quality (ADQs), (details provided in Section A7.1).
The Battelle QAO or her designee will perform the TSA. The first data set will be delivered within 30
days of test initiation.  Un-audited data will include the disclaimer have not been reviewed by Battelle QA
Manager. The first ADQ will review the first data set delivered. The second ADQ will assess the
remainder of the data, the draft report, and the verification statement as described in Section C.

A7.2       Verification Test Schedule
Laboratory testing of the SEA Ring is scheduled to begin in November 2012 and will be initiated upon final
EPA and technology representative approval of this QAPP. Testing will occur over approximately a two-
month period. Data will be evaluated and the verification report and verification statement will be
drafted. It is anticipated that the final EPA-approved verification report and verification statement will be
completed by September 2013.

A7.3       Verification Location
Laboratory testing will be conducted at the SSC Pacific Bioassay Laboratory, San Diego, CA. This
laboratory is equipped to perform sediment and aqueous toxicity testing in a controlled environment and
reduces the costs of shipping the technology to the Battelle laboratory in Columbus, Ohio.

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                                 A8 QUALITY OBJECTIVES

This verification test is designed to evaluate the performance of the SEA Ring for determining the
bioavailability and toxicity of contaminants in water and sediments on aquatic and benthic organisms.
This verification will vary sediment type, organism and toxicity endpoint type, and contaminant
concentration in the SEA Ring device under controlled and repeatable test conditions. Parallel standard
bench-top tests will be conducted.  Both the SEA Ring and bench-top tests will follow EPA and ASTM
testing methods, with minor modifications as necessary. Any deviations from protocols referenced will
be thoroughly documented on bench datasheets and in the final report. The test conditions and quality
indicators for this verification test lie in the performance parameters and the QC samples. Data quality
indicators (DQIs) ensure that the verification tests provide suitable data for a robust evaluation of
performance. DQIs have been established for organism age and water quality. The DQIs were
established to ensure that data used to support the SEA Ring technology tests are of sufficient quality.
Acceptance criteria for the DQIs and QC samples are  detailed in the Tables 1 through 5, and are specific
to each test species.

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 Table 1.  Toxicity Test Methodology and QA/QC Requirements for Water Column Toxicity Tests
                            Using the Mysid Shrimp Americamysis bahia
Test organism

Test organism source

Test organism age at initiation

Test duration; endpoint

Test solution renewal

Feeding

Test chamber

Test solution volume

Test temperature

Dilution water

Salinity

Test concentrations

Number of organisms/chamber

Number of replicates

Photoperiod

Aeration

Test Protocol

Test acceptability objective

Reference toxicant
Mysid shrimp -Americamysis bahia

Aquatic BioSystems - Laboratory culture (Fort Collins, CO)

3-5 days post-hatch; less than or equal to 24-h range in age (required)

96-hour; survival

80% volume renewal one time (48 hours)

Artemia nauplii, twice daily

0.5-L plastic cup (laboratory); 5 inch cellulose acetate butyrate (CAB) core tube
(SEA Ring)

Approximately 500 mL (laboratory and SEA Ring)

20 ฑ 1ฐC test-wide mean, 20 ฑ 3ฐC instantaneous

Filtered (0.45 um) natural seawater collected from near the mouth of San Diego
Bay at SSC Pacific Laboratory

32ฑ2%ppt

Lab control, 100, 200, 400 ug/L Cu


10
 16 hours light/8 hours dark., ambient laboratory lighting

None, unless DO < 4 mg/L

EPA-821-R-02-012 (EPA, 2002a)

> 90 % mean survival in natural seawater control

Copper sulfate (Standard EPA laboratory method only); five concentrations (3
replicates each)

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 Table 2.  Toxicity Test Methodology and QA/QC Requirements for Water Column Toxicity Tests
                                 Using TopsmeltAtherinops affinis
Test organism

Test organism source

Test organism age at initiation

Test duration; endpoint

Test solution renewal

Feeding

Test chamber

Test solution volume

Test temperature

Salinity

Dilution water

Test concentrations

Number of organisms/chamber

Number of replicates

Photoperiod

Aeration

Test Protocol

Test acceptability objective

Reference toxicant
Topsmelt -Atherinops affinis

Aquatic BioSystems - Laboratory culture (Fort Collins, CO)

9-15 days post-hatch

96-hour; survival

80% volume renewal at 48 hours

Artemia nauplii, twice daily

0.5-L plastic cup (laboratory); 5 inch CAB core tube (SEA Ring)

Approximately 500 mL (laboratory and SEA Ring)

20 ฑ 1ฐC test-wide mean, 20 ฑ 3ฐC instantaneous

32ฑ2%ppt

Filtered (0.45 um) natural seawater collected from near the mouth of San Diego
Bay at SSC Pacific Laboratory

Lab control, 100, 200, 400 ug/L Cu


10

5

16 hours light/8 hours dark, ambient laboratory lighting

None, unless D.O. < 4 mg/L

EPA-821-R-02-012 (EPA, 2002a)

> 90 % mean survival in natural seawater control

Copper sulfate (standard EPA lab method only); 96 hours, 48-hr renewal/five
concentrations (3 replicates each)

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   Table 3. Toxicity Test Methodology and QA/QC Requirements for Solid-Phase Toxicity Tests
                        Using the Marine Amphipod Eohaustorius estuarius
Test organism

Test organism source

Test organism age at initiation

Control sediment source

Test duration; endpoint

Test solution renewal

Feeding

Test chamber

Test sediment depth

Overlying water volume

Test temperature

Overlying water


Salinity

Test concentrations

Number of organisms/chamber

Number of replicates

Photoperiod


Aeration


Test Protocol

Test acceptability objective

Reference toxicant
Marine Amphipod - Eohaustorius estuarius

Northwest Aquatic Sciences (Newport, OR)

NA - Field collected (3-5 mm adult)

Sediment from amphipod collection site, Yaquina Bay, OR (YB)

10 days; survival

None

None

1-L glass jar (lab), 10 inch CAB core tube (SEA Ring)

2 cm (lab and SEA Ring)

750 ml (lab and SEA Ring) natural seawater

18 ฑ PC test-wide mean, 18 ฑ 3ฐC instantaneous

Filtered (0.45 um) natural seawater collected from near the mouth of San Diego
Bay at SSC Pacific Laboratory

32ฑ2%ppt

Undiluted sediment sieved to < 2.0 mm

20

5 (lab and SEA Ring)

Continuous light (24 hr), ambient laboratory lighting

Laboratory filtered air, continuous (1-2 bubbles per second delivered through a
Pasteur pipette in laboratory beaker, 1-2 bubbles per second from three Pasteur
pipettes in SEA Ring Chemtainer (outside exposure chambers)

EPA 600-R-94-025 (EPA,  1994a)

> 90 percent mean survival in control

Cadmium chloride (standard EPA lab method only); 96-h water only exposure

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    Table 4. Toxicity Test Methodology and QA/QC Requirements for Solid-Phase Toxicity and
           Bioaccumulation Tests Using the Marine Polychaete Neanthes arenaceodentata
Test organism

Test organism source

Test organism age at initiation

Control sediment source

Test duration; endpoint(s)

Test solution renewal

Feeding

Test chamber

Sediment depth

Overlying water volume

Test temperature

Overlying water


Salinity

Test concentrations

Number of organisms/chamber

Number of replicates

Photoperiod


Aeration


Test Protocol

Test acceptability objective

Reference toxicant
Marine polychaete, Neanthes arenaceodentata

Dr. Mary AnnRempel Hester, Aquatic Toxicity Support, Inc. (Bremerton, WA)

2 weeks

Sediment from the amphipod collection site, Yaquina Bay, OR (YB)

28 days; survival and growth

Twice-weekly (laboratory jar/SEA Ring Chemtainer)

1 ml of flake food slurry twice weekly after test solution renewal (slurry
comprised of 100 mL seawater: 1 g Tetraminฎ fish feed)

1-L glass jar (lab),  10 inch CAB core tube (SEA Ring)

2 cm

750 ml

18 ฑ 1ฐC test-wide mean, 18 ฑ 3ฐC instantaneous

Filtered (0.45 um)  natural seawater collected from near the mouth of San Diego
Bay at SSC Pacific Laboratory

32ฑ2%ppt

Undiluted sediment sieved to < 2.0 mm

20
16 hours light/8 hours dark, ambient laboratory lighting

Laboratory filtered air, continuous (1-2 bubbles per second delivered through a
Pasteur pipette in laboratory beaker, 1-2 bubbles per second from three Pasteur
pipettes in SEA Ring Chemtainer (outside exposure chambers)

ASTM 2000 E1611-00

> 90 percent mean survival in control

Copper Sulfate (standard ASTM laboratory method only); 96-hr water only
exposure

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Table 5.  Test Methodology and QA/QC Requirements for 28-Day Bioaccumulation Tests Using the
                                   Marine Clam Macoma nasuta
Test organisms

Test organism source

Test organism age at initiation

Control sediment source

Test duration

Test solution renewal

Feeding

Test chamber

Sediment depth

Overlying water volume

Test temperature

Overlying water

Salinity

Test concentrations

Number of organisms/chamber

Number of replicates

Photoperiod


Aeration


Test Protocol

Test acceptability objective

Reference toxicant
Marine clamMaco/wa nasuta

Brezina & Associates (Dillon Beach, CA)

~1" Small Adult (field collected)

Sediment collected from clam collection site, Dillon Beach, CA (DB)

28 days, + 24-hr depuration period

Three-times weekly with clean seawater

None

5 1-L glass beakers in 10 gallon aquarium (lab); 5 1-L CAB core tubes in
Chemtainer (SEA Ring)

5 cm (lab and SEA Ring chambers)

Approximately 750 mL (laboratory and SEA Ring)

18 ฑ 3 ฐC instantaneous

Filtered (0.45 um) natural seawater (salinity 32-34 ppt) collected from near the
mouth of San Diego Bay at SSC Pacific Laboratory

32 ฑ2% ppt

Undiluted  sediment sieved to <2.0 mm
16 hours light/8 hours dark, ambient laboratory lighting

Laboratory filtered air, continuous (1-2 bubbles per second delivered through a
Pasteur pipette in laboratory beaker, 1-2 bubbles per second from three Pasteur
pipettes in SEA Ring Chemtainer (outside exposure chambers)

EPA 503/8-91/001, ASTME-1688-10
> 90 percent mean survival in controls

None

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                          A9  SPECIAL TRAINING/CERTIFICATION

Documentation of training related to technology testing, field testing, data analysis, and reporting is
maintained for all Battelle technical staff in training files at their respective locations.  SPAWAR staff
will receive training in documentation and records management procedures required for ETV testing
during the kick-off meeting. The Battelle Quality Manager will verify the presence of appropriate
training records prior to the start of testing. Battelle and EPA staff involved in this verification will be
specifically trained on the operation of the SEA Ring technology.  Training in the use of the SEA Ring
will be conducted by the technology representative. Battelle will document this training with a consent
form, signed and dated by the technology vendor, which states which Battelle technical staff have been
trained to use the technology and can train other staff to do so as well. In the event that other staff
members are required to use the technology, they will be trained by either the operators that were trained
by the technology representative or the technology representative.

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                           A10 DOCUMENTATION AND RECORDS

The documents for this verification test will include the QAPP, vendor instructions, reference methods,
verification reports, verification statements, and audit reports. The project records will include laboratory
record books (LRBs) and data collection forms, supporting laboratory records, training records, electronic
files (both raw data and spreadsheets), and QA audit files. Section BIO summarizes data management  for
the test and the types of data to be recorded. Documentation of Battelle staff training by the technology
representative and copies of other project specific training will also be included in the project files. All of
these records will be maintained by the SPAWAR point of contact during the test, and will be transferred
to permanent storage at Batte lie's Records Management Office (RMO) at the conclusion of the
verification test. All Battelle LRBs are stored indefinitely with the project files by Battelle's RMO.
Section BIO further details the data management practices and responsibilities.

All data generated during this project will be recorded directly, promptly, and legibly in  ink.  All data
entries will be  dated on the date of entry, and signed or initialed by the person entering the data. Any
changes in entries will be made  so as not to obscure the original entry, will be dated and  signed or
initialed at the  time of the change and will indicate the reason for the change. Project specific data forms
will be developed prior to testing to ensure that all critical information is  documented in  real time.  The
draft forms will be provided to the Battelle QA Manager for review.

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                                          SECTION B
                         MEASUREMENT AND DATA ACQUISITION
                                Bl  EXPERIMENTAL DESIGN

This QAPP addresses the verification of the SEA Ring through laboratory testing.  Specifically, the SEA
Ring will be evaluated for the following performance parameters:
       •   Repeatability;
       •   Comparability;
       •   Intra-unit reproducibility; and
       •   Operational factors.

The verification test will be conducted in the laboratory over a period of two months. Prior to initiation of
the SEA Ring verification test, sediment samples will be collected for use in the experiment and testing
organisms obtained from vendors.  Collection records will include the collection date and location,
collector and storage conditions. Test organism records will include the source, date and location of
collection (if collected) or age (if cultured), and holding and acclimation conditions.

Bl.l       Test Procedures
The following sections describe the test procedures that will be used to evaluate each of the performance
parameters listed above.  Cost information will be provided by the technology vendor (i.e., price of
technology, operation and maintenance cost). The performance parameters are defined in detail in Tables
1 through 5. Figure 5 illustrates the sediment test design variables, and the WC test design is shown in
Figure 6.

Bl.1.1     Sediment and Water  Sources. Three different types of sediment will be  used in the ETV
verification of the SEA Ring. The laboratory water used by SSC Pacific Laboratory is 0.45 (im filtered
seawater collected from near the mouth of San Diego Bay on an incoming high tide, and has been used
successfully for a number of years  to conduct toxicity testing that regularly meets test acceptability
criteria for a number of different standardized laboratory tests. The laboratory seawater will be used as
the overlying water for sediment tests and as the dilution water for aqueous tests.

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Control Sediment (YB or DB):  Sediment from Yaquina Bay, OR (referred to as YB) will be used as the
control sediment for testing with E. estuarius and N. arenaceodentata. Yaquina Bay sand is commonly
used as a negative control in West Coast marine sediment toxicity testing. This sand will be obtained
from Northwestern Aquatic Sciences (Newport, OR), which also collects E. estuarius from the same
location for sediment toxicity testing.  Sediment from Dillon Beach, CA (referred to as DB) will be used
as the control sediment forM nasuta. This sediment is from the clam collection site and is more
organically rich and more suitable forM nasuta.

Metals Contaminated Sediment (MS): A fine-grained marine sediment from an undisclosed
(proprietary) site, contaminated primarily with Cu, zinc, and lead (referred to as MS) will be used for
toxicity testing only. Chemical analysis of this sediment will be performed as part of the test design.

PCB Contaminated Sediment (PSNS): A medium-fine grained field sediment from the Puget Sound
Naval Shipyard in Bremerton, WA (referred to as PSNS) that is contaminated with numerous classes of
chemicals will be used for both toxicity and bioaccumulation testing.  With the exception of PCBs,
concentrations of other contaminants in this sediment are not expected to be at toxic levels. Historical
data on the chemical profile of this material will be obtained. In addition, PCB, total organic carbon
(TOC), and grain size analysis of this sediment will be performed as part of the test design.

The MS and PSNS sediments are already in storage (4 ฑ 2 ฐC, in the dark) at the SSC Pacific Laboratory.
Before being introduced into the  test chambers, the sediments will be  re-homogenized and sieved to < 2.0
mm to remove shell hash and other indigenous material from interfering with the laboratory bioassays.
The solids content (percent solids), initial TOC concentration, and percentage of silt and clay sized
particles will be measured by ERDC.

Laboratory Dilution Water:  The laboratory dilution water used by SSC Pacific Laboratory is 0.45 (im
filtered seawater collected from near the mouth of San Diego Bay on an incoming high tide, and has been
used for a number of years in successful toxicity testing that meets test acceptability criteria for a number
of different standardized laboratory tests.  The laboratory dilution water will be used as the overlying
water for sediment tests and as the dilution water for aqueous tests.

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   Sediment Test  - SEA  Ring
                 YB Control Sediment
YB Control Sediment    Polychaete(20) - 28 days
Amphipod (20) -10 days  DB Control Sediment
                 Clam (3)-28 days
                                    MS Sediment -Toxlclty
                                    Amphipod(20) -10 days
                                    Polychaete (20) - 28 days
PSNS -Toxicity/Bioaccum
Amphipod(20) -10 days
PSNS- Toxicity/Bioaccum
Polychaete (20) - 28 days
Clam (3)- 28 days
   Sediment Test - Laboratory
                     Yaquina Bay - Control - Amphipod (20) - 10 days
                    Yaquina Bay - Control - Polychaete (20) - 28 days
                    Dillon Beach - Control - Clam (3) - 28 days
                 (   MS Sediment -Toxicity - Amphipod (20) - 10 days
                     MS Sediment -Toxicity - Polychaete (20) - 28 days
                   PSNS Sediment-Toxicity & PCB Bioaccumulation - Amphipod(20) -10 days |

                   PSNS Sediment-Toxicity & PCB Bioaccumulation - Polychaete (20) - 28 days I
                   PSNS Sediment-Toxicity & PCB Bioaccumulation -Clam (3) -28 days
Figure 5.  Overview of Sediment Toxicity and Bioaccumulation Testing Approach with Both SEA
                            Ring and Standard Laboratory Tests
                   (Number of test organisms per replicate in parentheses).

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    Water Column Toxicity Test - SEA Ring
    0 ppb - Control
    Mysid shrimp (10) &
    Topsmelt(lO)
100 ppb-Copper
Mysid shrimp (10) &
Topsmelt(lO)
200 ppb-Copper
Mysid shrimp (10) &
Topsmelt(lO)
400 ppb-Copper
Mysid shrimp (10) &
Topsmelt(lO)
      Repeat the 0% and 400 ppb for Repeatability Test

  Water  Column Toxicity Test - Laboratory
                    0 ppb - Control - Mysid shrimp (10) & Topsmelt (10)
                    100 ppb - Copper - Mysid shrimp (10) & Topsmelt (10)
                    200 ppb -Copper- Mysid shrimp (10) & Topsmelt (10)
                    400 ppb-Copper-  Mysid shrimp (10) & Topsmelt (10)
        All treatments in replicates of 5, number of organisms per chamber = 10
    Figure 6. Overview of Water Column Toxicity Testing Approach with Both SEA Ring and
                                Standard Laboratory Tests
                  (Number of test organisms per replicate is in parentheses)
Copper Spiking for Water Column Tests: Laboratory dilution water will be spiked with three

concentrations of Cu, bracketing the expected median lethal concentration (LC50) for each of the two WC

tests species.  Concentrations of Cu to be tested are 100, 200, and 400 parts per billion (ppb) as Cu. The

appropriate amount of Cu will be added to laboratory dilution water using a 1,000 parts per million (ppm)

verified stock solution made from reagent grade copper sulfate (CuSO4).  Organisms will be loaded into

one of four SEA Rings as depicted in Figure 6 and each ring will be exposed to a different Cu

concentration.
Bl.1.2     Benthic and Aquatic Organism Collection. Depending on availability, up to five different

types of organisms will be used in this ETV verification test. For sediment tests, three organisms will be

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used: a free burrowing deposit feeder (the marine amphipod, Eohaustorius estuarius), a deposit feeding
tube building organism (the marine polychaete worm, Neanthes arenaceodentata), and a facultative filter
feeding clam (the bent-nosed clam, Macoma nasutd).  Survivors from each test species will be analyzed
for PCB congeners from the PSNS sediment treatments, following test termination and a depuration
period (overnight) in uncontaminated seawater. All replicates from one organism will be analyzed for
PCB congeners in the YB control sediment. Only one organism will be analyzed because it is expected
that there will be no PCB congener detections in the control sediment organisms.

Two common west coast marine test organisms will be used for the WC tests depending on their
availability: Americamysis bahia (mysid shrimp) and Atherinops affinis (Pacific topsmelt). An alternative
vertebrate species, the inland silverside minnow Menidia beryllina, may be used should topsmelt not be
available.

The age/size and source information for the proposed test organisms is provided in Tables 1 through 5.
All test organisms will be acclimated to laboratory exposure conditions at the SSC Pacific Laboratory for
1 to 5 days prior to use, depending on species. Acclimation time will be taken into account when the
animals are ordered so that they will be within the acceptable age at the time of test initiation.  During the
acclimation period, water quality measurements of temperature, salinity, DO, and pH will be recorded
daily. Laboratory SOPs for water quality monitoring and frequency are provided in Appendix E.
Mortality of animals during holding should be no greater than 10% for all organism batches to ensure
high quality organisms are being used.

Bl.1.3     SEA Ring Preparation and Operation
Preparation- The SEA Ring hardware will be cleaned in a dilute (2%) detergent (Liquinox) overnight,
followed by conditioning in uncontaminated, filtered laboratory seawater, and a final soak in flowing
deionized water.  Disposable parts (pump tubing, bottom end caps, and inner exposure chambers) will be
replaced. SEA Rings will be placed into appropriate Chemtainers, and tested to ensure the pump and
water quality sensor is functioning properly by connecting to a laptop uploaded with appropriate sensing
software.

Initiation and Operation- The SEA Ring will be placed in a Chemtainer with enough water to be
completely submerged. The water in the Chemtainer outside of the SEA Ring will be aerated
continuously at a rate of one to two bubbles per second using trickle flow aeration in both the sediment

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and water toxicity tests. This will allow delivery of aerated water to the exposure chambers as the water
is pumped from the Chemtainer.

The required amount of sediment and/or clean seawater or Cu-spiked seawater water (Tables 1 through 5)
will then be added to each exposure chamber, followed by securing of the top chamber caps, and
initiation of the pump. The pump will be set to the desired turnover rate (approximately 10 exchanges
between the inner exposure chamber and the water in the Chemtainer per day). For sediment tests,
sediment will be allowed to settle overnight prior to organism addition.  For WC tests, organisms will be
added within 3 hours of addition of samples to the test chambers. Organisms will be arbitrarily selected
and added through the organism delivery port in the chamber caps.

Replacement of the overlying water in both water and sediment tests will occur at the same frequency as
the concurrent traditional laboratory methods according to the test method summaries in Tables  1 through
5.  Approximately 80% of the water will be replaced on water renewal days. Although feeding may not
take place in field exposures depending on species, organisms will be fed in laboratory trials according to
test conditions found in Tables  1 through 5 to ensure that any mortality is not as a result of lack of food.
Any required feeding will occur through the organism delivery port of each exposure chamber.

B1.2       Laboratory SEA Ring Test
The primary objective of the laboratory test is to evaluate the ability of the SEA Ring to provide
comparable data (using quantitative and qualitative criteria) to traditional EPA and ASTM-approved
laboratory methods under controlled laboratory conditions. It should be noted, however, that actual
application of the  SEA Ring device in situ is not expected to necessarily produce the  same results as
laboratory tests due to reasons already stated earlier in other sections of this document. For the purposes
of this comparison, SEA Rings  will be contained in the laboratory in containers using test conditions and
experimental designs that are similar to those used in traditional laboratory toxicity and bioaccumulation
tests. The containers are 17 gallon high density polyethylene (HDPE) containers (Chemtainer Industries,
Inc.), frequently used to transport the SEA Rings to field sites (Figure 7).

Both sediment toxicity and WC toxicity tests will be conducted. The objective of WC toxicity tests is to
determine the potential impact of dissolved and suspended contaminants on test organisms in the WC.
The objective of benthic toxicity tests is to determine the potential impact of whole sediment exposure on
benthic  organisms.

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                                                          ll

    Figure 7. The SEA Ring verification testing will be conducted in 17-gallon HOPE containers
(Chem-Tainer Industries; left), with concurrent standardized laboratory testing using glass beakers
                                  such as those shown at right
Bl.2.1     Repeatability (Replicate Variability). Variability in biological response will be evaluated
among the five replicate exposure chambers in the SEA Ring to provide a measure of repeatability within
a single trial.  This measure of repeatability will be assessed by quantifying biological responses at the
end of the exposure period (survival of all species tested and growth of polychaetes). A control will
consist of uncontaminated sediment from YB for comparison.
Sediment toxicity repeatability test - Two different organisms will be tested for the sediment toxicity
repeatability test: the marine amphipod Eohaustorius estuarius and the marine polychaete Neanthes
arenaceodentata. Three sediment types will be tested: 1) a sandy control sediment from Yaquina Bay,
OR, where the amphipods are collected (YB); 2) a fine-grained metals contaminated sediment (MS) that
has previously been shown to be toxic to the proposed test species; and 3) a medium-fine grained
moderately contaminated from Puget Sound Naval Shipyard in Bremerton, WA (PSNS). This third
sediment contains numerous classes of chemicals (e.g., metals, polycyclic aromatic hydrocarbons [PAHs],
PCBs), but is not expected to be toxic to the species tested for this verification study based on prior
studies.  The exposure period for the sediment toxicity tests will be 10 days for the amphipod test (acute)
and 28 days for the polychaete test (chronic).  Survivorship of both species will be evaluated at the end of
the exposure period. Growth of polychaetes will also be measured. Details of the test are provided in
Table 6.  Five replicate chambers with 20 organisms per replicate will be tested for each species.  The
reference toxicant for the solid phase sediment toxicity tests will be cadmium chloride (CdCl2) for the
amphipod and copper sulfate (CuSO4) for the polychaete. The reference toxicant tests (performed as

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                                                   Table 6. Summary of Tests and Testing Frequency
 Performance
  Parameter
         Objective
        Endpoint
 Comparison Based On
         Testing Frequency
Minimum number
    replicates
 Repeatability
 Comparability
Reproducibility
                  Determine the repeatability
                  among five replicates within
                  one SEA Ring
Determine the ability of the
SEA Ring to measure toxicity
of benthic and aquatic
organisms compared to
EPA/ASTM methods under
the same conditions
Determine the reproducibility
among three different SEA
Rings tested under the same
contaminant concentrations
and organisms
                              1) Organism survival, or
                                survival and growth2

                              2) Bioaccumulation of
                                contaminant within
                                organism tissue3
1) Organism survival and
   growth

2) Bioaccumulation of
   contaminant within
   organism tissue
                                               Organism survival
                          Survival, growth, and
                          bioaccumulation of
                          contaminants in organisms
                          among five replicates
                          within one SEA Ring
Survival (and growth),
and bioaccumulation of
contaminants in organisms
in the SEA Ring
compared to the bench
scale EPA and ASTM
methods
                          Survival of WC test
                          organisms in the SEA
                          Ring
1)  Survival in WC tests with four
    contaminant concentrations
    (including a control) and two test
    species.
2)  Survival (and growth) in sediment
    tests with three sediment types
    including a control and up to three
    test species.
3)  Bioaccumulation in sediment
    toxicity test of two test species,
    two sediment types including a
    control. Five replicates in each
    case.
Survival in WC tests with four
contaminant concentrations (including
a control) and two test species.
Survival and growth in sediment tests
of three sediment types including a
control and up to three test species.
Both WC and sediment toxicity tests
will be conducted in SEA Ring and in
laboratory tests.
Five replicates of each treatment.
WC tests of one toxic Cu
concentration (400 ppb) and one
control, both with two test species.
Five replicates of each.
Total of six SEA Rings required.
                                                               Survival = 25
                                                               Growth = 5
                                                               Bioaccumulation =
                                                               15
                                                                                                                                         Survival = 50
                                                                                                                                         Growth = 110
                                                                                                                                         Bioaccumulation =
                                                                                                                                         30
                                                               Survival = 40
      Survival will be determined in all species: mysid, topsmelt, amphipod, polychaete, and clam.
      2Growth will be determined for one species only, the polychaete.
      3Bioaccumulation of PCBs will be determined in amphipods, polychaetes, and clams.

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standard lab exposures only) will be conducted in water only for 96 hours, with five concentrations (three
replicates each), but otherwise follow the same testing conditions summarized for the relevant test
organisms.

Water Column toxicity repeatability test - Survival of two organisms will be evaluated for the WC
toxicity test, Americamysis bahia (mysid shrimp) andAtherinops affinis (Pacific topsmelt) under a range
of Cu concentrations. This test will also include five replicate chambers for each exposure concentration
and a clean seawater control (e.g., laboratory water used to acclimate test organisms) with 10 organisms
in each replicate. The exposure period for the WC toxicity tests will be 96 hours, standard for acute
exposures for these organisms (EPA, 2002a).  The details of these tests are presented in Tables 6 and  7.
Reference toxicant tests will be conducted following standard EPA methods using five dilutions of copper
sulfate in the lab concurrent to the limited Cu exposures in the SEA Ring. The three concentrations of Cu
tested in the SEA Ring will, however, allow for direct comparison to results in the standard reference
toxicant test.

Sediment bioaccumulation repeatability test - Bioaccumulation of total PCBs (as a sum of detected
congeners) will be evaluated in amphipods, polychaetes, and clams exposed to PSNS sediments in both
the SEA Ring and laboratory exposures.  Exposure periods for the different species are shown in Figure 6.
Each test treatment will consist of five replicates, with amphipod and polychaete chambers containing 20
organisms, and clam chambers containing three organisms. Organisms from three of the replicates will
be purged in clean seawater overnight and analyzed for PCB concentrations.  The remaining two
replicates will be purged and frozen/archived. Tissues will be analyzed by ERDC as described in Section
B4.2.

Bl.2.2     Comparability. Comparisons between results obtained from tests in the SEA Ring and
traditional EPA and ASTM  laboratory methods will be evaluated under controlled laboratory conditions
as described in Section B 1.2.1. Comparability will be evaluated between responses (survival, growth, and
bioaccumulation) obtained in the standard laboratory exposures.  Since both exposures will occur under
controlled laboratory conditions, results should be similar with a goal of ฑ 20% for this assessment.

Sediment toxicity comparability test - The sediment comparability test will be conducted concurrently
with the repeatability test, using the results derived from the  approach described in Section B1.2.1.

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WC toxicitv comparability test - The WC comparability test will be conducted concurrently with the
repeatability test, using the results derived from the approach described in Section B 1.2.1.

Sediment bioaccumulation comparability test - The bioaccumulation comparability test will be
conducted concurrently with the repeatability test, using the results derived from the approach described
in Section B 1.2.1.

Bl.2.3     Reproducibility. To determine if different SEA Rings are capable of producing the same
results, reproducibility among three different SEA Rings will be evaluated under the same environmental
test conditions (i.e., the same environment, contaminants and test species). The reproducibility test will
utilize the same conditions used in the repeatability and comparability tests.  This evaluation will be
conducted using the WC toxicity tests only (described in Section B1.1.2) using a single concentration of
Cu (400 (ig/L). This test will be conducted concurrently with the same batch of test organisms, Cu stock
solutions, dilution water batch, and test conditions to minimize these potential confounding factors.  Mean
responses will be derived for each SEA Ring with a goal of less than 20% difference in mean response
between all three, and no statistical difference among the three SEA Rings tested.

B1.3       EPA/ASTM Method Laboratory Comparability Tests
Water column toxicitv bench scale test - Pre-cleaned 500 mL plastic or 1 L glass chambers will be
prepared by washing with 2% dilute detergent (Liquinox), rinsing five times  with tap water, placing in a
clean 10% HNO3 acid bath for a minimum of 4 h, followed by rinsing with acetone and five subsequent
rinses with deionized water. The final step consists of a thorough flushing with deionized water.  Salinity
for marine/estuarine organisms will be kept stable within ฑ 2 parts per thousand (ppt) of the target 32 ppt;
temperature will be stable within ฑ 1ฐC throughout the exposure period. DO concentration will be kept
above a minimum threshold of 4 mg/L as feasible with the current methods described.  The water quality
parameters (DO, salinity, pH and temperature) will be measured daily throughout the experiment in a
surrogate laboratory beaker.

Three concentrations  of the Cu spiked seawater will be tested: 100 ppb (sublethal), 200 ppb (possibly
lethal), and 400 ppb (likely lethal) with five replicates for each concentration. Five replicates of a
negative (uncontaminated seawater) control will also be tested.  The same organisms and same number of
organisms used in the SEA Ring will be used in the laboratory test.  The test chambers will be capped and

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placed in an incubator or recirculating water bath held under constant conditions for 96 hours. Survival
will be assessed at the end of the exposure.

Sediment toxicity bench-scale test - Tests will be conducted in 1 L containers that have been washed with
detergent (2% Liquinox), rinsed with acetone, five times with tap water, placed in a clean 10% nitric acid
bath for a minimum of 4 h, rinsed five times with deionized water, soaked in filtered, uncontaminated
seawater, and then thoroughly flushed with either distilled or deionized water. The final step consists of a
thorough flushing with deionized water.  Salinity for marine/estuarine organisms will be kept stable
within ฑ 2 ppt of the target 32 ppt; temperature will be stable within ฑ 1ฐC throughout the exposure
period.  DO concentration will be kept above a minimum threshold of 4 mg/L as feasible with the current
methods described.  The water quality parameters (DO, salinity, pH and temperature) will be measured
daily throughout the experiment in a surrogate laboratory beaker. The test sediments will be thoroughly
homogenized and press-sieved (< 2.0 mm) to remove any naturally occurring benthic organisms.
Sediment will be allowed to settle  overnight before introducing the organisms.

Flow rate: Per Tables 1 through  5,  laboratory exposures will be conducted as static  (amphipod) or static-
renewal (mysid, topsmelt, polychaete, clam) tests. The 17-gallon Chemtainers holding the SEA Rings
will follow the same renewal rate of the concurrent laboratory tests. It should be noted that although the
SEA Ring's on-board pump will be programmed to circulate the overlying water within the Chemtainer
(i.e., between the SEA Ring exposure chamber replicates and the water inside the Chemtainer outside the
replicates), there will be  no actual  replacement of the water from the system until the renewal is
conducted per the relevant laboratory-based protocol.  It is possible that the circulation of the overlying
water between the outside and inside of the SEA Ring exposure chambers could result in a different
exposure to the samples than the standard laboratory tests, but this difference is expected to be minimal.

Other observations:  During the exposure period, daily records will be  kept of observable test species'
mortality, emergence of infaunal organisms, formation of tubes or burrows, and any other or unusual
behavior. Daily records  of water quality (e.g., DO, salinity temperature, and pH) will be recorded in one
of the test replicates. Water quality within SEA Rings will also include continuous water quality sensing
within one  replicate chamber for each treatment using a Troll 9500 (In Situ, Inc.) datasonde (Figure 8).
Ammonia concentration will be  determined in the overlying water at test initiation and test end for each
test type.

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               Twist-Lock Connector
                                          TROLL 9500
                                                                              Nosecone
  Figure 8. A Troll 9500 datasonde (In Situ, Inc.) will be used to continuously measure and record
     water quality parameters in one of the SEA Ring exposure chambers associated with each
                                        treatment type
B1.4       Operational Factors
The operational factors to be evaluated include the training required to operate the SEA Ring. The
technology representative will train one Battelle staff member on the use of the SEA Ring. The Battelle
staff member, as well as the technology representative, will individually use the SEA Ring during the
tests.  The Battelle staff member will then document the ease of training and use of the SEA Ring. The
SEA Ring will also be compared to the EPA/ASTM approved method in terms of its practicality,
implementation and sustainability (i.e., the sampling time, waste produced, and the amount of protective
equipment required by the individual operating the technology). This will be evaluated visually by the
Battelle staff member and recorded.  Examples of information to be recorded include (1) effort during
training, (2) ease of preparation of site and technology for use, (3) actual use and repair of the technology,
(4) cost associated with maintenance and repair of the technology, (5) overall convenience of the
technology, (6) safety issues when using the technology, (7) number of samples that can be tested per day,
and (8) clarity of the technology representative's instructions. Battelle will summarize these observations
to aid in describing the technology performance in the Technology Verification Report.
B1.5       Supporting Analyses
Several supporting measurements will be performed by SPAWAR during testing. Table 7 summarizes
the measurements, equipment and analytical methods or SOP.

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                             Table 7.  Test Methods and Equipment
          Parameter
                      EPA Reference Test Method and
                     	Equipment	
SEA Ring Method and Equipment
Temperature
Dissolved oxygen
pH
Salinity

% solids
Total Organic Carbon (sediment)
Silt and clay content
PCB Congeners (sediment)

PCB Congeners (tissue)

Copper (seawater)
Ammonia (overlying water)
                     Oakton pH 11 meter
                     Orion 830A D.O. Meter
                     Oakton pH 11 Meter
                     Orion A+ conductivity meter
Troll 9500 Datasonde (In Situ, Inc.)
Troll 9500 Datasonde (In Situ, Inc.)
Troll 9500 Datasonde (In Situ, Inc.)
Troll 9500 Datasonde (In Situ, Inc.)
                                                EPA 1311
                                Modified Corp Eng. 81 and EPA 9060 procedures
                                          ASTM Method D422-63
                                        Extraction: EPA Method 3545
                                        Analysis: EPA Method 8082B
                                        Extraction: Jones et al. (2006)
                                        Analysis: EPA Method 8082B
                                             EPA Method 6020
                                           HACH Method 10031
B1.6
Statistical Analysis
Sediment toxicity data:  A total of six test groups (two organisms, and three test sediment types) including

a reference sediment group (controls) will be assessed.  Each group will be assessed in replicates of five.

General descriptive characteristics will be provided in the form of n, mode, mean, standard deviation,

median, minimum and maximum for continuous measures (test conditions, initial number of organisms,

concentration of contaminants, and the number and percent of organisms surviving in each of the replicate

chambers at the test) (EPA, 2002b).
Mean mortality in the control sediment of less than 10% will indicate acceptability of the test (organisms

are not affected by stressors other than the contaminants being tested) (EPA, 1994, 2002a). For

comparison purposes, the distribution of the proportion of surviving organisms and the homogeneity of

variances will be examined. If the data do not satisfy the assumptions of normality and constant variance,

they will be transformed using the arcsine/square root transformation or any other transformation that

increases normality and stabilizes the variance, such as the log transformation. The primary comparisons

of the number of organisms surviving between the replicates within a SEA Ring and between SEA Rings

will be performed using the analysis of variance (ANOVA) and the Dunnett's test (each test versus

control) or other suitable multiple comparison method.  A secondary comparison of the number of

organisms surviving in all the test groups combined with that in the reference sediment (uncontaminated)

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will be performed using the t-test.  A non-parametric test such as Kruskal-Wallis test may also be
explored on the untransformed data. Test results that are significantly different than the controls will be
determined using these statistical tests.

The LC50, defined as the concentration at which 50% lethality occurs, will be calculated for the reference
toxicant tests.  The statistical package CETIS (Comprehensive Environmental Toxicity Information
System) to calculate the LC50. The LCSOs will also be compared to historical data available at SPAWAR
and Nautilus to see if sensitivity of the test species/method is similar to that historically observed under
controlled laboratory conditions.

Water column toxicity data:  Test groups (two organisms, three Cu concentrations) and a clean seawater
group (control) will be assessed. Each group will be assessed in replicates of five  (ASTM, 2008; EPA,
2002a). General descriptive characteristics will be provided in the form of n, mode, mean, standard
deviation, median, minimum and maximum for continuous measures (test conditions, initial number of
organisms, concentration of contaminants, and the number and percent of organisms surviving in each of
the replicate chambers at the test).

A mean mortality in the control group of less than 10% will indicate acceptability  of the test (organisms
are not affected by stressors other than the contaminants being tested) (EPA, 1994, 2002a). For
comparison purposes, the distribution of the proportion of surviving organisms and the homogeneity of
variances will be examined.  If the data do not satisfy the assumptions of normality and constant variance,
they will be transformed using the arcsine/square root transformation or any other transformation that
increases normality and stabilizes the variance, such as the log transformation. The primary comparisons
of the number of organisms surviving between the groups will be performed using the ANOVA and the
Dunnett's test (each test versus control) or other suitable multiple comparison method. The test group
with the highest Cu concentration will be compared to the control group. A secondary comparison of the
number of organisms surviving in all the test groups combined with that in the control group will be
performed using the t-test. A non-parametric test such as Kruskal-Wallis test may also be explored on the
untransformed data. Test results that are significantly different than the controls will be determined using
these statistical tests.

The LC50 will be calculated for the standard laboratory reference toxicant tests, as well as the concurrent
Cu dilutions series conducted in the SEA Rings.

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Bioaccumulation test data: PCB concentrations will be measured in tissues from organisms exposed to
sediment from YB/DB controls and PSNS.

Student t-tests will be used to compare the differences between the groups (a = 0.05) in order to
determine whether organisms exposed in the SEA Rings bioaccumulated PCBs differently than in the
laboratory tests. Dunnett's test may be used to compare individual test groups with the reference sediment
group.

Repeatability: Repeatability, assessed as replicate variability in this case, will be evaluated for the
sediment toxicity, WC toxicity and bioaccumulation tests.

The outcome (the number of organisms surviving in each of the replicate chambers at the end of the test
period or the bioaccumulation) will be calculated overall across all test groups  and within each test group
(one of two organisms, and one of three sediment types) using descriptive statistics.

Precision will be evaluated using the standard deviation and the standard error of the sample mean ( se ),
calculated as the sample standard deviation  (a) divided by the square root of the sample size (n):

                                          se = a/^Jn

The smaller the  se, the greater the precision.

The coefficient of variation ( CV ) will be calculated as the percentage of the sample standard deviation
(a) divided by  the sample mean (x ):
                                               fa\
                                         CV =   -  100
                                               \x/

Similar measurements will be conducted for the organisms in the reference sediment (uncontaminated)
and will be  considered a measure of stability of the  SEA Ring device. A CV of less than 25% will be a
goal.

Differences in the outcome between the groups within the same SEA Ring will be explored using
ANOVA and the Tukey method. The number of organisms surviving (or the uptake of contaminants in

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case of the bioaccumulation test) in each test group will be compared to that in the control group using
ANOVA and the Dunnett's test or other suitable multiple comparison method. A non-parametric test
such as Kruskal-Wallis test may also be explored on the untransformed data.

Comparability:  The purpose of this analysis is to ensure that the SEA Ring will provide comparable data
to the traditional EPA/ASTM methods under controlled laboratory conditions. Thus, the concurrently
conducted traditional EPA/ASTM methods will be considered the gold standard in this analysis.

Comparability will be assessed for the same tests used to evaluate replicate variability. The general
analytical approach will be to compare the difference between all test groups with the corresponding
traditional EPA methods, followed by between group comparisons.

For the sediment and WC toxicity tests, the overall difference in the number of organisms surviving in the
SEA Ring will be compared to that observed using traditional EPA methods.  Comparisons will be
performed using the t-test or a non-parametric analog as discussed above for two sample comparisons.
Between group differences (with more than two groups) will be explored using ANOVA and the
Dunnett's test.

Uptake of contaminants in tissues during the bioaccumulation exposures conducted in the SEA Ring will
be compared to that obtained following the traditional EPA/ASTM methods using the t-test or a non-
parametric analog.

Other tests may be conducted as appropriate. For example, within the sediment toxicity and WC toxicity
tests, each test group result may be standardized by the corresponding control and that standardized result
compared to the standardized result obtained using the traditional EPA methods.

Deviation of the sediment toxicity and WC toxicity test results  from the traditional EPA methods may be
assessed in terms of bias. Bias will be calculated as average percent difference (%D) of each of the
sediment toxicity, WC toxicity and bioaccumulation test results from the traditional EPA methods both
overall and within each test group, as shown below:
                                                        100

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where k is the number of valid comparisons, and x is the sample mean and X is the mean of the
traditional EPA methods.

Reproducibility: The purpose of this analysis is to determine if different SEA Ring units have a similar
performance under controlled experimental conditions. At least three different SEA Rings will be
compared under the same experimental conditions (same environment, contaminant concentration and test
organism). The test will be conducted for the WC toxicity tests only (described in Section B1.1.2), and
will be conducted concurrently with the same batch of test organisms, the highest Cu stock solution,
dilution water batch, and test conditions to minimize these as potential confounding factors.

The general analytic approach will be to compare the results among all SEA Rings deployed. The overall
difference in the number of surviving organisms will be compared among the SEA Rings using ANOVA
and between group differences will be explored using  multiple comparison tests.

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                        B2 SAMPLING METHOD REQUIREMENTS

B2.1       Toxicity Test Breakdown - Collection of Test Organisms
The exposure chambers in the SEA Ring are held in place with a retaining pin. Upon completion of the
exposure period, the retaining pin is removed and the chamber freed from the chamber holder.  Test
organisms from both SEA Ring exposure chambers and laboratory beakers will be recovered by sieving
sediment through a 500 (im mesh sieve, which will retain the survivors.

B2.2       Collection and Analysis of Tissue Samples
At the conclusion of each sediment toxicity test, organisms will be recovered from the sediment with a
500 (im mesh size stainless steel  sieve, enumerated, and transferred to clean seawater to purge ingested
sediment overnight. Whole amphipods  and polychaetes, and soft body portions from clams from each
replicate will then be quickly rinsed in deionized water, weighed (for wet weight/growth assessment), and
frozen (-20 ฐC) in  2 mL plastic micro-centrifuge vials until chemical analysis.

B2.3       Collection and Analysis of Water and Sediment Samples
The concentration of Cu in WC toxicity tests will be confirmed through quantitative analysis. Water
samples of each Cu test concentration (control, 100 ppb, 200 ppb, and 400 ppb) will be collected for
analysis.  Samples will be  collected using trace metals techniques (Method EPA 6020) in 500 mL HDPE
or fluorocarbon bottles acidified with HNO3 to pH < 2.  Samples will be stored at 0 to 4ฐC for up to 6
months, and shipped to the laboratory under COC.

The concentration of PCBs in sediment toxicity and bioaccumulation tests (YB/DB and PSNS sediments)
will be confirmed through quantitative analysis. Prior to dispensing the homogenized sediments to test
chambers, a 500 g sample  will be collected into a wide-mouth glass with a Teflonฎ-lined lid and chilled to
0 to 4ฐC.  Sediment will be extracted using EPA SW846, Method 3545, and  analyzed using Method
8082B.

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                 B3 SAMPLE HANDLING AND CUSTODY REQUIREMENTS

B3.1       Handling of Aquatic Organisms
All test organisms will be acquired from commercial vendors from either laboratory culture or field
collection, and shipped overnight to the SSC Pacific Laboratory. Upon arrival, all test organisms will
immediately commence acclimation to laboratory test water quality conditions. Organisms will be
observed for abnormal behavior and mortality prior to use in tests.  A mortality rate of 5% will be used as
a threshold for organism quality prior to use in verification testing.

Organism handling will follow laboratory or above-mentioned procedures for addition to the SEA  Ring
apparatus. Following the appropriate exposure duration, all organisms from the bioaccumulation tests
will be purged in uncontaminated SSC Pacific Laboratory seawater overnight, weighed, and frozen in
preparation for shipment to ERDC. A subsample of organisms will also be frozen at the beginning of the
test without any exposure to assess time zero concentrations, if needed.

B3.2       Sample Custody
Sample custody will be maintained for all water, sediment, and tissue samples. Each sample will have a
unique project identification number.  This identification number will be recorded on a sample collection
form along with the other information specified on the form. After the labeled sample containers are
inspected, the sample custodian will complete the analysis request on the COC form.  The COC form will
include details about the sample, such as the time, date, location, and person collecting the sample. The
COC form will track sample release from the sampling location to the testing laboratory. The COC form
will be signed by the person relinquishing samples once that person has verified that the COC form is
accurate. Samples will be sent to the  appropriate laboratory via Federal  Express Next or Second Day
Service (or equivalent service).

The COC procedures emphasize careful documentation of constant secure custody of samples during the
laboratory, transport, and analytical stages of project. The sample custodian (and alternate) responsible
for the proper COC during this project is:

Sample Custodian:
               Gunther Rosen
               SPAWAR Systems Center Pacific

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              53475 Strothe Rd., Bldg. 111, Rm 216
              San Diego, CA 92152
              Tel: (619) 553-0886
              Cell: (619) 890-9692
              E-mail: gunther.rosen@navy.mil

Alternate custodian:
              Marienne Colvin
              SPAWAR Systems Center Pacific
              53475 Strothe Rd., Bldg. Ill
              San Diego, CA 92152
              Tel: (619) 553-5615
              Cell: (858) 349-2926
              E-mail: marine.colvin.ctr@navy.mil
B3.3       Sample Receipt
The laboratory's sample clerk will examine the shipping container and each sample cassette or sample
container to verify sample numbers and check for any evidence of damage or tampering. The COC form
will be checked for completeness and signed and dated to document receipt. Any changes will be
recorded on the original COC form and then the form will be forwarded to the VTC.  The sample clerk
will log in all samples and assign a unique laboratory sample identification number to each sample and
sample set.  COC procedures will be maintained in the analytical laboratory.

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                       B4  ANALYTICAL METHOD REQUIREMENTS

B4.1       Water Analysis
Cu analysis will be conducted at the SSC Pacific Laboratory.  Samples associated with the WC testing
will be analyzed for Cu in duplicate. Cu concentrations in the exposure water will be verified using a
Perkin Elmer ELAN DRC IIICP-MS.  The lab will use EPA Method 6020 for quantification. Actual
detection limits will be determined by the laboratory and the method used to calculate them will be
reported with the test data. Duplicate samples as well as spike samples will be measured as a QA/QC
measure. The SSC Pacific Laboratory technical point of contact for Cu measurements will be Brandon
Swope (brandon.swope@navy.mil). He will provide SOPs and appropriate QA reporting for the
verification test.

The contact information for the SSC Pacific Laboratory representative is:
Brandon Swope
SPAWAR SSC Pac Chemistry Laboratory
53560 Hull Street
San Diego, CA 92152-5001

B4.2       Sediment and Tissue Analysis
PCB congeners will be analyzed in  both sediment and tissues of relevant tests. Following the appropriate
exposure duration, all necessary organisms will be purged in uncontaminated (SSC Pacific Laboratory
dilution water) seawater overnight,  weighed, and frozen in preparation for shipment to ERDC.  ERDC
will be responsible for analyzing the samples for PCB congeners. The 18 National Oceanic and
Atmospheric Administration Status & Trend Congeners will be quantified for this test: PCBs 8,  18, 28,
52,44,66, 101, 118, 153, 105, 138, 187, 128, 180, 170, 195, 206, and 209.  The handling of the sediment
and tissue samples by ERDC is outlined in its SOP. Sediment samples will be extracted using pressurized
fluid extraction (EPA Method 3545), and analyzed using gas chromatography (GC) following EPA
Method 8082B. Reporting limits for PCB congeners in sediment are expected to be <0.6 (ig/kg dry wt.
Tissue analysis will be  conducted using a micro-extraction technique for use with small masses (150-500
mg wet weight; Jones et al., 2006).  Tissue extracts will be analyzed for PCB congeners by GC (EPA
Method 8082B). Reporting limits for tissue are expected to be less than 2 (ig/kg on a wet weight basis.
Sediment and tissue PCB concentrations will be expressed as the sum of all detected PCB congeners, or

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as the sum of PCB homologs.  The actual detection limits and the method used to calculate them will be
reported with the test data.

Three tissue samples for each species will be analyzed for both laboratory and SEA Ring exposures,
providing QA of the measurement and sufficient data with which to make statistical comparisons between
the laboratory and SEA Ring exposure methods. Ms. Patricia Tuminello will be the point of contact at
ERDC.  She will provide SOPs and appropriate QA reporting for the verification test.

The contact information for the SSC Pacific ERDC Chemistry Laboratory representative is:
Patricia Tuminello
USAGE ERDC Chemistry Laboratory
3909 Halls Ferry Road
Vicksburg, MS 39180-6199

B4.3       Tissue Lipid Analysis
Polychaete lipid concentrations will be analyzed by the ERDC toxicology laboratory with a
spectrophotometer at 490 nm following homogenization and chloroform/methanol extraction, and
calibrated using stock solutions of soybean oil according to Van Handel (1985).

The contact information for the USAGE ERDC Environmental Laboratory Risk  Assessment Branch
representative is:
Dr. Jacob Stanley
3909 Halls Ferry Road
Vicksburg, MS 39180-6199
jacob.k.stanley@us.army.mil

B4.4       Instrument Calibration Requirements
The inductively coupled plasma mass spectrometry (ICP-MS) calibration requirements are presented
below.  If criteria are not met, analysis will stop, corrective action taken, the instrument recalibrated, and
samples not bracketed by a passing initial calibration (ICAL) or continuing calibration verification (CCV)
reanalyzed:
        •   For copper measurements using ICP-MS a multi-point (no less than  five) calibration curve
           will be generated using Perkin Elmer multi-element solution 3 (Part No. N9300233) diluted

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           with IN optima grade nitric acid. The standard curve is rejected if the R2 value is less than
           0.995.  The range of the calibration curve is constructed based on the best estimate of copper
           concentrations being measured.  If a measured value falls outside of the standard curve range,
           the sample will be re-run with a different dilution factor or using a standard curve with a
           greater concentration range.
       •   ICAL: Prior to analysis a minimum of one high standard and a calibration blank; if more than
           one calibration standard is used, r > 0.995.
       •   CCV: After every 10 field samples and at the end of the analysis sequence.  All analytes
           within ฑ10% of true value.
The GC calibration requirements are presented below. If criteria are not met, analysis will stop,
corrective action taken, the instrument recalibrated, and samples not bracketed by a passing ICAL or
CCV reanalyzed:
       •   ICAL: Prior to analysis a minimum of five standard standards; r > 0.995.
       •   Second source calibration verification (ICV): Immediately following ICAL; all project
           analytes within ฑ20% of expected value from
       •   CCV: Prior to sample analysis, after every 10 samples, and at the end of the analysis
           sequence. All project analytes within ฑ20% of expected value.

B4.5       Quality Control
Laboratory QC samples will be processed with each analytical batch to demonstrate analytical control. If
criteria are not met, the sample should be re-analyzed and/or re-extracted and re-analyzed. If re-analysis
is not possible due to available sample mass or holding time, then the data should be reported with a "J"
qualifier to indicate that the value is an estimated value, typically outside of the calibration range. This is
a common EPA data qualifier used in data analysis.

The ICP-MS QC requirements for Cu analysis are presented below.
       •   Method blank: One per batch of <20  samples; no target analyte detected at > detection limit.
       •   Laboratory control sample (LCS): One per batch of <20 samples; recovery within laboratory
           control limits or 80 to 120%.
       •   Matrix spike sample: One per batch of <20 samples; used to assess matrix interference
           recovery within laboratory control limits or 25 to 145% as determined by the laboratory. If
           LCS passes, re-analysis is not required.

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The GC QC requirements for PCB analysis are presented below.
       •   Method blank: One per batch of <20 samples; no target analyte detected at > detection limit.
       •   LCS: One per batch of <20 samples; recovery within laboratory control limits or 25 to 145%
           (based on PCB congener or Aroclor)
       •   Matrix spike sample:  One per batch of <20 samples; used to assess matrix interference;
           recovery within laboratory control limits or 25 to 145% (based on PCB congener or Aroclor).
           If LCS passes, re-analysis is not required.
       Surrogate recovery: One or more surrogates spiked into each sample prior to sample processing
       and extraction; recovery within laboratory control limits. The acceptable percent recoveries for
       the surrogates are: for water samples -TMX, 25 to 140% and decachlorobiphenyl, 40 to 135%;
       for sediment samples -TMX, 40 to 125%, decachlorobiphenyl, 50 to 125%; and  for tissue
       samples - TMX, 45 to 125% and decachlorobiphenyl, 45 to 125%. The surrogate recoveries are
       defined by the laboratory based on historical experience with the extraction and  analysis method
       in tissue. In particular, it should be noted that the tissue sample size  (150 to 500  mg) is
       significantly less than the standard amount (30 g) and that will reduce extraction efficiency.
       •   Acceptance criteria for the PE sample  will be assessed as the percent recovery vs. the actual
           value defined by the PE supplier. The MS recovery criteria will be applied (25  to 145%) as
           acceptance criteria. This is well within acceptable control limits because due to  interferences
           from the matrix itself (tissue samples)  it may not be possible to obtain a clean chromatogram
           to accurately and specifically integrate a specific PCB peak. EPA method  8082A shows a
           similar range  for the fish tissue  Standard Reference Material: 33 to 133% recovery.

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                         B5 QUALITY CONTROL REQUIREMENTS

QC measures are included to ensure quality data are provided by the verification test. This includes
reference toxicant tests, acceptable results in control treatments, documentation that test conditions were
within required conditions, sufficient replication to demonstrate repeatability and ability to detect
significant differences among treatments.

B5.1       Reference Toxicant Test
Reference toxicant tests (also known as positive controls) are typically conducted concurrently with each
batch of test organisms to ensure organism and laboratory technical quality.  Reference toxicants for the
selected test types are Cu or cadmium,  depending on the species (Tables 1 through 5). Five
concentrations and a control will be prepared from verified stock solutions consisting of CuSO4 or CdCl2.
LC50 values generated from the dose response curves should be within two standard deviations of the
running mean for the testing laboratory. The proposed concentrations for the reference toxicity tests are
within the same range as that used for the WC toxicity test (100 to 400 ppb) and include an additional
concentration of 800 ppb. Where insufficient data are available, LCSOs should be comparable (within a
factor of 2) to published values for tests conducted under the same conditions. The control charts are
provided in Appendix B.

B5.2       Control Performance
Control survival is frequently used as a measure of test acceptability/QC.  Where  denoted in Tables 1
through 5, survival requirements will  be  used to assess overall QC, typically 90% survival in exposed
organisms.

B5.3       Test Conditions Acceptability
Each test has specific water quality  acceptability criteria, including measures for pH, temperature,
salinity, and DO.  These data will be recorded daily on the attached data sheets (Appendix A), and
compared with the acceptable ranges shown in Tables 1 through 5.  Deviations from the acceptable ranges
will be considered during data interpretation. Ammonia concentration (a confounding factor in some
sediment toxicity tests) will also be  measured in the overlying water prior to test initiation and test end for
each test type, using a HACH DR/2400 Spectrophotometer (Colorimetric Method, Method 10031).  If
ammonia concentrations exceed published thresholds for the test species, a renewal of the overlying water
prior to organism addition will be considered and/or resulting data will be  flagged prior to acceptance as

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part of the verification study. It should be noted that ammonia is also considered a naturally occurring

toxicant.


B5.4       Comparison to Background Tissue Levels

PCB bioaccumulation in the polychaete and clam will be used as a means of assessing repeatability within

SEA Ring tests, and comparability between laboratory and SEA Ring tests. Tissue concentrations in the

PSNS sediment will be compared statistically with the YB control sediment.

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      B6  INSTRUMENT/EQUIPMENT TESTING, INSPECTION, AND MAINTENANCE
When Battelle staff operate and maintain the SEA Ring undergoing testing, those activities will be

performed as directed by the technology representative. Otherwise, operation and maintenance of the

samplers will be the responsibility of the technology representative.  The manual for the SEA Ring is

provided in Appendix D.

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                    B7  INSTRUMENT CALIBRATION AND FREQUENCY


The SEA Ring will be cleaned as specified above, disposable parts replaced, batteries charged, and tested

for proper function prior to test initiation.  Prior to and during (daily) the test, SEA Ring pumping

operation will be verified  using the on board hardware and connection to a laptop computer. Water

quality monitoring, which will be recorded continuously aboard SEA Rings, will be checked several

times during the exposures to ensure proper operation. All bench-top meters and probes (e.g., pH, DO,

salinity and temperature) used to measure water quality in the laboratory tests will be calibrated daily.

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           B8 INSPECTION/ACCEPTANCE OF SUPPLIES AND CONSUMABLES


All materials, supplies, and consumables will be ordered by the technology vendor.  Reagents and

standards used by SPAWAR in preparation of analytical standards, spiking solutions, and reference

toxicant tests will be reagent grade or better and used within the expiration date assigned by the

manufacturer.

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                            B9 NON-DIRECT MEASUREMENTS


Data published previously in the scientific literature will not be used to evaluate the vendor's technology

during this verification test.

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                                  BIO  DATA MANAGEMENT

Various types of data will be acquired and recorded electronically or manually by Battelle and vendor
staff during this verification test. Table 8 summarizes the types of data to be recorded.  All maintenance
activities, repairs, calibrations, and operator observations relevant to the operation of the sampling
systems being tested will be documented by Battelle or vendor staff in the project-specific LRB or
dedicated data collection forms. During testing, raw data (records of test setup, measurements,
observations, etc.) will be held by the SPAWAR point of contact. Once testing is complete, these  raw
data forms and records will be submitted to the VTC.  Report formats will include all necessary data to
allow traceability from the raw data to final results. A dedicated shared folder within the ETV AMS
Center SharePoint site will be established for all project records.

Records received by or generated by any Battelle or subcontractor staff during the verification test will be
reviewed by a Battelle staff member within 5 days of receipt or generation, respectively, before the
records are used to calculate, evaluate, or report verification results.  If a Battelle staff member generated
the record, this review will be performed by a Battelle technical staff member involved in the verification
test, but not the staff member who originally received or generated the record.  The review will be
documented by the person performing the review by adding their initials and date to the hard copy of the
record being reviewed. In addition, any calculations performed by Battelle will be spot-checked by
Battelle technical staff to ensure that calculations are performed correctly.  Some of the checks that will
be performed include:
        •   QC samples and calibration standards were analyzed according to the QAPP and the
           acceptance criteria were met.  Corrective action for exceedances was taken;
        •   100% hand-entered and/or manually calculated data were checked for accuracy;
        •   Calculations performed by software are verified at a frequency sufficient to ensure that the
           formulas are correct, appropriate, and consistent;
        •   For each cut and paste function, the first and last data value was verified versus the source
           data;
        •   Data are reported in the units specified in the QAPP;
        •   Results of QC samples are reported; and
        •   Any statistical calculations  described in this QAPP.

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Battelle will provide technology test data and associated reference data (including records; data sheets;

notebook records) from the first day of testing within one day of receipt to EPA and the vendor for

simultaneous review.  The goal of this data delivery schedule is prompt identification and resolution of

any data collection or recording issues. These data will be labeled as preliminary and will not have  had a

QA review before their release.

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                                                Table 8. Summary of Data Recording Process
     Data to Be Recorded
   Where Recorded
    How Often Recorded
      By Whom
       Disposition of Data
Dates, times, and details of test
events
ETVLRBs, test forms
Real time data recording
throughout testing
Battelle staff
Used to organize/check test results;
manually incorporated in data
spreadsheets as necessary
SEA Ring operating conditions,    ETV LRBs, or
maintenance, downtime, etc.       electronically

Cu concentration in water and      Obtained from
PCB concentration in sediment     laboratory
Water quality parameters
                         When performed
                         After each sampling event
Read electronically from   Initially and daily
instrument and recorded
in laboratory notebook
                               Technology
                               Representative and
                               Battelle staff
                               Battelle Staff
                               Technology
                               Representative and
                               Battelle staff
                       Incorporated in verification report
                       as necessary

                       Converted to spreadsheet for
                       statistical analysis and comparisons

                       Converted to spreadsheet for
                       statistical analysis and comparisons
Final dry weight of polychaetes
Number of surviving organisms
PCB concentration in tissue
samples
Obtained from
laboratory

Obtained from
laboratory

Obtained from
laboratory
After each sampling event


After each sampling event


After each sampling event
Technology
Representative

Technology
Representative

Technology
Representative
Converted to spreadsheet for
statistical analysis and comparisons

Converted to spreadsheet for
statistical analysis and comparisons

Converted to spreadsheets for
statistical analysis and comparisons

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                                          SECTION C
                               ASSESSMENT AND OVERSIGHT
                        Cl ASSESSMENT AND RESPONSE ACTIONS

Every effort will be made in this verification test to anticipate and resolve potential problems before the
quality of performance is compromised. One of the major objectives of this QAPP is to establish
mechanisms necessary to ensure this.  The procedures described in this  QAPP, which is peer reviewed by
a panel of outside experts, implemented by the technical staff and monitored by the VTC, will provide
information on data quality on a day-to-day basis.  The responsibility for interpreting the results of these
checks and resolving any potential problems resides with the VTC. Technical staff has the responsibility
to identify problems that could affect data quality or the ability to use the data. Any problems that are
identified will be reported to the VTC, who will work with the Battelle  Quality Manager to resolve any
issues. Action will be taken to control the problem, identify a solution to the problem, minimize losses,
and correct data, where possible.  Independent of any EPA QA activities, Battelle will be responsible for
ensuring that the audits described below are conducted as part of this verification test.

Cl.l       Performance Evaluation Audit
PE audits provide an independent assessment of the accuracy of laboratory analyses.  For the ERDC
laboratory,  which is analyzing PCB congeners in sediment and tissue samples, a PCB congener standard
reference material will be obtained from the National Institute of Standards and Technology and sent to
the ERDC laboratory for analysis. The PE sample will be a blind, independent standard reference
material supplied to the laboratory by  Battelle. The range of potential congeners will encompass the
congeners of interest; however, the actual congeners are blind so that both false positives and false
negatives can be assessed. The acceptance criteria will be based on the  actual concentrations which are
blind at this time. Battelle will evaluate whether the laboratory has passed or failed the PE. The results of
the PE sample will be reported to Battelle and EPA management.  If the laboratory PE results are not
acceptable, the laboratory will be informed as to whether the results are biased high or low.  Corrective
action will include an examination by  the laboratory of instrument, sample handling, and sample analysis
procedures.  A second PE will be supplied once the laboratory feels its analytical system is in control.
Sample analysis will not begin until PE results are acceptable. Alternatively, another laboratory will be
identified.  Routine analysis will not be initiated until the laboratory demonstrates the ability to analyze
the sample with acceptable results.

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C1.2       Technical Systems Audits
The Battelle QAO or delegate will perform a TSA during testing at SPAWAR. The purpose of the audit
is to ensure that the verification test is being performed in accordance with the AMS Center QMP and this
QAPP. The reference laboratories are not expected to be assessed during a separate TSA, provided
acceptable performance on the PE audits. The TSA may be designated to an independent person by
providing a checklist to be completed on site.  During the TSA, the Battelle QAO or designee will
compare  actual test procedures to those specified or referenced in this plan and review data acquisition
and handling procedures.  A project-specific checklist based on the QAPP requirements will be prepared
to guide the TSA, which will include a review of the test and analytical procedures, use of the SEA Ring
technology and general testing conditions and review of test records and documentation. The Battelle
QAO will also check data acquisition procedures, and may confer with the vendor staff.  The Battelle
QAO will prepare an initial TSA report and submit the report to the EPA Quality Manager (with no
corrective actions documented) and VTC within 10 business days after completion of the audit. A copy
of the final TSA report (with corrective actions documented) will be provided to  the EPA AMS Center
Project Officer and Quality Manager within 20 business days after completion of the audit. At EPA's
discretion, EPA QA staff may also conduct an independent on-site TSA during the verification test. The
TSA findings will be communicated to technical staff at the time of the audit and will be documented in a
TSA report.

C1.3       Data Quality Audits
The Battelle QAO, or designee, will audit at least 10% of the sample results acquired in the verification
test and 100% of the calibration and QC data versus the QAPP requirements.  Two ADQs will be
conducted for this project: The first will be conducted on the data set delivered within 30 days of test
initiation. The ADQ will be completed within 10 business days of receipt using a project-specific
checklist. The second ADQ will assess the remainder of the data, the draft report, and the verification
statement. During these audits, the Battelle QAO, or designee, will trace the data from initial acquisition
through reduction and statistical comparisons, to final reporting. All calculations performed on the data
undergoing the ADQ will be checked. Data must undergo a 100% validation  and verification by technical
staff (i.e., VTC, or designee) before it will be assessed as part of the data quality  audit. All QC data and
all calculations performed on the data undergoing the audit will be checked by the Battelle QAO. Results
of each ADQ will be documented using the checklist and reported to the VTC and EPA within 10
business  days after completion of the audit. These reports will not include documented corrective actions.
The completed ADQs with corrective actions documented will be provided to EPA within 10 business

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days of receipt from the VTC. A final ADQ that assesses overall data quality, including accuracy and
completeness of the technical report, will be prepared as a narrative and distributed to the VTC and EPA
within 10 business days of completion of the audit.

C1.4       QA/QC Reporting
Each assessment and audit will be documented in accordance with Section 3.3.4 of the AMS Center
QMP.  The results of all audits will be submitted to EPA within 10 business days as noted above.
Assessment reports will include the following:
       •   Identification of any adverse findings or potential problems;
       •   Recommendations for resolving problems (If the QA audit identifies a technical issue, the
           VTC or Battelle AMS Center Manager will be consulted to determine the appropriate
           corrective action);
       •   Response to adverse findings or potential problems;
       •   Confirmation that solutions have been implemented and are effective; and
       •   Citation of any noteworthy practices that may be of use to others.

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                              C2 REPORTS TO MANAGEMENT

During the laboratory evaluation, any QAPP deviations will be reported immediately to EPA. The
Battelle Quality Manager and/or VTC, during the course of any assessment or audit, will identify to the
technical staff performing experimental activities any immediate corrective action that should be taken.
A summary of the required assessments and audits, including a listing of responsibilities and reporting
timeframes, is included in Table 9.  If serious quality problems exist, the Battelle Quality Manager will
notify the AMS Center Manager, who is authorized to stop work. Once the assessment reports have been
prepared, the VTC will ensure that a response is provided for each adverse finding or potential problem
and will implement any necessary follow-up corrective action. The Battelle Quality Manager will ensure
that follow-up corrective action has been taken. The QAPP and final report are reviewed by the EPA
AMS Center Quality Manager and the EPA AMS Center Project Officer. Upon final review and
approval, both documents will then be posted on the ETV Web site (www.epa.gov/etv).
                           Table 9.  Summary of Assessment Reports(a)
    Assessment
Prepared By   Report Submission Timeframe
         Submitted To
       TSA
       ADQ
        PE
  Battelle     10 business days after TSA is
              complete
  Battelle     ADQ will be completed within 10
              business days after receipt of the
              initial data batch and then after all
              data for a phase is submitted
  Battelle     10 business days after receiving
              results of PE samples
EPA ETV AMS Center
EPA ETV AMS Center
EPA ETV AMS Center
(a)  Any QA checklists prepared to guide audits will be provided with the audit report.

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                                         SECTION D
                           DATA VALIDATION AND USABILITY
         Dl DATA REVIEW, VERIFICATION, AND VALIDATION REQUIREMENTS

The key data review and data verification requirements for this test are stated in Section BIO of this
QAPP. In general, the data review requirements specify that data generated during this test will be
reviewed by a Battelle technical staff member within 5 days of generation of the data. The reviewer will
be familiar with the technical aspects of the verification test but will not be the person who generated the
data. This process will serve both as the data review and the data verification, and will ensure that the
data have been recorded, transmitted and processed properly.  Furthermore, this process will ensure that
the monitoring systems data were collected under appropriate testing.

The data validation requirements for this test involve an assessment of the quality of the data relative to
the DQI (organism age and water quality) and QC results for this test referenced in Tables 1 through 5.
Any deficiencies in these data will be flagged and excluded from any statistical comparisons to the SEA
Ring being tested, unless these deviations are accompanied by descriptions of their potential impacts on
the data quality.

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                     D2 VERIFICATION AND VALIDATION METHODS

Data verification is conducted as part of the data review as described in Section BIO of this QAPP. A
visual inspection of handwritten data will be conducted to ensure that all entries were properly recorded
or transcribed, and that any erroneous entries were properly noted (i.e., single line through the entry, with
an error code, such as wn for wrong number, and the initials of the recorder and date of entry).
Instrument parameters and laboratory data collected during the test will be inspected to ensure proper
transfer from the data-logging system. All calculations used to transform the data will be reviewed to
ensure the accuracy and the appropriateness of the calculations. Calculations performed manually will be
reviewed and repeated using a handheld calculator or commercial software (e.g., Excel).  Calculations
performed using standard commercial office software (e.g., Excel) will be reviewed by inspection  of the
equations used for the calculations and verification of selected calculations by handheld calculator.
Calculations performed using specialized commercial software (i.e., for analytical instrumentation) will
be reviewed by inspection and, when feasible, verified by handheld calculator, or standard commercial
office software.

To ensure that the data generated from this test meet the goals of the test, a number of data validation
procedures will be performed.  Sections B and C of this QAPP provided a description of the validation
safeguards employed for this verification test. Data validation efforts include the completion of QC
activities and the performance of a TSA as described in Section C. The data from this test will be
evaluated relative to the measurement DQIs described in Section A8 of this QAPP. Data failing to meet
these criteria will be flagged in the dataset and not used for evaluation of the SEA Ring, unless these
deviations are accompanied by descriptions of their potential impacts on the data quality.
An ADQ will be conducted by the Battelle Quality Manager to ensure that data review, verification, and
validation procedures were completed, and to  ensure the overall quality of the data.

The PE  sample will be used as verification that the  laboratory analytical  system is in control to correctly
identify and quantify the PCB congeners of interest.

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                                                               Sediment Ecotoxicity Assessment Ring
                                                                                         QAPP
                                                                                   Page 71 of 73
                                                                                       Version 1
                                                                                   May 16, 2012
                   D3 RECONCILIATION WITH USER REQUIREMENTS

The purpose of this verification test is to evaluate the performance of the SEA Ring in situ technology
relative to standard laboratory-based EPA/ASTM Methods for evaluating sediment and WC toxicity to
aquatic and benthic organisms. To meet the requirements of the user community, input on the tests
described in this QAPP has been provided by external experts. Additional performance data regarding
operational characteristics of the  SEA Ring will be collected by verification test personnel.  To meet the
requirements of the user community, these data will include thorough documentation of the performance
of the samplers during the verification test. The data review, verification, and validation procedures
described above will ensure that data meeting these requirements are accurately presented in the
verification reports generated from this test, and will ensure that data not meeting these requirements will
be appropriately flagged and discussed in the verification reports.

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                                                            Sediment Ecotoxicity Assessment Ring
                                                                                      QAPP
                                                                                Page 72 of 73
                                                                                   Version 1
                                                                                May  16, 2012
                                        SECTION E
                                      REFERENCES
ASTM. 2008. Standard Practice for Statistical Analysis of Toxicity Tests Conducted Under ASTM
    Guidelines. El847-96.

ASTM. 2000. "Standard Guide for Conducting Sediment Toxicity Tests with Marine and Estuarine
    Polychaetous Annelids," E 1611-00. In: Annual Book of ASTM Standards. Vol. 11.05. Philadelphia,
    PA, pp 991-1016.

ASTM. 2010. "Standard Guide for Determination of the Bioaccumulation of Sediment-Associated
    Contaminants by Benthic Invertebrates," Designation: E1688 - 10. July.

Battelle. 2011. Quality Management Plan for the  ETV Advanced Monitoring Systems Center, Version 8.
    U.S. Environmental Technology Verification Program, April.

EPA. 1994a. Methods for Measuring the Toxicity and Bioaccumulation of Sediment-associated
    Contaminants with Estuarine and Marine Amphipods. U.S. Environmental Protection Agency. Office
    of Research and Development. EPA-600-R-94-025

EPA. 1994b. Method 200.8. Revision 5.4. Determination of Trace Elements in Waters and Wastes by
    Inductively Coupled Plasma-mass Spectrometry. Environmental Monitoring Systems Laboratory.
    USEPA-ORD. Cincinnati, OH.

EPA. 2000. Method Guidance and Recommendations for Whole Effluent Toxicity (WET) Testing (40
    CFRPart 136). United States Environmental  Protection Agency. Office of Water (4303). EPA 821-B-
    00-004. July.

EPA. 2002a. Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater
    and Marine Organisms" Fifth Edition. EPA 821/R-02/012, October.

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                                                             Sediment Ecotoxicity Assessment Ring
                                                                                       QAPP
                                                                                 Page 73 of 73
                                                                                    Version 1
                                                                                 May 16, 2012
EPA 2002b. Short-term Methods for Estimating Chronic Toxicity to Freshwater Organisms, EPA 821-R-
    02-013, October.

EPA. 2008. Environmental Technology Verification Program Quality Management Plan (ETV QMP).
    January (EPA/600/R-08/009).

EPA and USAGE. 1998. Evaluation of Dredged Material Proposed for Discharge in Waters of the U.S.-
    Testing U.S. Army Corps of Engineers Manual. Inland Testing Manual. EPA-823-B-98-O04
    Environmental Protection Agency & US Army Corps of Engineers, February 1998, Office of Water
    (4305).

Jones, R.P., R.N. Millward, R.A. Karn, and A.H. Harrison. 2006. "Microscale Analytical Methods for the
    Quantitative Detection of PCBs and PAHs in Small Tissue Masses," Chemosphere 62: 1795-1805.

Van Handel, E. 1985. "Rapid Determination of Total Lipids in Mosquitoes," J. Am. Mosquito Control
    Assoc. 1, 302-304.

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                       Sediment Ecotoxicity Assessment Ring
                                               QAPP
                                             Version 1
                                          May 16, 2012
    APPENDIX A
TEST DATA SHEETS

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10-Day Marine Sediment Bioassay
Static Conditions
                      Sediment Ecotoxicity Assessment Ring
                                             QAPP
                                           Version 1
                                        May 16, 2012

               Water Quality Measurements
Client:
Sample ID:
Test Day
0
1
2
3
4
5
6
7
8
9
10



Salinity
(ppt)











Temperature
(ฐC)











Dissolved
Oxygen (mg/L)











Test Species:
Start Date/Time:
End Date/Time:
PH
(units)











Technician
Initials











E. estuarius



Comments











 QC Check:
Final Review:

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28-Day Marine Sediment Bioassay
Static-Renewal Conditions
Sediment Ecotoxicity Assessment Ring
                              QAPP
                           Version 1
                       May 16, 2012

          Water Quality Measurements
Client:

Sample ID:
    Test Species:

   Start Date/Time:

   End Date/Time:
Test Day
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
Salinity
(PPt)





























Temperature
(ฐC)





























Dissolved
Oxygen (mg/L)





























pH
(units)





























Fed





























Water
Change





























Technician
Initials





























Comments





























  QC Check:
                                                                            Final Review:

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Sediment Ecotoxicity Assessment Ring
                           QAPP
                        Version 1
                     May 16, 2012
Marine Acute Bioassay
Static-Renewal Conditions
Project:
Sample ID:
Test No.:
Concentration
ppb
Lab Control



50



100



200



400



800







Initial Counts
QC'd by:



Re
P
A
B
C
D
A
B
C
D
A
B
C
D
A
B
C
D
A
B
C
D
A
B
C
D
A
B
C
D
Number of Live
Organisms
0
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5





24





























48




























72




























96




























Test Species:
Start Date/Time:
End Date/Time:
Salinity
(PPt)
0




























24




























48
1
f


i
f


i
f


i
f


i
f


i
f


i
f



Animal Source/Date Received:
72




























96































Water Qi
&Tes
Counts:
Readings:
Dilutions made by:
Temperature
0




























24




























Age at Initiation:
48
'
f


'
f


'
f


'
f


'
f


'
f


'
f


72




























96




























Dissolved Oxygen
mg/L)
0




























24





























48
1
f


i
f


i
f


i
f


i
f


i
f


i
f



Comments: i = intial reading in fresh test solution, f = fina read ng in test chamber prior to renewal
QC Check:

Organisms fed prior to initiation, circle one ( y / n)
Tests aerated? Circle one ( y / n ) if yes, sample ID(s): Duration:
Aeration source:


72




























96




























AM:
PM:
Final Review:
jality Measurements
t Organism Survival
Tech Initials
0



24



48



72



96





0




























24




























PH
units)
48
1
f



f



f



f



f


i
f



f


72




























96





























Feeding Times
0


24


48


72


96






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                                                                    Sediment Ecotoxicity Assessment Ring
                                                                                                 QAPP
                                                                                              Version 1
                                                                                          May 16, 2012
Marine Chronic Bioassay
           Project:
        Sample ID:
          Test No.:
                                                       Water Quality Measurements
                                                   Test Species:
                                                 Start Date/Time:
                                                  End Date/Time:
Concentration
(%)
Lab Control
Brine Control
6.25
12.5
25
50

Salinity
(PPt)
V •







•24"







• 49







Temperature
(ฐC)
1 B •







54 •







•48"







Dissolved Oxygen
(mg/L)
• CF







1 ฃ4 •







*48"







pH
(pH units)
• 0"







• 2* '







*8 •







                                         24
                              48
Technician Initials:    WQ Readings:
                Dilutions made by:

Animal Source/Date Received:
Comments:
 Ohrs:
24 hrs:"
48 hrs:"
QC Check:
                                                    Final Review:

-------
                                                                                                     Sediment Ecotoxicity Assessment Ring
                                                                                                                                QAPP
                                                                                                                              Version 1
                                                                                                                          May 16, 2012
Marine Chronic Bioassay
         Project:
      Sample ID:
        Test No.:
                                                                                           Water Quality Measurements
                                                                     Test Species: S. purpuratus
                                                                   Start Date/Time:	
                                                                   End Date/Time:
Concentration
%








Salinity
(PPQ
•0 •








ซ4 •








i8 '








TO '








96








Temperature
PC)
1 9








• 29








•49








•72"








•96'








Dissolved Oxygen
(mg/L)
•0 ป








24"








18 •








TO '








•96" '








PH
(pH units)
0








1 21








1 48








•7?








•96"








                                         24    48   72    96
Technician Initials:     WQ Readings:
                 Dilutions made by:
Animal Source/Date Received:
Comments:
 Ohrs:
24hrs:
48hrs:
72hrs:
       QC Check:
                                                                              Final Review:

-------
                       Sediment Ecotoxicity Assessment Ring
                                                QAPP
                                             Version 1
                                          May 16, 2012
   APPENDIX B

CONTROL CHARTS

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                                                                                                     Sediment Ecotoxicity Assessment Ring
                                                                                                                                 QAPP
                                                                                                                              Version 1
                                                                                                                          May 16, 2012
                (SSC SD -Lab 123) CONTROL CHART FOR (Americamvsis bahia survival (96h) EC25/EC50) AND CV
  600.00 n	r  110
  500.00 -•
(3
   100.00 --
                                                                                                                             -•  100
                                       •   M.-4-4-A   '
                                                                                                                                   s?
                                                                                                                                   o
     0.00 -I	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1-  0
           1.0  2.0  3.0 4.0  5.0  6.0  7.0  8.0  9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0 25.0 26.0 27.0 28.0 29.0 30.0

                                                              TEST NUMBER
                -•— LC50 MG/L
                -A—- LAB LCL
                     FLAT LCL
-->--• LAB UCL
—•— Running Mean EC50
-•— LAB CV
• EPA Max UCL
 MEAN LC50
 EPA CV% BENCH
• EPA Max LCL
 FLAT UCL

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                                                                                      Sediment Ecotoxicity Assessment Ring
                                                                                                               QAPP
                                                                                                            Version 1
                                                                                                         May 16, 2012
350.00
300.00
  0.00
                                       Control Chart for Atherinops affinis
                2.0     3.0     4.0     5.0
 6.0
7.0     8.0     9.0

  TEST NUMBER
                                                                    110
                                            •••••••••<
10.0    11.0    12.0     13.0    14.0    15.0
                LC50 MG/L
                LAB LCL
                FLAT LCL
LAB UCL
Running Mean EC50
LAB CV
                   • EPA Max UCL
                    MEAN LC50
                    EPA CV% BENCH
                          •EPA Max LCL
                          FLAT UCL

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                                     Sediment Ecotoxicity Assessment Ring
                                                                QAPP
                                                             Version 1
                                                          May 16, 2012
Table B-l. 96 hr Reference Toxicity Test Data



Species & Endpoint
E. estuarius
96-hr survival
E. estuarius
96-hr survival
M. galloprovincialis
48-hr development
M. galloprovincialis
48-hr development
S. purpuratus
Fertilization



Test Period
6/10-6/14/08
6/17-6/21/08
6/6 - 6/8/08
6/12-6/14/08
6/13/2008
LC50 or
EC50
(mg/L Cd or
|jg/L Cu)
7.0
7.9
8.9
10
20.5
Historical
mean ฑ 2
SD (mg/L
Cd or |jg/L
Cu)
6.4 ฑ4.8
6.1ฑ4.3
6.5 ฑ4.1
6.7 ฑ4.3
18.8 ฑ15.5

95% Lower
Confidence
Limit
6.2
6.6
4.1
4.3
19.5

95% Upper
Confidence
Limit
7.8
9.5
10.6
10.9
21.6



CV (%)
37
35.2
31.5
32
41.2

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                           Sediment Ecotoxicity Assessment Ring
                                                   QAPP
                                                Version 1
                                             May 16, 2012
        APPENDIX C

CHAIN OF CUSTODY FORMS

-------
              ENVIRONMENTAL SCIENCES AND
              APPLIED SYSTEMS BRANCH, CODE 71750
              53605 HULL STREET
              SAN DIEGO, CA 92152-5000
Chain of Custody Record
Systems Center
  San Diego
                             Date:	

                             Page	of
Project Title/Project Number:
Remarks/Air Bill:
Samplers): (Signature)
Tel:
Fax:
Email:
Special Instructions:
Field Sample
Identification
















Date
















Relinquished by: (Signature)
Relinquished by: (Signature)
Time
















Matrix
















Type
















Temp (ฐC)
















Project Leader:
Contact:
Contact Tel:
Requested Analyses

















Received by: (Signature)
Received by: (Signature)





















































































Date:
Date:




































































Time:
Time:

-------
                        Sediment Ecotoxicity Assessment Ring
                                                QAPP
                                             Version 1
                                          May 16, 2012
   APPENDIX D

SEA RING MANUAL

-------
               ZEBRA-TECH LTD
                  www.zebra-tech.co.nz
SEA Rings
Operation Manual
     Version 1.0

-------
                                                                       SEA Rings Operation Manual
Contents
1. Overview	1
  Pump	1
  Control module	2
  Chamber cap	3
2. Software Installation	4
3. Charging	4
4. On-Off Switch	4
5. Status Indicator LED's	5
6. Operation	6
  Chamber cap removal	6
  Software	6
7. Datafile	9
8. Serial Debug	9
  Fitting Exposure Chambers	9
9. Servicing	10
  Changing the pump tubing	10
  O-rings	10
10. Firmware Upgrade	  11
11. Connector Pin Outs	11
                                                                        ZEBRA-TECH LTD
                                                                                www.zebra-tech.co.nz

-------
                                                                 SEA Rings Operation Manual
1. Overview
Pump

The pump consists of a pump motor housing and a pump housing.
The pump motor housing contains the pump motor, control electronics, and battery pack.
       Warning:
       The pump has a very powerful motor that can cause personal harm.  Keep fingers away
       from the pump rotor and always switch off before removing the pump cover plates.
         Pump rotor
         support bar
    Pump roller
       Pump motor
         housing
  Pump

Pump rotor


 Inlet manifold
                                                                   Control module
                                                                 ZEBRA-TECH LTD
                                                                        www.zebra-tech.co.nz

-------
SEA Rings Operation Manual

Control module

The control module features 2 status indicator LED's, an on/off switch, and the charging/communication
connector.

         Battery
       status LED
Mode status
    LED
                                                                          Connector cap
                                                                           On/off switch
    ZEBRA-TECH LTD
                                          2|
           tvww.zebra-tech.co. nz

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                                                                  SEA Rings Operation Manual
Chamber cap
      Inlet connector

     Duck bill valve
Syringe port
  stopper
                                                                          Cap
                                                                      retaining pin
                                                                  Inlet filter
                                                                 Outlet filter
                                         3 |
                                                                   ZEBRA-TECH LTD
                                                                          www.zebra-tech.co.nz

-------
SEA Rings Operation Manual
2.  Software  Installation
The SEA Rings are supplied with a USB flash drive. This contains the SEA Rings communication software
installation package.  Double clicking this should launch the installer. When upgrading to a more recent
version, the previous version does not need to be removed prior to installation.

The latest software is available for the Zebra-Tech web site;

http://www.zebra-tech.co.nz/downloads
3.  Charging
The SEA Rings have an on-board Metal Hydride battery pack. The pack can be re-charged using the
supplied charger. Allow 24 hours for a full charge.

The charger model number is the Universal charger, part number BPNC112900. This can be powered
from an AC adaptor. The AC adaptor can be obtained from Radio Shack, part number 273-318, with an
adaptor plug, part number 273-344. (Note: Align "tip" on the adaptor plug with "+" on the charger).

After disconnecting the charger, do not replace the corns connector cap on the SEA Rings for 1 hour.
This enables any gas discharged by the battery pack to vent through the corns connector.

Metal Hydride batteries self-discharge at a rate of around 1% per day. Always charge the SEA Rings as
close to the deployment date as possible.
4. On-Off Switch
The SEA Rings control module has an On-Off switch. In the off position, the SEA Rings pump will not
operate, although the SEA Rings will still communicate with a PC whilst in the "off" position.

When the switch is turned on, if the start time/date has not been reached, the SEA Rings will sleep until
the start time/date rolls over. The first flush then occurs after the flush interval has expired.

If the start time/date has expired when the switch is turned on, the first flush occurs after the flush
interval has expired.
    ZEBRA-TECH LTD
            www.zebra-lech.co.nz

-------
                                                                     SEA Rings Operation Manual
5. Status Indicator LED's
The SEA Rings control module has 2 status indicator LED's, that blink every 15 seconds.
Battery status indicator:
      LED Blink Sequence:
           One flash
          Two flashes
         Three flashes

Operation mode indicator:
I      LED Blink Sequence:
           One flash
          Two flashes
         Three flashes
      Status Description:
             Ok
Low battery warning (< 7.3 volts)
Low battery shutdown (6.5 volts)
      Status Description:
             Off
   Delayed start countdown
         Operational
                                          15 |
                                                                     ZEBRA-TECH LTD
                                                                             www.zebra-tech.co.nz

-------
SEA Rings Operation Manual
6.   Operation
Chamber cap removal

The chamber caps are secured in the Chamber Holder with a retaining pin. The retaining pin is secured
by a keyhole style locking mechanism. To remove the retaining pin, rotate it so that the black dot is
uppermost. The pin can then be pulled out of the chamber holder.
   Keyhole lock
   Chamber cap
   retaining pin
                                                                         Chamber holder
                                                                         Orientation dot
Software

Ensure the SEA Rings are charged. Connect the corns cable to the SEA Rings and a USB port on the PC.
Start the SEA Rings communication application. Provided the SEA Rings are correctly connected and
operational, the min window should open (Figure 1).
    ZEBRA-TECH LTD
                                          6|
           tvww.zebra-tech.co. nz

-------
                                                                            SEA Rings Operation Manual
                                                17
                                           1    13  12
                                           15   20

                                           1    13  12
Start time (HH:MM)

Start date (MM:DD:YY)

Stop time (HH:MM)

Stop date (MM:DD:YY)

Chamber flush
duration (Minutes)

Chamber flush interval
(Minutes)
                       SEA Rings time/date  15:14:36 01/16/2012

                       PC time/date       15:14:37 01/16/2012
                                                                  About
                                                                Test pump
Offload
                                           1
                                                               Upload settings
                                                                Delete data
                                          Set time
                                                                  Close
                    Voltage: 1 AAV- Memory capacity status:1 %
                            Figure 1: SEA Rings application main program window
Test Pump

Pressing this button switches on the pump. The pump remains on until the button is pressed again, or
the SEA Rings application is closed.
Offload

The Offload button downloads data from the SEA Rings to a user selected file on the PC. The file format
is ASCII, comma separated, and can be opened in Excel.

The data in the SEA Rings is stored in non-volatile memory.  If the battery goes flat, data is not lost.
Upload Settings

Once the operating parameters have been set, they are sent to the SEA Rings by pressing the "Upload
settings" button.
                                               17 |
                                                                             ZEBRA-TECH LTD
                                                                                     www.zebra-tech.co.nz

-------
SEA Rings Operation Manual

Delete Data

Data can be deleted off the SEA Rings using the "Delete data button".


Set Time

The current time and date of the SEA Rings can be synchronised with the PC time and date. The SEA
Rings time will be reset if the battery goes completely flat.


Chamber Flush Duration

This field is the number of minutes that the pump will be operating for each flush cycle.


Chamber flush interval

This field is the number of minutes that the pump is not operating between flush cycles.
Voltage

This field indicates the battery voltage. Around 9 volts is fully charged, 7.5 volts is mid-charge, and 6.5
volts is flat. If the battery voltage drops lower than 6.5 volts, the SEA Rings will cease functioning, and
enter a low power shutdown mode. The pump will not operate until the batteries have been recharged.
Memory status

This is the percentage of the memory used.
    ZEBRA-TECH LTD
            www.zebra-lech.co.nz

-------
                                                                     SEA Rings Operation Manual
7.   Datafile
SEA Rings serial number: 1231
PC download time 13/01/2012 15:30
Start  15:17  13/1/2012   8.4   5
Stop  15:18  13/1/2012   8.4   4
Start  15:19  13/1/2012   8.4   5
Stop  15:20  13/1/2012   8.4   5

The fields are:
Start/stop, time (HH:MM), date (MM: DD: YY), battery voltage, number of pump revolutions.
8. Serial Debug
The SEA Rings can be optionally supplied with a wet pluggable connector on the side of the pump
housing. This can be used to monitor the pump operation in a laboratory test situation.

To display the serial debug, connect the cable onto a PC and start a terminal emulator, such as "Term",
which is included on the Zebra-Tech USB flash drive. Set the serial  port to the appropriate number and
set the baud rate to 19200. The parity is None, data bits 8, stop bits 1.

Whenever the pump starts or stops, the time and date will  be displayed, together with the number of
pump revolutions.

When the serial debug cable is disconnected, the dummy connector MUST be fitted to protect the
connector.

Never connect both the serial debug cable and the  main corns cable onto SEA Rings at the same time.

Fitting Exposure Chambers

The exposure chambers can be made out of Butyrate tube. The size is 2.75 OD x 2.625 ID.

The tube can be sourced from K-Mac Plastics, 3821 Clay Ave SW, Wyoming, Michigan 49548,
Tel: 616-406-0671.

The part number is KM-2340 - CAB- Hollow Tubes- Clear- Tenite- 2.75 OD x 2.625 ID.

A cross hole for the chamber cap pin needs to be drilled through the tube:

   1.  Install the exposure chamber onto the Chamber Jig, available from Zebra-Tech. Alternatively fit
       the chamber onto a chamber cap.
   2.  Using a 9mm drill, drill the cross hole through the walls of the tube, using the cap or jig as the
       guide.
   3. De-burr the 2 holes, particularly the internal side of the holes.
                                          19 |
                                                                     ZEBRA-TECH LTD
                                                                             www.zebra-tech.co.nz

-------
SEA Rings Operation Manual
9. Servicing
Changing the pump tubing

The SEA Rings use around 3m of silicone tube, 8mm ID x 10mm OD. When replacing the tube, replace all
the tubes using tube from a single roll. This ensures the tube wall thickness will be consistent. If tubes
with inconsistent wall thickness are used, the pump performance maybe compromised.

To change the peristaltic pump tubing:

   1.  Switch off the SEA Rings.
   2.  Disconnect the pump tubing from the tube connectors on the chamber caps, and the inlet
      manifold.
   3.  Remove the 4 nuts around the top of the pump, and lift off the pump cover plate.
   4.  Lift the pump housing off the pump motor housing.
   5.  Unscrew the 2 slotted nylon countersunk screws and remove the pump rotor support bar.
   6.  Gently ease the pump rotor up, out of the pump housing.
   7.  Remove the old pump tubing from the pump housing.
   8.  Replace the pump rotor and the rotor support bar.
   9.  Systematically thread the new pump tube pieces into the pump housing, rotating the pump rotor
      to aid insertion.
   10. Connect the tubes onto the corresponding port on the inlet manifold.  Manually rotate the pump
      rotor to ensure the tubing is correctly positioned.
   11. Hook the outlet end of the pump tubes onto the connector on the corresponding chamber caps.
   12. Replace the pump onto the pump motor housing ensuring the drive train engages correctly.
   13. Switch on the SEA Rings, and using the setup application, test run the pump, checking the tubes
      remain correctly positioned. Stop the pump.
   14. Replace the pump cover plate.

O Rings

Chamber Cap: 2 3/8" x 3/32" Nitrile (Optionally silicone)

Syringe port: 15/16" x 3/32" Nitrile (Optionally silicone)
    ZEBRA-TECH LTD
                                          I 101
           www.zebra-lech.co.nz

-------
                                                                           SEA Rings Operation Manual
10. Firmware Upgrade
The firmware inside the SEA Rings can be updated using the boot-loader application provided on the
Zebra-Tech USB flash drive. Consult Zebra-Tech prior to performing a boot-load.

   1.  Ensure SEA Rings application is closed.  Connect the SEA Rings to the corns cable and plug the
       cable into the PC.
   2.  Start the boot-loader application.
           chip45boot2 GUI
           Version 1.11
            Main   Automator |  Command Shell
                          chip45
             Select COM Port
RS455    iBaudratei     ] Show Non-Standard Baudrates
              COM24
              COM23
              C.Q.M7
              COM22
             Rash Hexfile  	
             c:\Pnojects\Sea Rings DevXExeXSea Ring .hex
             Eeprom Hexfile
                              Select Rash Hexfile
                                                               Select Eeprom Hexfile
                Send This Pre-String Before Connect and wait •*•
                                                 90
                      msec.
             •BOOTLOADER/
              Connect to Bootloader
                Start Application
   Program Rash
Program Eeprom
            Show Communication Log
           (C)chip45GmbH&Co. KG
      http://www .chip45.com
                               Ascii   <  Hex
Read Eeprom
                                   Status
                 better embedded.
                                              I HI
                                                                            ZEBRA-TECH LTD
                                                                                    www.zebra-tech.co.nz

-------
SEA Rings Operation Manual
 11.  Connector Pin Outs
Charging/Communication Cable:
I       Pin number:
            i
 Function:
  Charge
  Ground
PC Transmit
PC Receive
Optional Serial Debug Connector:
       Pin number:
            1
            2
 Function:
  Ground
PC Receive
   ZEBRA-TECH LTD
                                       I 12
          www.zebra-lech.co.nz

-------
                               Sediment Ecotoxicity Assessment Ring
                                                      QAPP
                                                   Version 1
                                                May 16, 2012
            APPENDIX E

STANDARD OPERATING PROCEDURES

-------
       SPfWAR
        Systems Center
           PACIFIC
Standard Operating Procedures
      November 10, 2011

             For
SSC Pacific Bioassay Laboratory
      Bldg. Ill Rm. 116
          53475 Strothe Road
          Bldg. Ill Room 116
        San Diego, CA 92152-5000
        619-553-0886 • 619-553-2766

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                                 Table of Contents

Section                                                                          Page

 1.0 PROCEDURES FOR CONDUCTING TOXICITY TESTS	4
   1.1 Bivalve embryo-larval development test	4
   1.2 Acute toxicity test with bioluminescent dinoflagellates (QwikLite)	8
   1.3 Reference toxicant test with marine amphipods	11
   1.4 Sediment toxicity test with marine amphipods	14
   1.5 Embryo-larval development test with sand dollars	18
   1.6 Embryo-larval development test with purple sea urchins	22
   1.7 Acute toxicity test with juvenile mysid shrimp	26
   1.8 Acute toxicity test with topsmelt larvae	30
 2.0 TEST CONDITIONS AND ACCEPTABILITY CRITERIA	34
   2.1 Bivalve embryo-larval development test (chronic)	34
   2.2 Sediment-water interface (SWI) toxicity test with bivalve embryos	35
   2.3 Marine Amphipod Reference Toxicity Test	36
   2.4 Sediment toxicity test with marine amphipods (acute)	37
   2.5 Bioluminescence Inhibition Test (Qwiklite) with Dinoflagellates	38
   2.6 Echinoderm embryo-larval development test (chronic)	39
   2.7 Mysid shrimp Survival Test (Acute)	40
   2.8 Topsmelt Larval Survival Test (Acute)	41
 3.0 PROCEDURES FOR EQUIPMENT	42
   3.1 Protocol for autoclave	42
   3.2 Calibration and use of the Orion 720A ISE meter/ ammonia probe	43
   3.3 Calibration and use of the Accumet pH meter	46
   3.4 Calibration and use of the Orion (model 840) dissolved oxygen probe	48
   3.5 Measuring ammonia with the HACK DR/2000  spectrophotometer	Error! Bookmark not
   defined.
   3.6 Measureing ammonia with the HACK DR/2400 spectrophotometer	50
   3.7Barnstead e pure water purification system	52
   3.8 Calibration and use of the Orion aplus (105a+) basic conductivity meter	53
   3.9 Calibration and use of the Orion (model 830A)  Portable dissolved oxygen probe	55
   3.10 Percival Scientific 136LL Incubator	57
   3.11 Calibration and use of the Oakton pH 11 meter	59
 4.0 STANDARD OPERATING PROCEDURES- MISCELLANEOUS	60
   4.1 Glassware and plasticware cleaning	60
   4.2 Receiving and holding test organisms	63
   4.3 Maintaining dinoflagellate cultures	65
   4.4 Preparation of enriched seawater medium (ESM)1	67
   4.5 Hatching brine shrimp and their use as test organism food	69
   4.6 Hypersaline brine and artificial sea salt use	70
   4.7 Reference toxicant test dilutions	72
   4.8 Acquisition, Reduction, and Reporting of Data	75
   4.9 Recording and handling data	76
   4.10 Statistical analysis of data	78
   4.11 Hazardous material storage, disposal and safety information	80

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  4.12 Counting sperm with a hemocytometer	85
  4.13 Counting mussel/oyster larvae using an inverted microscope	88
5.0 LOGS AND DATA SHEETS	90
  5.1 Echinoderm embryo-larval development test - water quality data	90
  5.2 Bivalve embryo-larval development test - water quality data	91
  5.3 Embryo-larval development test calculations	92
  5.4 Embryo-larvae development test results RAW data sheeT	93
  5.5 Dinoflagellate PMT count sheet for copper reference toxicant test	94
  5.6 Dinoflagellate PMT count sheet	95
  5.7Neanthes 28 day water chemistry data sheet	98
  5.8 Neanthes survival data sheet	99
  5.9 Amphipod 10 day water chemistry datasheet	101
  5.10 Amphipod survival data sheet	102
  5.11 Acute fish/mysid survival sheet	103
  5.12 Dinoflagellate maintenance log	104
  5.13 Brine Dilution Worksheet	105
6.0 REFERENCES	107

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1.0 PROCEDURES FOR CONDUCTING TOXICITY TESTS

1.1 BIVALVE EMBRYO-LARVAL DEVELOPMENT TEST
TESTING FACILITY:  SPAWAR SYSTEMS CENTER
                       BIOASSAY LABORATORY (RM 116)
                       CODE 71750
                       53475 STROTHE RD.
                       BLDG. 111
                       SAN DIEGO, CA 92152

I.      OBJECTIVE: This method estimates the chronic toxicity of effluent and receiving waters to the
       embryos and larvae of bivalve molluscs. The test endpoint is normal shell development and should
       also include mortality1.

II      NECESSARY MATERIALS AND SUPPLIES

          Brillo pads - to clean exterior of mussels
          Beakers- 400-600ml - with dilution water held at 15ฐC  for mussels once spawning is induced,
          and 1 L beaker for egg solution.
          Plastic holding tanks - 3 to 6L
          Ippt copper stock solution- for reference toxicant test
          Graduated cylinders - Class A, borosilicate glass or non-toxic plastic labware, 5 0-1000ml for
          making test solutions.
          pH meter - for measuring test solutions
          Dissolved oxygen meter - for measuring test solutions
          Refractometer - for determining salinity of test solutions
          Thermometer - digital or laboratory grade
          Test chambers - 20ml glass scintillation vials and caps - pre-conditioned in dilution water.
          Colored labeling tape
          Dilution water - natural seawater or hypersaline brine made from natural seawater and diluted
          with deionized water
          Light microscope and slides
          Pipets, automatic -  adjustable, to cover a range of 0.01 to 5 ml and pipette tips
          Calculator
          Volumetric flasks- Class A, borosilicate glass  or non-toxic plastic labware, 250ml for making
          test solutions
          Wash bottles - for reagent water, dilution water, for topping off graduated cylinders, for
          rinsing small glassware and instrument electrodes and probes
          Inverted microscope - for inspecting gametes and counting embryos and larvae.
          Counter, two unit, 0-999 - for recording counts of embryos and larvae
          80 jam screen
          54 jam screen
          37 jam screen
          22 jam screen
          Hemocytometer- for counting sperm
          Data sheets

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PROTOCOL FOR 48 HOUR BIVALVE EMBRYO-LARVAL DEVELOPMENT TEST WITH THE BLUE
MUSSELS OR PACIFIC OYSTER (Mytilus galloprovincialis or Crassostrea gigas) Cont'd

III     METHODS

    A. OBTAINING AND HOLDING ORGANISMS

       1.   Purchase mussels or oysters from Carlsbad Aquafarm and hold in tanks (cold room) with raw
           flowing seawater (ideal conditioning temperature is 12 - 14 ฐC) until they are needed for
           testing.  Holding and conditioning tanks should be drained and sprayed with fresh water at least
           once weekly to prevent accumulation of organic matter and bacteria. Dead animals should be
           removed daily.

       2.   Clean the exterior of approximately 50 mussels or oysters with a brillo pad and filtered
           seawater. Hang remaining mussels or oysters off research pier or place in cold room tanks,
           depending on space availability and testing requirements.

   B.  SPAWNING AND FERTILIZATION

       1.   Place approximately 10-15 mussels/oysters in a single layer at the bottom of the spawning
           chambers (3 or 6 L plastic holding tanks). Plug sink and fill with hot water. Place 2 L
           Erlenmeyer flasks filled with dilution water into the hot water. Remove flask when temperature
           reaches 25-30 ฐC, or approximately 10 ฐC above the holding temperature. Pour enough of the
           25-30 ฐC water on mussels/oysters so that they are covered completely. When individuals
           begin to spawn, remove from the holding tank and place each in a separate beaker at testing 15-
           20 ฐC) temperature in filtered seawater. During spawning, a sub-sample of gametes from each
           beaker should be observed for quality under the microscope and then labeled "eggs" or
           "sperm".

       2.   If no animals spawn within 30 minutes, the water should be returned to conditioning
           temperature (15 ฐC for mussels, 20 ฐC for oysters) for 15 minutes and the stimulation process
           repeated. In addition to heat treatment, mussels can be injected in the posterior adductor
           muscle with 1.0 ml of 0.5 M KC1. When individuals begin to release gametes,  immediately
           isolate in a 200-300 ml glass beaker with filtered dilution water held at 15 ฐC in the incubator.

       3.   Eggs should be passed through an 80-|om screen. The eggs will pass through the screen and
           debris retained. Pool quality eggs from different females together in a 1 L beaker, and fill with
           dilution water to approximately the 600 ml mark. Poor quality eggs will be vacuolated, small,
           or abnormal in shape. The concentration of the egg stock can be determined by counting a 1 ml
           sample at 400X. The pooled egg density should be adjusted to 5,000 to 8,000 eggs/ml before
           adding sperm.

       4.   Sperm should be passed through a 37-|o,m screen to remove feces and other material. Sperm
           will pass through the screen while debris will be retained. Sperm counts can be made  with a
           hemacytometer. Sperm should be added so there are 105 to 107 sperm/ml in the final mixture.

       5.   Add sperm to beaker with eggs. Hold at  15 ฐC, gently mixing solution with a stirring
           rod every few minutes.

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PROTOCOL FOR 48 HOUR BIVALVE EMBRYO-LARVAL DEVELOPMENT TEST WITH THE BLUE
MUSSELS OR PACIFIC OYSTER (Mytilus galloprovincialis or Crassostrea gigas) Cont'd

       6.  After fertilization (10 to 15 minutes), pass the embryo suspension through a 54-|om screen to
           remove debris.  Excess sperm, bacteria, and protozoans should be removed  by pouring
           embryos onto a 22-|am screen, washing delicately with dilution water, then  backwashing into a
           suitable container with dilution water. Adjust embryo density to about 1500 to 3000
           embryos/ml. Maintain the resulting mussel embryo suspension at 15 ฐC, and oyster embryo
           solution at 20 ฐC. Keep embryos suspended by stirring frequently, and begin test within 4
           hours.

    C. CONDUCTING THE TEST

       1.  About  1 hour after fertilization, a 1 ml sample should be placed in a Sedgwick-Rafter cell and
           the number of embryos developing to a 2-cell stage or beyond counted.

       2.  In addition to test materials or effluents, a reference toxicant test with copper should be
           conducted.  Copper concentrations should include 0, 2.9, 4.1, 5.9, 8.4, 17.2, 25 ppb Cu for
           mussels.  Concentrations should  include 0, 4.1, 5.9, 8.4,  17.2, 25, 35 ppb Cu for oysters.  Allow
           these solutions to equilibrate for  at least one hour before testing. *Please refer to the "Bivalve
           embryo development data sheet"' for calculations.

       3.  Within 4 h of fertilization, distribute embryos to test containers already containing 10 ml of test
           solution in a random order using an automatic pipette. Be sure to keep embryo suspension well
           mixed. This is achieved by use of a perforated plunger or gently alternating between swirling
           and back and forth motions of the flask. The concentration of embryos in the test solutions
           should be about 20 embryos/ml.  This typically requires an addition of 100 (il of a 2000
           embryo/ml suspension.

       4.  Cap or cover (with acrylic plates) scintillation vials to prevent evaporation.  Keep vials in an
           incubator at 15 ฐC (mussels) or 20  ฐC (oysters) on a 12 hr light/12 hr dark cycle.

       5.  Initial embryo density is measured in five additional scintillation vials, which are immediately
           preserved by adding 1 ml of concentrated formaldehyde  to each vial.

       6.  Water quality parameters (pH, temperature, salinity, dissolved oxygen) are  measured
           for an additional replicate with test organisms, but not used to assess larval  development, at test
           initiation and termination.

       7.  After 48 h (or up to 54 h if development in clean water controls is not complete),
           preserve test organisms by adding 1 ml concentrated formaldehyde and capping vials.


IV     DATA COLLECTION

    A. Within 7 days, count larvae using an inverted microscope, noting normally developed (those that
       have achieved the D-hinge stage) vs. abnormally developed.

    B. Refer to  "Protocol for counting mussel larvae"2 for tips on counting.

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PROTOCOL FOR 48 HOUR BIVALVE EMBRYO-LARVAL DEVELOPMENT TEST WITH THE BLUE
MUSSELS OR PACIFIC OYSTER (Mytilus galloprovincialis or Crassostrea gigas) Cont'd

V.      ANALYZING DATA

    A.  Using CETIS or Toxcalc, enter data retrieved from enumeration of larvae to determine the EC50,
        LOEC, NOEC, or other appropriate toxicity metric.  Please refer to "Protocol for Statistical
        Analysis of Toxicity Data3".

    B.  In accordance with USEPA (2002), all Toxcalc-generated concentration-response curves will be
        evaluated for acceptability.
'Modified from "US EPA's Short-Term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Marine and Estuarine
Organisms. First edition. EPA/600/R-95/136. August 1995.

2 "Bivalve embryo development data sheet" and "Protocol for counting mussel larvae" can be found in the sub-directory :
C:\WINDOWS\Desktop\Laboratory 116\Protocols and logs

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3 "Protocol for Protocol for Statistical Analysis of Toxicity Data" can be found in the sub-directory : C:\WINDOWS\Desktop\Laboratory
116\Protocols and logs
1.2 ACUTE TOXICITY TEST WITH BIOLUMINESCENT DINOFLAGELLATES
(QWIKLITE)
TESTING FACILITY:  SPAWAR SYSTEMS CENTER
                       BIOASSAY LABORATORY (RM 116)
                       CODE 71750
                       53475 STROTHE RD.
                       BLDG. 111
                       SAN DIEGO, CA 92152
I.      OBJECTIVE:  This method is used to estimate toxicity of effluent and receiving waters to
       dinoflagellates. When specific species of bioluminescent Dinoflagellates are exposed to toxicants, a
       measurable reduction in bioluminescence is observed following mechanical stimulation when
       compared to controls1.

II      NECESSARY MATERIALS AND SUPPLIES

           500 ml flasks to maintain dinoflagellate cultures.
           Temperature controlled light chamber (e.g. Percival Scientific Model I-35LLVL) capable of
           maintaining test conditions at 19 ฐC with a 12h light: 12h dark photoperiod
           Test chambers - 4.5 ml clear plastic cuvettes - pre-soaked in dilution water.
           Cuvette rack
           Colored labeling tape
           Dilution water - natural seawater (i.e.  Scripps) or hypersaline brine made from natural
           seawater
           Light microscope and slides
           Pipets, automatic - adjustable, to cover a range of 0.01 to 5 ml and pipette tips
           Calculator
           Volumetric flasks- Class A, borosilicate glass  or non-toxic plastic labware, 250ml  for making
           test solutions
           Ippt copper stock solution
           Graduated cylinders - Class A, borosilicate glass or non-toxic plastic labware, 5 0-1000ml for
           making test solutions.
           pH meter - for measuring test solutions
           Dissolved oxygen meter - for measuring test solutions
           Refractometer - for determining salinity of test solutions
           Thermometer - digital or laboratory grade
           Wash bottles - for reagent water, dilution water, for topping off graduated cylinders, for
           rinsing small glassware and instrument electrodes and probes
           Qwiklite testing equipment - either SeaLite or NRaD testing units
           Data sheets
           Black felt or box to cover dinoflagellates while testing
           Red light for minimal illumination for experimenter during testing

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PROTOCOL FOR CONDUCTING A 24 HOUR TOXICITY TEST WITH BIOLUMINESCENT
DINOFLAGELLATES (Ceratocorys horrida) Cont'd

III     METHODS

    A. DETERMINING CULTURE DENSITIES

       1.  Select a culture that is  1 to 2 weeks old.

       2.  To identify a healthy culture, look for a flask with a high density of cells (high density ensures
       less culture needed, which lowers the chance of EDTA and trace metals from interfering with the
       test), low levels of debris, and one that illuminates brightly when agitated (in a dark room).

       3.  After ensuring the culture is well homogenized, pipette a 20 \i\ aliquot onto a slide and count
       cells. Cultures are homogenized by gentle mixing, alternating between swirling and side to side,
       and back and forth movement of the flask.  If cells are moving too fast on the slide, add a drop of
       formalin. Repeat 2 more times with additional 20 \i\ aliquots.

       4.  To determine the density in cells/ml, take the mean count of the three 20 jol aliquots and use the
       following formula:

       (X cells/20 nl) x (1000 nl/ml) = 	cells/ml, where X= mean of 3 aliquots

IV     CONDUCTING THE TESTS

    A. Calculate the volume of culture to add to each flask (reference toxicant or effluent dilution) using
       the following formula:

       CiVi=C2V2

       For example, if your culture has 2000 cells/ml, your desired cell concentration is 100 cells/ml and
       the total desired volume of each flask is 50ml;

       (2000cells/ml) (X) = (100cells/ml)(50 ml)
       X= 2.5 ml of culture be added to 47.5 ml of solution.

    B. Prepare reference toxicant dilutions using the table described in  "protocol for reference toxicant
       dilutions1". Prepare effluent dilutions as well. Allow solutions to calibrate for a least one half-hour.

    C. Add calculated volume  of cell culture to test solutions, remembering to agitate culture
       adequately so cells are evenly distributed.  Pipet from the same place in the flask each
       time.

    D. Pipette five replicates of 3 ml test solution for each concentration into cuvettes, swirling test
       solution every three replicates.

    E. Store cuvettes in 19 ฐC incubator for 24 hrs.

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PROTOCOL FOR CONDUCTING A 24 HOUR TOXICITY TEST WITH BIOLUMINESCENT
DINOFLAGELLATES (Ceratocorys horrida) Cont'd

V      DATA COLLECTION

    A.  Turn the lights off in the laboratory, gently remove test cuvettes from incubator- approximately 4
        hours after initiation of the dark phase (typically between 11 am - 2 pm, depending on how lights
        are set in incubator).

    B.  Be very gentle. Do not shake, bump or swing cuvettes around (this will cause them to illuminate
        and lose potential light productivity).

    C.  Carefully take each cuvette and place into SeaLite or SPAWAR testing unit, press appropriate start
        button and record data onto pre-printed data sheet. Be sure to keep cuvettes covered with black felt
        as they await reading of light output. If using the SeaLite unit, the "QwikLite" software it uses can
        record data automatically, but will not take into account randomization of cuvette readings,  which
        is recommended.

    D.  After measuring bioluminescence in each cuvette, empty solution into beaker and discard
        cuvette.

VI      ANALYZING DATA

    A.  QwikLite software will  compute statistics (e.g. EC50 values where appropriate).

    B.  If SPAWAR unit is being used, PMT counts can be entered into an MS Excel spreadsheet called
        "NRaD analysis^ .

    C.  After opening the Excel file, enter PMT counts into appropriate cells. Calculations  will be made
        automatically.

    D.  If an EC50 value is desired, it can be determined with CETIS or ToxCalc software. Alternatively,
        the "Toolkit" program found on the LAB 116 computer desktop can be used to determine an EC50
        by linear interpolation.
1 Modified from ASTM Standard Guide for Conducting Toxicity Tests With Bioluminescent Dinoflagellates. Designation E 1924 - 97
2 Protocol for reference toxicant dilutions can be found in the sub-directory : C:\WINDOWS\Desktop\Laboratory 116\Protocols and logs
3 Nard analysis can be found in the sub-directory: C:\WINDOWS\Desktop\Laboratory 116\Sealite and NRaD.
                                                10

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1.3 REFERENCE TOXICANT TEST WITH MARINE AMPHIPODS
TESTING FACILITY:  SPAWAR SYSTEMS CENTER
                       BIOASSAY LABORATORY (RM 116)
                       CODE 71750
                       53475 STROTHE RD.
                       BLDG. 111
                       SAN DIEGO, CA 92152
I.      OBJECTIVE: This method estimates the acute (96 h) toxicity of a reference toxicant to
       amphipods using 3-5 mm individuals, in a 96 h, water only, non-renewal exposure. Reference
       toxicant tests are used to evaluate quality of the test organisms1.

II      NECESSARY MATERIALS AND SUPPLIES

           Plastic holding tanks - 3 to 6L
           Reference toxicant solution - Ammonia stock solution
           graduated cylinders - Class A, borosilicate glass or non-toxic plastic labware, 5 0-1000ml for
           making test solutions
           pH meter - for measuring test solutions
           Dissolved oxygen meter - for measuring test solutions
           Refractometer - for determining salinity of test solutions
           Thermometer - digital  or laboratory grade
           Dissolved Ammonia meter and probe - (prepare 24 hours in advance)
           Test chambers -  1 L glass beakers or jars with lids
           Colored labeling tape
           Dilution water - natural seawater or hypersaline brine made from natural seawater and diluted
           with deionized water
           Pipets, automatic - adjustable, to cover a range of 0.01 to 5 ml and pipette tips
           Calculator
           Beakers- Class A, borosilicate glass or non-toxic plastic labware, 1 to 2 L for making test
           solutions
           Wash bottles - for reagent water, dilution water, for topping off graduated cylinders, for
           rinsing small glassware and instrument electrodes and probes
           Data sheets
           Siphon tubes - for acclimation water changes
           Pasteur pipettes - for collection of amphipods
           Light box - for examining organisms
           Glass dishes for counting and transferring amphipods
                                             11

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PROTOCOL FOR CONDUCTING A 96 H REFERENCE TOXICANT TEST WITH AMPHIPODS
(Eohaustorius estuarius OR Rhepoxynius abronius) CONT'D

III     METHODS

       A.     OBTAINING, FEEDING AND HOLDING ORGANISMS

           1.  Amphipods should be ordered within a week and at least three days prior to testing date to
              allow for acclimation to testing conditions. Approximately 20% more amphipods than
              needed for the test should be ordered.

           2.  Acclimation rates to test salinity and temperature should not exceed 3 ฐC and 3%o per 24
              hours.

           3.  Determine arriving temperature, pH, and salinity.

           4.  Transfer amphipods to a large plastic container with sediment at the bottom in a 15 ฐC
              temperature controlled room, incubator, or water bath. A squirt bottle filled with filtered
              seawater can be used to help get amphipods off plastic bags or containers and into the
              holding tanks. Remove dead by siphoning out of tank with a small rubber hose.

           5.  Each day prior to distribution of amphipods into  beakers/jars, remove any dead, record
              physical parameters (temp, pH, salinity, D.O.), perform a 50% water change with seawater
              of the appropriate salinity and 15 ฐC seawater.


       B     CONDUCTING THE  TEST

       Day 0 (Hour 0)

       1.  Mix up the appropriate salinity seawater with the appropriate amount of reference toxicant (e.g.
           cadmium, ammonia). Add 750 mL to each of at least 2 replicates per concentration.

       2.  Record water quality parameters (temperature, salinity, and D.O.) from one replicate of each
           treatment on Day 0 and Day 4 of test.

       3.  Sieve amphipods from holding tray and place in a smaller plastic or glass container with test
           seawater. Fill glass dishes with approximately 150 mL of test seawater. Select healthy and
           active individuals with a transfer pipette and distribute in batches of 10 to glass dishes. The
           number of amphipods in each dish should be verified by recounting before adding to test
           chambers. Add one dish of 10 to each replicate.

       4.  Cover chambers with an opaque material or place in  a dark room or enclosure and hold at
           15ฐC.
                                               12

-------
PROTOCOL FOR CONDUCTING A 96 H REFERENCE TOXICANT TEST WITH AMPHIPODS
(Eohaustorius estuarius OR Rhepoxynius abronius) CONT'D

         Day 1 (Hour 24)

           1.  Measure and record temperature in one test chamber from each treatment every day
              thereafter.

           2.  Note and remove any mortalities.

           3.  Lights must remain off or chambers must remain in the dark during the entire
              exposure.
       C. TEST TERMINATION

       Day 4 (Hour 96)

               1.  Count surviving amphipods and record. Amphipods will occasionally "play dead".
                  Look for movement in the pleopods (back legs).

IV   ANALYZING DATA

    Using CETIS or Toxcalc 5.0, enter data retrieved from the survival endpoint to determine the LC50 or
    other relevant toxicity metric. Please refer to "Protocol for Statistical Analysis ofToxicity Data"2.
'Modified from "U.S. EPA Methods for Assessing the Toxicity of Sediment-associated Contaminants with Estuarine and Marine Amphipods" June
1994 EPA 600/R-94/025

2 "Protocol for Statistical Analysis of Toxicity Data" can be found in the sub-directory : C:\WINDOWS\Desktop\Laboratory
116\Protocolsand log
                                               13

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1.4 SEDIMENT TOXICITY TEST WITH MARINE AMPHIPODS

TESTING FACILITY:  SPAWAR SYSTEMS CENTER
                       BIOASSAY LABORATORY (RM 116)
                       CODE 71750
                       53475 STROTHE RD.
                       BLDG. 111
                       SAN DIEGO, CA 92152
I.      OBJECTIVE: This method estimates the acute (10 day) toxicity of whole sediment to amphipods
       using 3-5 mm individuals, in 10 d non-renewal exposures. Amphipods are intimately associated
       with sediment by nature of their burrowing or tube-dwelling and feeding habits, thus making them
       suitable species for sediment toxicity testing1.

II      NECESSARY MATERIALS AND SUPPLIES

           Plastic holding tanks - 3 to 6L
           Graduated cylinders - Class A, borosilicate glass or non-toxic plastic labware, 5 0-1000ml for
           making test solutions
           pH meter - for measuring test solutions
           Dissolved oxygen meter and probe - for measuring test solutions
           Dissolved Ammonia meter and probe - (prepare 24 hours in advance)
           Refractometer - for determining salinity of test solutions
           Thermometer - digital or laboratory grade
           Test chambers - 1 L glass beakers or jars with lids
           Colored labeling tape
           Dilution water - natural seawater or hypersaline brine made from natural seawater and diluted
           with deionized water
           Pipets, automatic - adjustable, to cover a range of 0.01 to 5 ml and pipette tips
           Calculator
           Beakers- Class A, borosilicate glass or non-toxic plastic labware, 1 to 2 L for making test
           solutions
           Wash bottles - for reagent water, dilution water, for topping off graduated cylinders, for
           rinsing small glassware  and instrument electrodes and probes
           Data sheets
           Siphon tubes - for acclimation water changes
           Pasteur pipettes - for collection of amphipods
           Light box - for examining organisms
           Glass dishes for counting and transferring amphipods
           Turbulence reducer - to prevent disturbance of sediment when adding overlying water
           Air grid and filter
           Plastic tubing
           1 mm sieves
           Plastic buckets - for sieving sediment
           Spatulas - nylon, fluorocarbon or polyethylene
                                              14

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PROTOCOL FOR CONDUCTING A 10 D SEDIMENT SURVIVAL TEST WITH AMPHIPODS Cont'd

III     METHODS

       A.     OBTAINING, FEEDING AND HOLDING ORGANISMS

              1.  Amphipods should be ordered within a week and at least three days prior to testing
                  date to allow for acclimation to testing conditions. Approximately 20% more
                  amphipods than needed for the test should be ordered.

              2.  Acclimation rates to test salinity and temperature should not exceed 3 ฐC and 5%o per
                  24 hours.

              3.  Determine arriving temperature, pH, and salinity.

              4.  Transfer amphipods to a large plastic container with sediment at the bottom in a 15 ฐC
                  temperature controlled room, incubator, or water bath.  A squirt bottle filled with
                  filtered seawater can be used to help get amphipods off plastic bags or containers and
                  into the holding tanks.  Remove dead by siphoning out of tank with a small rubber
                  hose.

              5.  Each day prior to distribution of amphipods into beakers/jars, remove any dead, record
                  physical parameters (temp, pH, salinity, D.O.), perform a 50% water change with
                  seawater of the appropriate salinity and 15 ฐC seawater.

    B.  CONDUCTING THE TEST

              1.  TEST PREPARATION
                  Day-1

                  a.   Sediments should be stored at 4ฐC and be tested within two weeks after collection1.
                      Press-sieving (1mm) all sediments (including control and reference) should be
                      performed if there  is concern about the presence of predatory organisms, large
                      debris, or organisms taxonomically similar to the test species.  Ensure that nearly
                      all sediment is pressed through sieve to prevent composition change in sediment.
                      Wash sieves between samples with acetone sparingly, then rinse well with
                      deionized water. Also  rinse spatulas, spoons and other utensils between samples.

                  b.   Take note of sediment homogeneity and grain size.

                  c.   Add 2 cm of homogenized sediment to each beaker/jar. Settle the sediment by
                      either tapping the side of the test chamber against the hand or smoothing with a
                      nylon, fluorocarbon or polyethylene spatula.
                  d.   Add 750 mL of 20%o seawaterto each replicate. To minimize disruption of
                      sediment as seawater is added, use a turbulence reducer. Position the turbulence
                      reducer just above  the  sediment surface and raised slowly as seawater is added.

                  e.   Cover all replicates and ensure gentle (approx. 100 bubbles/minute) aeration.
                                               15

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PROTOCOL FOR CONDUCTING A 10 D SEDIMENT SURVIVAL TEST WITH AMPHIPODS Cont'd

              2.  ADDITION OF AMPHIPODS
                  DayO

                  f.   Measure and record physical parameters (temp., salinity, DO, pH, ammonia) for
                      overlying water in one replicate. Pour off overlying water of the same replicate
                      and remove sediment for centrifugation to make Day 0 pore water measurements if
                      required.

                  g.   Sieve amphipods from sediment in holding tray and place in a smaller plastic or
                      glass container with test seawater. Fill glass dishes with approximately 150 mL of
                      test seawater. Select healthy and active individuals with a transfer pipette and
                      distribute in batches of 10 to glass dishes. The number of amphipods in each dish
                      should be verified by recounting before adding to test chambers. Add two dishes of
                      10 to each replicate (20 animals total). Be sure to select dishes randomly.

                  h.   After addition of animals, examine beakers/jars for animals that have been injured
                      or stressed. These individuals will not burrow into sediment and should be
                      removed and replaced. Eohaustorius estuarius generally burrows in 5 - 10
                      minutes. Record the number of amphipods that are replaced.

              3.  TEST MAINTENANCE
                  Days 1-10

                  a.   On Day 1, salinity, pH, D.O., and temperature from overlying water should be
                      measured from a replicate of each treatment every day thereafter.
                  b.   Note and remove any mortalities.
                  c.   Check aeration in each chamber.
                  d.   Lights must remain on during the entire exposure.

       C. TEST TERMINATION

              1.  On Day 10, measure water quality (salinity, pH, D.O. and temperature) and ammonia
                  from overlying and pore water (if needed) from one replicate of each treatment.

              2.  Pour approximately half of the overlying water over a 1  mm sieve. Use remaining
                  water to loosen sediment by swirling gently. Place the 1 mm sieve over a plastic bucket
                  and pour sediment onto sieve. Using a spray bottle with seawater of the appropriate
                  salinity, wash sediment through sieve.

              3.  Transfer amphipods into a counting dish.  Be very careful not to leave any amphipods
                  on the sieve during this process.

              4.  Count surviving amphipods and record. Amphipods will occasionally "play dead".
                  Look for movement in the pleopods (back legs).
                                              16

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PROTOCOL FOR CONDUCTING A 10 D SEDIMENT SURVIVAL TEST WITH AMPHIPODS Cont'd

VI      ANALYZING DATA

                        A.  Two sample comparisons can be done using a t-test to detect a significant
                            departure from the control and each treatment. * Please refer to "Protocol for
                            Statistical Analysis ofToxicity Data"2.
'Modified from "U.S. EPA Methods for Assessing the Toxicity of Sediment-associated Contaminants with Estuarine and Marine Amphipods" June
1994 EPA 600/R-94/025

 Protocol for Statistical Analysis ofToxicity Data can be found in the sub-directory : C:\WINDOWS\Desktop\Laboratory
116\Protocols and logs



                                                   17

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1.5 EMBRYO-LARVAL DEVELOPMENT TEST WITH SAND DOLLARS
TESTING FACILITY:  SPAWAR SYSTEMS CENTER
                       BIOASSAY LABORATORY 116
                       CODE 71750
                       53475 STROTHE RD.
                       BLDG. 111
                       SAN DIEGO, CA 92152
I.      OBJECTIVE: This method estimates the chronic toxicity of effluent and receiving waters to the
       embryos and larvae of sand dollars (Dendraster excentricus). The test endpoint is normal larval
       development and may include mortality.

II      NECESSARY MATERIALS AND SUPPLIES

           Refractometer - for determining salinity
           Thermometers - glass or electric, laboratory grade for measuring water temperatures
           DO and pH meters  - for routine physical and chemical measurements
           Balance - Analytical, capable of accurately weighing to O.OOOlg.
           Graduated cylinders - Class A, borosilicate glass or non-toxic plastic labware, 5 0-1000ml for
           making test solutions.
           Volumetric flasks - Class A, borosilicate glass or non-toxic plastic labware, 100-lOOOml for
           making test solutions.
           Plastic holding tanks - 3 to 6L
           Test chambers - 20ml glass scintillation vials and caps - pre-soaked in dilution water.
           Colored labeling tape
           Dilution water - natural seawater or hypersaline brine made from natural seawater and diluted
           with deionized water
           Biological microscope and slides
           Pipets, automatic - adjustable, to cover a range of 0.01 to 5 ml and pipette tips
           Calculator
           Wash bottles - for reagent water, dilution water, for topping off graduated cylinders, for
           rinsing small glassware and instrument electrodes and probes
           Inverted microscope - for inspecting gametes and counting embryos and larvae.
           Counter, two unit, 0-999 - for recording counts of embryos and larvae
           Beakers, 5-10ml borosilicate glass for collecting sperm from sand dollars
           Beakers, 1,000 ml for rinsing and settling sea urchin eggs.
           Vortex mixer - to mix sea urchin semen in tubes prior to sampling.
           Hemocytometer - for counting sperm
           Siphon hose -  for removing water from settled eggs
                                              18

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PROTOCOL FOR 72 HOUR ECHINODERM LARVAL DEVELOPMENT TEST WITH SAND DOLLARS
(Dendraster excentricus) Cont'd

III.    METHODS

    A. OBTAINING AND HOLDING ORGANISMS

       1.  Obtain ripe sand dollars from an uncontaminated subtidal area (i.e. mouth of Mission Bay) on
           morning of test setup and hold in tanks (e.g. cold room) with raw flowing seawater (target
           holding/conditioning temperature is 10 - 14 ฐC) until they are needed for testing. Holding and
           conditioning tanks should be drained and sprayed with fresh water at least once weekly to
           prevent accumulation of organic matter and bacteria. Dead animals should be removed daily.

    B. SPAWNING AND FERTILIZATION

       1.  Pour 20-30 ml seawater into 100 ml beakers for females and 25 ml in 25-50 ml beakers for
           males and place in 15 ฐC  incubator.

       2.  Carefully place sand dollars in a container lined with moist paper towels.

       3.  Inject 0.5 ml of 0.5 M KC1 into oral cavity of each sand dollar, cleaning needle with hot water
           between injections if sex of sand dollar is not known to prevent cross contamination. Record
           injection time on data sheet.

       4.  Swirl sand dollar for a few seconds then place back on moist paper towels.

       5.  When gametes begin to shed, note time, and separate sexes. Place males onto 25-50  ml
           beakers and females onto 100 ml beakers both oral side up. Spray eggs and sperm of sand
           dollar into beaker with  a wash-bottle. It is optimal to obtain gametes from at least 3 spawning
           individuals of each sex.

       6.  After confirming good  motility of each sperm sample under the microscope, combine equal
           quantities from up to four males, and store in refrigerator or on ice until use within 4  h.

       7.  Observe egg quality under the microscope for each spawning female. Pool quality eggs (i.e.
           normal size, regular shaped and absence of germinal vesicle) into a 100 ml or 250 ml graduated
           cylinder, bring volume  up and cover with parafilm and keep at 15 ฐC.

       8.  Confirm fertilization success by placing a drop of eggs onto a well slide with a small amount of
           sperm. Check for fertilization membrane. If no fertilization membrane present isolate new
           eggs.

       9.  To determine the egg density the egg stock will need to be diluted. Always cut pipette
           tip so that it is at least 2 mm wide. First, label two scint vials A and B, then fill  each
           with 9 ml of filtered seawater. Next, add 1ml of the concentrated  egg stock to vial A  invert
           gently several times, then add 1ml of vial A to vial B. Count 1 ml from vial B in a
           Sedgewick-Rafter counting cell. If the count is less than 30, count Vial A. Vial A
           represents a 1:10 dilution and vial B represents a 1:100 dilution.
                                               19

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PROTOCOL FOR 72 HOUR ECHINODERM LARVAL DEVELOPMENT TEST WITH SAND DOLLARS
(Dendraster excentricus) Cont'd

        10. Using the volume of concentrated egg stock determined by the equation on the Egg/Sperm
           count page, prepare egg stock in dilution water at the final target concentration of 1000
           eggs/ml. Check prepared solution by counting eggs again.

        11. Recommended sperm to egg ratio for fertilization is 500:1.tests.

    C.   SPERM DILUTION

        Note: If able to decant overlying water the final sand dollar sperm density is usually
        between 2x10A9 and 2xlOA10 sperm/ml.

        See the Protocol for Counting Sperm with a Hemocytometer for instructions on how to count
        sperm. With experience, the amount of sperm required for successful fertilization can be
        estimated, avoiding the need for precise cell counts.

    D.   FERTILIZATION

        Add calculated volume of sperm dilution to the egg dilution for a 500 sperm: legg ratio and mix
        gently. Wait 10 minutes and check for fertilization. If fertilization is not at least 90%, add a second
        volume of sperm dilution, wait 10 minutes and re-check. If fertilization is still not 90%, test must
        be restarted with different gametes. Once again, with experience, the amount of sperm to add can
        be estimated eliminating the need for precise counts.
IV     CONDUCTING THE TEST

    A. REFERENCE TOXICANT TESTS

       1.  Prepare reference toxicant stock and dilutions. Make up a 1 ppm stock using 200 \i\ of 1 ppt
           copper solution in 199.8 mL dilution water. Make dilutions according to species sensitivity and
           add 10 mL of each concentration to scintillation vials. In general, five replicates per treatment
           are used, plus one additional vial for water chemistry.  Cover and place in 15 ฐC to equilibrate
           for at least 30 minutes.
       2.  New, seawater leached scintillation vials containing 10 mL of test solution are pre-cooled to 15
           ฐC. To each vial, inject 0.25 mL fertilized eggs. It is important to be sure that the eggs are
           homogenized during additions.  This is accomplished by frequently mixing the contents of the
           flask with a combination of gentle swirling and back and forth motions, or using a perforated
           plunger.

       3.  The embryos should be incubated for 72 hours in the test chambers at 15 ฐC at ambient light
           level (16 h light and 8 h dark). If controls have not achieved the pluteus stage after 72 h, the
           exposure can be extended up to 96 hours.

       4.  Terminate test by addition of ImL of concentrated Formaldehyde and record the time.
                                               20

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PROTOCOL FOR 72 HOUR ECHINODERM LARVAL DEVELOPMENT TEST WITH SAND DOLLARS
        (Dendraster excentricus) Cont'd

    B.  EFFLUENTS, RECEIVING WATERS, AND OTHER SAMPLES

        1.  Test should begin within 36 hours of sample collection (USEPA 2002).

        2.  Prepare sample dilutions. Add 10 mL of each concentration to scintillation vials. In general,
           five replicates per treatment are used, plus one additional vial for water chemistry.  Cover and
           place in 15 ฐC chamber to equilibrate.

        3.  Once scintillation vials have reached 15 ฐC, add embryos using a cut pipette tip. Be sure that
           embryo stock is always homogenized. This is accomplished by frequently mixing the contents
           of the flask with a combination of gentle swirling and back and forth motions, or using a
           perforated plunger.

        4.  The embryos should be incubated for 72 hours in the test chambers at 15 ฐC and at ambient
           laboratory light levels (16 h light and 8 h dark). If controls have not achieved the pluteus stage,
           the exposure can  be extended up to 96 hours.

        5.  Terminate test by addition of 1ml of concentrated Formaldehyde. Record the time.

        6.  Additional notes:

               •   Additional  vials with site samples may need to be collected for water quality and
                   chemistry.
               •   Be sure to adequately homogenize sample before addition to vials and before taking
                   water quality measurements.
               •   If samples are salted up with hypersaline brine, be sure to incorporate a brine control.


V.      DATA COLLECTION

    A.  Observe embryos within one week of preservation. For each test replicate, the proportion of normal
        to abnormal larvae will be determined. Please refer to "Protocol for counting larvae with an
        inverted microscope1".

VI      ANALYZING DATA

    A.  Using CETIS or Toxcalc, enter data retrieved from counting to determine the EC50, LOEC,
        NOEC, or other appropriate toxicity metric. Please refer to "Protocol for Statistical Analysis of
        Toxicity Data3".

    B.  In accordance with USEPA (2002), all Toxcalc-generated concentration-response curves will be
        evaluated for acceptability.

'Modified from "Short-Term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Marine and Estuarine Organisms".
First edition. EPA/600/R-95/136. August 1995.
 "Protocol for Counting larvae with an inverted microscope" can be found in the sub-directory : C:\WINDOWS\Desktop\Laboratory 116\Protocols
and logs
3 "Protocol for Statistical Analysis of Toxicity Data" can be found in the sub-directory : C:\WINDOWS\Desktop\Laboratory 116\Protocols and logs


                                                 21

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1.6 EMBRYO-LARVAL DEVELOPMENT TEST WITH PURPLE SEA URCHINS
TESTING FACILITY:  SPAWAR SYSTEMS CENTER
                       BIOASSAY LABORATORY (RM 116)
                       CODE 71750
                       53475 STROTHE RD.
                       BLDG. 111
                       SAN DIEGO, CA 92152
I.      OBJECTIVE: This method estimates the chronic toxicity of effluent and receiving waters, pore
       water, and other seawater samples to the embryos and larvae of echinoderms (the sea urchin
       Stronglyocentrotus purpuratus) relative to control or reference samples. The test endpoint is
       normal larval development and may include mortality.

II      NECESSARY MATERIALS AND SUPPLIES

           Refractometer - for determining salinity
           Thermometers - glass or electric, laboratory grade for measuring water temperatures
           D.O. and pH meters - for routine physical and chemical measurements
           Balance - Analytical, capable of accurately weighing to O.OOOlg.
           Graduated cylinders - Class A, borosilicate glass or non-toxic plastic labware, 5 0-1000ml for
           making test solutions.
           Volumetric flasks - Class A, borosilicate glass or non-toxic plastic labware, 100-1000ml for
           making test solutions.
           Plastic holding tanks - 3 to 6L
           Test chambers - 20ml glass scintillation vials and caps - pre-soaked in dilution water.
           Colored labeling tape
           Dilution water - natural seawater or hypersaline brine made from natural seawater and diluted
           with deionized water
           Biological microscope and slides
           Pipets, automatic - adjustable, to cover a range of 0.01 to 5 ml and pipette tips
           Calculator
           Wash bottles - for reagent water, dilution water, for topping off graduated cylinders, for
           rinsing small glassware and instrument electrodes and probes
           Inverted microscope - for inspecting gametes and counting embryos and larvae.
           Counter, two unit, 0-999 - for recording counts of embryos and larvae
           Beakers, 5-10ml borosilicate glass for collecting sperm from sand dollars
           Beakers, 1,000 ml for rinsing and settling sea urchin eggs.
           Vortex mixer - to mix sea urchin semen in tubes prior to sampling.
           Hemocytometer - for counting sperm
           Siphon hose - for removing water from  settled eggs
           Sieves - 80 jam, 20 jam and 25 jam
                                             22

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PROTOCOL FOR 72 HOUR ECHINODERM LARVAL DEVELOPMENT TEST WITH SEA URCHINS
(Strongylocentrotus purpuratus) Cont'd

III     METHODS

    A. OBTAINING AND HOLDING ORGANISMS

       1. Obtain ripe sea urchins from an uncontaminated subtidal area (e.g. mouth of Mission Bay) on
          morning of test setup and hold in tanks (e.g. cold room) with raw flowing seawater (target
          holding/conditioning temperature is 12 - 14 ฐC) until they are needed for testing. Kelp should
          be added to tanks as a food supply. Holding and conditioning tanks should be drained and
          sprayed with fresh water at least once weekly to prevent accumulation of organic matter and
          bacteria.  Dead animals should be removed daily.

    B. SPAWNING

       1. Pour 0.45 um filtered seawater into 100 mL beakers for females and place in 15 ฐC incubator.
          Smaller (e.g. 25-50 mL) beakers can be used for males.

       2. Carefully remove urchins from holding tanks to prevent damage to tube-feet, and  place in a
          container lined with moist paper towels to prevent reattachment.

       3. Inject 0.5 mL of 0.5 M KC1 into soft periostomal membrane of each urchin, rinsing the needle
          with hot water between injections if sex of urchins is not known to prevent cross contamination.
          Record injection time on data sheet.

       4. Swirl urchin for a few seconds, then place onto the beakers, oral side down.

       5. When gametes begin to shed, note time, and separate sexes. Let females shed eggs into
          seawater-filled beakers oral side down. It is optimal to obtain at least 3 spawning individuals
          from each sex.

       6. Collect sperm from each male in 25-50 mL beakers, with minimal dilution. After confirming
          good motility of each sperm  sample under the microscope,  combine equal quantities from three
          to four males and use within 4 h.

       7. Observe egg quality under the microscope for each spawning female. Pool quality eggs (i.e.
          normal size, regular shaped and absence of germinal vesicles) into a 1 L beaker. Pass eggs
          through an 80 jam mesh screen (the eggs will pass through and debris is retained on screen).

       8. Pass sperm stock through a 25 jam mesh screen (sperm will pass through and debris are retained
          on screen).

       9. Confirm fertilization success by placing a drop of eggs onto a well slide with a small amount of
          sperm. Check for fertilization membrane. If no fertilization membrane is present, isolate new
          eggs.

       10. To determine the egg density, the egg stock will need to be diluted. Always cut pipette tip so
          that it is  2 mm wide to prevent damage to eggs. First, label two scint vials A and B, then fill
          each with 9 mL of filtered seawater. Next, add 1 mL of the egg stock to vial A invert
                                               23

-------
PROTOCOL FOR 72 HOUR ECHINODERM LARVAL DEVELOPMENT TEST WITH SEA URCHINS
(Strongylocentrotus purpuratus) Cont'd

           gently several times, then add 1ml of vial A to vial B. Count 1 mL from vial B in a Sedgewick-
       Rafter counting cell. If the count is less than 30, count Vial A. Vial A represents a 1:10 dilution and
       vial B represents a 1:100 dilution.

       11. Using the volume of concentrated egg stock determined by the equation on the
           Egg/Sperm count page, dilute to 20-50 eggs / mL for fertilization.

    C.  FERTILIZATION

       1.  Add sperm to the diluted egg stock at 15  ฐC. Sperm should be added at a density of
           approximately 105 to 107 sperm/mL in the final mixture.  Sperm density can be confirmed with
           a hemacytometer (see Protocol for Counting Sperm with a Hemocytometer).  With experience,
           precise sperm counts are not necessary (sperm should make diluted egg stock very slightly
           cloudy). Wait 10-15 minutes and check for complete fertilization. If fertilization is not at least
           90%, add a second volume of sperm stock, wait 10 minutes and re-check. If fertilization is still
           not 90%, test must be restarted with different gametes.

       2.  After adequate fertilization has been achieved, gently pour embryo stock over a 20 jam mesh
           screen to remove any excess sperm and debris  (embryos will be retained on screen while sperm
           and debris will pass through). Gently rinse embryos on screen with filtered seawater.

       3.  Re-concentrate embryo  stock solution to  desired density (e.g. 2000 embryos / mL).
IV     CONDUCTING THE TEST

       7.  REFERENCE TOXICANT TESTS

           a.   Prepare reference toxicant stock and dilutions. Make up a 1 ppm sub-stock using 200 joL of
               1 ppt Copper stock in 199.8 mL dilution water. Make dilutions according to species
               sensitivity and add 10 mL of each concentration to scintillation vials. In general, five
               replicates per treatment are used, plus one additional vial for water chemistry.  Cover and
               place in  15 ฐC to equilibrate for at least 30 minutes.

           b.   Scintillation vials containing 10 mL of each test concentration should have been pre-cooled
               to 15 ฐC. To each vial, add 100 \\L embryos being sure that the embryo stock is always
               homogenized. This is accomplished by frequently mixing the contents of the flask with a
               combination of gentle swirling and back and forth motions, or using a perforated plunger.

           c.   The embryos should be incubated for at least 72 hours in the test chambers at 15 ฐC at
               ambient  laboratory light levels (16 h light and 8 h dark). If controls have not achieved the
               pluteus stage by 72 hours, the exposure can be extended up to 96 hours.

           d.   Terminate test by addition of 1 mL of concentrated Formaldehyde. Record the time.
                                               24

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PROTOCOL FOR 72 HOUR ECHINODERM LARVAL DEVELOPMENT TEST WITH SEA URCHINS
(Strongylocentrotus purpuratus) Cont'd

        8.  EFFLUENTS, RECEIVING WATERS, AND OTHER SAMPLES

           e.   Test should begin within 36 hours of sample collection (USEPA 2002).

           f.   Prepare sample dilutions. Add 10 mL of each concentration to scintillation vials. In
               general, five replicates per treatment are used, plus one additional vial for water chemistry.
               Cover and place in 15 ฐC to equilibrate.

           g.   Once  scintillation vials have reached 15 ฐC, add embryos using a cut pipette tip. Be sure
               that embryo stock is always homogenized during the additions. This is accomplished by
               frequently mixing the contents of the flask with a combination of gentle swirling and back
               and forth motions, or using a perforated plunger.

           h.   The embryos should be incubated for 72 hours in the test chambers at 15 ฐC and at ambient
               laboratory light levels (16 h light and 8 h dark). If controls have not achieved the pluteus
               stage, the exposure can be extended up to 96 hours.

           i.   Terminate test by addition of 1ml of concentrated Formaldehyde. Record the time.

               •   Additional vials with site samples  may need to be collected for water quality and
                   chemistry.
               •   Be sure to adequately homogenize sample before addition to vials and before taking
                   water quality measurements.
               •   If samples are salted up with brine addition, be sure to incorporate a brine control.

V.      DATA COLLECTION

    A.   Observe embryos within one week of preservation. For each test replicate, the proportion of normal
        to abnormal larvae will be determined. Please refer to  "Protocol for counting larvae with an
        inverted microscope ".

VI      ANALYZING DATA

    A.   Using  CETIS  or Toxcalc, enter data retrieved from counting to determine the EC50, LOEC,
        NOEC, or other appropriate toxicity metric. Please refer to "Protocol for Statistical Analysis of
        Toxicity Data3".

    B.   In accordance with USEPA (2002), all Toxcalc-generated concentration-response curves will be
        evaluated for acceptability.

1 Modified from "Short-Term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Marine and Estuarine Organisms".
First edition. EPA/600/R-95/136. August 1995.
 "Protocol for Counting larvae with an inverted microscope" can be found in the sub-directory : C:\WINDOWS\Desktop\Laboratory 116\Protocols
and logs
3 "Protocol for Statistical Analysis of Toxicity Data" can be found in the sub-directory : C:\WINDOWS\Desktop\Laboratory 116\Protocols and logs
                                                25

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1.7 ACUTE TOXICITY TEST WITH JUVENILE MYSID SHRIMP
Testing Facility:        SPAWAR SYSTEMS CENTER
                       BIOASSAY LABORATORY (RM 116)
                       CODE 71750
                       53475 STROTHE RD.
                       BLDG. 111
                       SAN DIEGO, CA 92152
I.      OBJECTIVE: This method estimates the acute (96 h) toxicity of effluents and receiving waters to
       the mysid using three to five day old juveniles, in a 96 h static-renewal exposure1. Mysids are
       exposed to effluent samples via dilution series experiments (typically 5 concentrations plus a
       control). Receiving water tests are conducted using undiluted receiving water alongside a negative
       control.

II      NECESSARY MATERIALS AND SUPPLIES

          Plastic holding tanks - 3 to 6L
          Reference toxicant solution -  Ippt copper stock solution
          graduated cylinders - Class A, borosilicate glass or non-toxic plastic labware, 5 0-1000ml for
          making test solutions
          pH meter - for measuring test solutions
          Dissolved oxygen meter - for measuring test solutions
          Refractometer - for determining salinity of test solutions
          Thermometer - digital or laboratory grade
          Test chambers - 300ml glass  beakers
          Watch glasses - for covering  test chambers
          Colored labeling tape
          Dilution water - natural seawater or hypersaline brine made from natural seawater and diluted
          with deionized water
          Pipets, automatic - adjustable, to cover a range of 0.01 to 5 ml and pipette tips
          Calculator
          Beakers- Class A, borosilicate glass or non-toxic plastic labware, 1 to 2 L for making test
          solutions
          Wash bottles - for reagent water, dilution water, for topping off graduated cylinders, for
          rinsing small glassware and instrument electrodes and probes
          Data sheets
          Brine Shrimp, Artemia, culture unit
          Separatory funnels, 2L - two  for culturing Anemia
          Siphon tubes (Tygon tubing) - for test solution renewal
          Light box - for examining organisms
          Parafilm
                                              26

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PROTOCOL FOR CONDUCTING A 96 HOUR SURVIVAL TEST WITH THE MYSID (Americamysis bahia)
Cont'd

III.    METHODS

       A.     OBTAINING, HOLDING AND FEEDING ORGANISMS

               1.   Mysids should be 1-3 days old at time of shipping, so that they will be approximately
                   3-5 days old at start of test.

               2.   Prepare Artemla culture on day of mysid order so that there are freshly hatched nauplii
                   available when the mysids arrive. Hatching takes 24-36 h at 20 ฐC. Please refer to
                   "Protocol for preparing Artemia nauplif'2.

               3.   Upon receipt of mysids, open plastic bag and determine arrival temperature, pH, D.O.
                   and salinity. Record values on the "Organism Arrival Log" sheet.

               4.   Provide gentle aeration by placing an airstone in the bag.

               5.   Transfer mysids  to a large plastic holding tank (3-6 L) in a 20 ฐC temperature
                   controlled room, incubator, or water bath.  The easiest way to transfer is to place open
                   plastic bag in holding tank and cut open bottom of bag with a razor blade.  Gently pull
                   up on bag,  releasing mysids into the container. A squirt bottle filled with filtered
                   seawater can be used to help get mysids off plastic and into the tank. Remove dead by
                   siphoning out of tank with Tygon tubing.

               6.   Perform approximately 50% water change with filtered  (0.45 jam) seawater adjusted to
                   20 ฐC. Be  sure seawater is within 2 %o and 2 ฐC of the arriving conditions. If the
                   salinity is below the desired level (usually 34 %o), adjust by no more than 2 %o per day.

               7.   Collect newly  hatched Artemla nauplii.  Pipette nauplii so that each mysid receives
                   about 100 nauplii per day.

               8.   Each day prior to distribution of mysids into beakers, remove any dead, record physical
                   parameters (temp, pH, salinity, DO), perform a 50% water change with filtered 20 ฐC
                   seawater of the appropriate salinity, and feed.
       B.      TEST SETUP

               1.  Test should begin within 36 hours of sample collection (USEPA 2002).

               2.  Randomly distribute 10 larvae to each 300 ml glass beaker, using a 5 ml plastic pipette
                  with the lowest 0.5 cm cut off to prevent injury. Be sure beaker has a few ml of
                  filtered seawater to cushion entry. It is generally easiest to track and count mysids
                  with the holding tank and beakers on a light table.
                                               27

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PROTOCOL FOR CONDUCTING A 96 HOUR SURVIVAL TEST WITH THE MYSID (Americamysis bahia)
Cont'd

              3.  After all required beakers have been filled with test solutions, mark them either with
                 numbers from a randomization chart or with the test concentration and replicate (e.g.
                 A, B, or C). If using random numbers, ensure that identification of each number is
                 written down and stored in a safe place for referral after mortality assessment. Test
                 results will be meaningless if you don't know what the exposure was!

              4.  Feed all replicates with Anemia.

       C. TEST MAINTENANCE

              Day 0 (Hour 0)

              1.  Measure and record physical parameters (temp., salinity, DO, pH, ammonia) for all
                 samples and concentrations.

              2.  Make up dilutions. Dilutions for samples lower in salinity than  desired are
                 generally "salted up",  or adjusted to the testing salinity with synthetic sea salt
                 (Crystal Sea MarineMix, Bioassay Grade). The copper reference dilutions are
                 made from clean, filtered 0.45-|j,m seawater (i.e. Scripps)  and a 5 ppm copper
                 solution prepared in filtered seawater on the day of test  setup (from Ippt master
                 stock solution).

              3.  Siphon off as much water from beakers as possible without stressing the
                 mysids.  Replenish with 200 ml of the appropriate dilution just  prepared.
                 Siphon and replenish one beaker before moving on to next one  to reduce stress
                 on mysids.

              4.  Measure and record physical parameters from one replicate from each test
                 solutions or test concentration. If D.O. is below 4.0 mg/L in any concentration
                 for a test, aerate all beakers for that test.  Provide a gentle bubble rate (no more
                 than 100 bubbles/minute).

              5.  Be sure test organisms have been fed.

       DAY 1 (Hour 24)

              1.  Measure and record physical parameters.
              2.  Note and remove any mortalities.
              3.  Feed mysids with Artemia nauplii.

       DAY2 (Hour 48)

              1.  Measure and record physical parameters.
              2.  Note and remove any mortalities.
              3.  Prepare fresh dilutions as on Day 0 using same effluent sample.
                                             28

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PROTOCOL FOR CONDUCTING A 96 HOUR SURVIVAL TEST WITH THE MYSID (Americamysis bahia)
Cont'd

                4.  Siphon off all but approx. 10% of sample, and replenish beakers with appropriate
                   dilution that was just prepared.
                5.  Feed mysids with Artemia nauplii.

        DAY3 (Hour 72)

                1.  Measure and record physical parameters.
                2.  Note and remove any mortalities.
                3.  Feed mysids with Artemia nauplii.

        DAY4 (Hour 96)

                1.  Measure and record physical parameters.
                2.  Make final mortality observations and record.
                3.  Terminate tests by pouring contents of beakers through a sieve into sink. Surviving
                   mysids should be  sacrificed by freezing or other humane methods.

IV.     ANALYZING DATA

Using CETIS or Toxcalc, enter mortality data obtained from the test at 48- and/or 96-hour exposure periods
        to determine the LC50, LOEC, NOEC, or other relevant toxicity metrics. Please refer to "Protocol
       for Statistical Analysis of Toxicity Data"3.

In accordance with USEPA (2002), all Toxcalc-generated concentration-response curves will be evaluated
        for acceptability.
1 Modified from "Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms". Fifth
Edition. EPA/821/R/02/012. October 2002.

2 "Protocol for preparing Artemia nauplii" can be found in the sub-directory : C:\WINDOWS\Desktop\Laboratory 116\Protocols and logs

3 "Protocol for Statistical Analysis of Toxicity Data" can be found in the sub-directory : C:\WINDOWS\Desktop\Laboratory 116\Protocols and logs
                                                  29

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1.8 ACUTE TOXICITY TEST WITH TOPSMELT LARVAE
TESTING FACILITY:  SPAWAR SYSTEMS CENTER
                       BIOASSAY LABORATORY (RM 116)
                       CODE 71750
                       53475 STROTHE RD.
                       BLDG. 111
                       SAN DIEGO, CA 92152
I.      OBJECTIVE: This method estimates the acute (96 h) toxicity of effluents and receiving waters to
       topsmelt (Atherinops affinis) larvae static- renewal exposure. Topsmelt are exposed to effluent
       samples via dilution series experiments (typically 5 concentrations plus a control). Receiving water
       tests are typically conducted using full strength (100%) sample, and are compared with control
       performance.

II      NECESSARY MATERIALS AND SUPPLIES

           Plastic holding tanks - 3 to 6L
           Reference toxicant solution -  Ippt copper stock solution
           Graduated cylinders - Class A, borosilicate glass or non-toxic plastic labware, 5 0-1000ml for
           making test solutions
           pH meter - for measuring test solutions
           Dissolved oxygen meter - for measuring test solutions
           refractometer - for determining salinity of test solutions
           Thermometer - digital or laboratory grade
           Test chambers - 400ml glass  beakers
           Watch glasses - for covering  test chambers
           Colored labeling tape
           Dilution water - natural seawater or hypersaline brine made from natural seawater and diluted
           with deionized water
           Pipets, automatic - adjustable, to cover a range of 0.01 to 5 ml and pipette tips
           Calculator
           Beakers- Class A, borosilicate glass or non-toxic plastic labware, 1 to 2 L for making test
           solutions
           Wash bottles - for reagent water, dilution water, for topping off graduated cylinders, for
           rinsing small glassware  and instrument electrodes and probes
           Data sheets
           Brine Shrimp, Artemia,  culture unit
           Separatory funnels, 2L - two  for culturing Anemia
           Siphon tubes - for test solution renewal
           Light box - for examining organisms
           Parafilm
                                             30

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PROTOCOL FOR CONDUCTING A 96 HOUR SURVIVAL TEST WITH TOPSMELT LARVAE
(Atherinops affinis) Cont'd

III     METHODS

       A.      OBTAINING HOLDING AND FEEDING ORGANISMS

           1.   Fish should be approximately 7-10 days old at time of shipping, so that they will fall within
               the 9-15 day requirement for conducting the test.

           2.   Prepare Anemia culture so that there are freshly hatched nauplii available when the fish
               arrive. Hatching takes 24-36 h at 20 ฐC.  Please refer to "Protocol for preparing Artemia
               nauplii"2.

           3.   Upon receipt of fish, open plastic bag and determine arriving temperature, pH, salinity, and
               D.O.

           4.   Provide gentle aeration by inserting an airstone into the bag.

           5.   Transfer fish to one or two large plastic holding tanks (6 L) in a 20 ฐC temperature
               controlled room, incubator, or water bath. One easy way to transfer from the bag is to
               place the open plastic  bag in plastic holding tank and cut open bottom of bag with a razor
               blade.  Gently pull up  on bag, releasing fish into the container. A squirt bottle filled with
               filtered seawater can be used to retrieve any fish adhered to the bag. Remove dead by
               siphoning out of tank with a small rubber hose.

           6.   Perform a -50% water change with filtered (0.45 jam) seawater adjusted to 20 ฐC. Be sure
               seawater is within 2 %o and 2 ฐC of the arriving conditions. If the salinity is below desired,
               adjust by no more than 2 %o per day.

           7.   Collect newly hatched Anemia nauplii. Pipette nauplii so that each fish larva receives
               about 40 nauplii at each feeding.  Feed two times a day.

           8.   Each day prior to  distribution offish into beakers, remove any dead, record physical
               parameters (temp, pH, salinity, DO), perform -50% water change with filtered 20 ฐC
               seawater, and feed.

       B. TEST SETUP

           1.   Test should begin within 36 hours of sample collection (USEPA 2002).

           2.   Randomly distribute 5 larvae to each 400 ml glass beaker using a 5 ml plastic pipette with
               the lower 0.5 cm cut off to prevent injury. Be sure beaker has a few ml of filtered seawater
               to cushion entry.  Fill to 200 ml marking on beaker with test solution. It is generally
               easiest to track and count fish with holding tank and beakers on a light table (there is one in
               Rm. 116).
                                               31

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PROTOCOL FOR CONDUCTING A 96 HOUR SURVIVAL TEST WITH TOPSMELT LARVAE
(Atherinops affinis) Cont'

           3.  After all required beakers have been filled, mark them either with numbers from a
              randomization chart or with the test concentration and replicate (i.e. A, B, C, or D).
              If using random numbers, ensure that identification of each number is written down and
              stored in a safe place  for referral after mortality assessment. Test results will be
              meaningless if you don't know what the exposure was!

           4.  Distribute Artemia to all replicates.

       C. TEST MAINTENANCE

           DAY0 (Hour 0)

           1.  Measure and record physical parameters (temp., salinity, DO, pH, ammonia) for all
              samples and test concentrations.

           2.  Make up dilutions.  800 mL of each test concentration will be required to fill four
              replicates. The copper reference dilutions are made from clean, filtered 0.45-|om seawater
              (i.e. Scripps) and a 5 ppm copper stock solution prepared in filtered seawater on the day of
              test setup (from Ippt stock solution).

           3.  Siphon off as much water from beakers as possible without stressing fish. Replenish with
              200 ml of the appropriate dilution just prepared. Siphon and replenish one beaker before
              moving on to next one to reduce stress on fish.

           4.  Measure and record physical parameters for one replicate from each test concentration or
              test sample.  If D.O. is below 4.0 mg/L in any concentration for a test, aerate all beakers for
              that test. Provide a gentle bubble rate (no more than 100 bubbles/minute).

           5.  Be sure test organisms are fed two times a day.

           DAY 1 (Hour 24)

           1.  Measure and record physical parameters.
           2.  Note and remove any mortalities.
           3.  Feed two times as usual (morning and evening).

           IMF2 (Hour 48)

           1.  Measure and record physical parameters.
           2.  Note and remove any mortalities.
           3.  Prepare fresh dilutions as on Day 0 using same  effluent sample.
           4.  Siphon off all but approx. 25 ml of sample, and replenish beakers with appropriate dilution
              that was just prepared.
           5.  Feed two times as usual (morning and evening).
                                              32

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PROTOCOL FOR CONDUCTING A 96 HOUR SURVIVAL TEST WITH TOPSMELT LARVAE
(Atherinops affinis) Cont'd

            DAY3 (Hour 72)

            1.  Measure and record physical parameters.
            2.  Note and remove any mortalities.
            3.  Feed two times as usual (morning and evening).

            DAY4 (Hour 96)

            1.  Measure and record physical parameters.
            2.  Make final mortality observations and record.
            3.  Terminate tests by pouring contents of beakers through a sieve into sink.  Surviving fish
               should be sacrificed by freezing or other humane methods.

IV      ANALYZING DATA

Using CETIS or Toxcalc, enter mortality data obtained from the test at 48- and/or 96-hour exposure periods
        to determine the LC50, LOEC, NOEC, or other relevant toxicity metrics. Please refer to "Protocol
       for Statistical Analysis of Toxicity Data"3.

In accordance with USEPA (2002), all Toxcalc-generated concentration-response curves will be evaluated
        for acceptability.
1 Modified from "Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms". Fifth
Edition. EPA/821/R/02/012. October 2002.

2 "Protocol for preparing Artemia nauplii" can be found in the sub-directory : C:\WINDOWS\Desktop\Laboratory 116\Protocols and logs

3 "Protocol for Statistical Analysis of Toxicity Data" can be found in the sub-directory : C:\WINDOWS\Desktop\Laboratory 116\Protocols and log
                                                 33

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 2.0 TEST CONDITIONS AND ACCEPTABILITY CRITERIA
 2.1 BIVALVE EMBRYO-LARVAL DEVELOPMENT TEST (CHRONIC)

 Oyster (Crassostrea gigas) or Mussel (Mytilus galloprovincialis)
Test Type
Salinity
Temperature
Light quality
Light intensity
Photoperiod
Test Chamber type/size
Test solution volume
No. Larvae/test chamber
No. of replicate chambers/concentration
Dilution water
Test Concentrations

Dilution factor
Test Duration
Test acceptability criteria
Endpoint measured
static-nonrenewal
30 ฑ 2 ppt
20 ฑ 1 ฐC (oysters) and 15 or 18 ฑ 1 ฐC (mussels)
ambient laboratory illumination
10-20 uE/m2/s (Ambient laboratory levels)
16 h light/ 8 h darkness
20ml
10ml
150-300
4 or 5
Uncontaminated lum filtered natural seawater or
hypersaline brine prepared from natural seawater
Effluent: Minimum of 5 and a control; 0, 6.25,12.5, 25, 50, 100%
Copper Ref. Tox.; 0, 4.1, 8.4, 12, 17.2, 24, 35 ppb
Receiving waters: 100% receiving water and a control.
Effluents: > 0.5 Receiving waters: > 0.5
48h
> 70% survival in controls (oysters), and
> 50% survival in (Mussels); > 90% normal
development of shell with surviving controls. MSD of <25%.
Survival and normal shell development
Criteria from EPA's Short Term Methods for Estimating the Chronic Toxicity of Effluents and Receiving
Waters To West Coast Marine and Estuarine Organisms. EPA/600/R-95/136. August 1995
                                               34

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 2.2 SEDIMENT-WATER INTERFACE (SWI) TOXICITY TEST WITH BIVALVE
 EMBRYOS

 For mussel (Mytilus galloprovincialis)
Test Type
Salinity
Temperature
Light quality
Light intensity
Photoperiod
Test Chamber type/size
Test solution volume
No. Larvae/test chamber
No. of replicate chambers/concentration
Dilution water
Test Concentrations
Dilution factor
Test Duration
Test acceptability criteria
Endpoint measured
static -nonrenewal
30 ฑ 2 ppt
15 or 18 ฑ 1 ฐC
ambient laboratory illumination
10-20 uE/m2/s (Ambient laboratory levels)
16 h light/ 8 h darkness
Polycarbonate tubing with polyethylene mesh
300-500 ml
150-300
4 or 5
Uncontaminated lum filtered natural seawater or
hypersaline brine prepared from natural seawater
Copper Ref. Tox.; 0, 4.1, 5.9, 8.4, 12, 17.2, 24, 35 ppb
Receiving waters: 100% receiving water and a control.
Receiving waters: None or > 0.5
48h
> 70% normal survival
Survival and normal shell development
Criteria from EPA's Short Term Methods for Estimating the Chronic Toxicity of Effluents and Receiving
Waters To West Coast Marine and Estuarine Organisms. EPA/600/R-95/136. August 1995
                                                 35

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2.3 MARINE AMPHIPOD REFERENCE TOXICITY TEST
For Eohaustorius estuarius or Rhepoxynius abronius
Test Type
Salinity
Temperature
Light quality
Photoperiod
Test Chamber type/size
Test solution volume
No. of organisms/chamber
No. of replicate chambers/concentration
Dilution water
Test Concentrations
Aeration
Test Duration
Test acceptability criteria
Endpoint measured
Water-only test
20 ppt (E. estuarius); 30 ppt (R. abronius), ฑ 1 ppt
15 ฑ 1ฐC
Chambers should be kept in dark or covered with opaque material
24 hours dark : 0 hours light
1 L glass beaker or jar with ~10 cm ID.
750 mL (minimum)
10 (minimum) / chamber
1 minimum : 2 recommended
Uncontaminated sand filtered natural seawater or
hypersaline brine prepared from natural seawater
Ammonia: 0, 37.5, 75, 150, 300, 600 mg/L for E. estuarius
Ammonia: 0, 18.75, 37.5, 75, 15, 300 mg/L for ft abronius
Cadmium: 0, 1.5, 3, 6, 12 mg/L forE. estuarius
Cadmium: 0, 0.125, 0.25, 0.5, 1, 2 mg/L for ft abronius
Control and at least 5 test concentrations (0.5 dilution factor)
Recommended; but not necessary if >90% D.O. saturation can
be achieved without aeration
96 hours
minimum mean control survival > 90% Survival
Survival
Criteria from EPA's Methods for Assessing the Toxicity of Sediment-associated
Contaminants with Estuarine and Marine Amphipods. (EPA/600/R-94/025 June 1994)
                                                36

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2.4 SEDIMENT TOXICITY TEST WITH MARINE AMPHIPODS (ACUTE)
For Eohaustorius estuarius or Rhepoxynius abronius
Test Type
Salinity
Temperature
Light quality
Light intensity
Photoperiod
Test Chamber type/size
Test solution volume
No. of organisms/chamber
No. of replicate chambers/concentration
Dilution water
Aeration
Test Duration
Test acceptability criteria
Endpoint measured
whole sediment toxicity test - static non-renewal
20 ppt (E. estuarius) ฑ 1 ppt; 30 ppt (R. abronius) ฑ 1 ppt
15ฑ1ฐC
wide-spectrum fluorescent lights
50-1000 lux
24 hours light : 0 hours dark
1 L glass beaker or jar with ~10 cm ID.
2cm sediment : 750 mL overlying water
20 / chamber
at least 4, with at least one additional for chemistry
Uncontaminated sand filtered natural seawater or
hypersaline brine prepared from natural seawater
Recommended; but not necessary if >90% D.O. saturation can
be achieved without aeration
10 day
minimum mean control survival > 90% survival
Survival
Criteria from EPA's Methods for Assessing the Toxicity of Sediment-associated
Contaminants with Estuarine and Marine Amphipods. (EPA/600/R-94/025 June 1994)
                                                37

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2.5 BIOLUMINESCENCE INHIBITION TEST (QWIKLITE) WITH
DINOFLAGELLATES

For Ceratocorys horrida
Test Type
Salinity
Temperature
Light quality
Light intensity
Photoperiod
Test Chamber type/size
Test solution volume
Age of test organism

No. of replicate chambers/concentration
Dilution water
Test Concentrations
Dilution factor
Test Duration
Test acceptability criteria
Endpoint measured
static, non-renewal
34 ฑ 2 ppt
19 ฑ 1ฐC
ambient laboratory illumination
10-20 uE/m /s (Ambient laboratory levels)
12 h light/ 12 h darkness
4.5 ml cuvettes
3 ml / replicate
12-20 days
50-100 cells/ml
at least 4
Uncontaminated lum filtered natural seawater or
hypersaline brine prepared from natural seawater
Effluent: Minimum of 5 and a control; 0, 6.25,12.5, 25, 50, 100%
Copper Ref. Tox.; 0, 15.6, 31.3, 62.5, 125, 250 ppb
Receiving waters: 100% receiving water and a control.
Effluents: > 0.5 Receiving waters: None or > 0.5
24 hours
at least 106 PMT counts in controls
bioluminescence inhibition
Modified from ASTM Standard Guide for Conducting Toxicity Tests With Bioluminescent Dinoflagellates. Designation E 1924 - 97.
                                            38

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2.6 ECHINODERM EMBRYO-LARVAL DEVELOPMENT TEST (CHRONIC)




For Strongylocentrotuspurpuratus or Dendraster excentricus
Test Type
Salinity
Temperature
Light quality
Light intensity
Photoperiod
Test Chamber type/size
Test solution volume
No. of replicate chambers/concentration
Dilution water
Test Concentrations
Dilution factor
Test Duration
Test acceptability criteria
Endpoint measured
static non-renewal
34 ppt ฑ 2 ppt
15ฑ1ฐC
ambient laboratory illumination
10-20 uE/m /s (Ambient laboratory levels)
16 h light: 8 h darkness
20 ml Scintillation vials
10ml
at least 4
Uncontaminated lum filtered natural seawater or
hypersaline brine prepared from natural seawater
Effluent: Minimum of 5 and a control; 0, 6.25,12.5, 25, 50, 100%
Copper Ref. Tox.; 0, 4.1, 8.4, 12, 17.2, 24, 35 ppb
Receiving waters: 100% receiving water and a control.
Effluents: > 0.5 Receiving waters: 100% and a control
72-96 h
at least 80% Normal development in the controls (USEPA 1995);
MSD <25%
Normal development; mortality can be included
                                        39

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2.7 MYSID SHRIMP SURVIVAL TEST (ACUTE)
For Americamysis bahia
Test Type
Salinity
Temperature
Light quality
Light intensity
Photoperiod
Test Chamber type/size
Test solution volume
Renewal of test solutions
Age of test organism
No. Larvae/test chamber
No. of replicate chambers/concentration
Source of food
Feeding regime
Cleaning
Aeration
Dilution water
Test Concentrations
Dilution factor
Test Duration
Test acceptability criteria
Sample volume required
Endpoint measured
static-renewal
5-34 (ฑ 2 ppt)
20 ฑ 1 ฐC
ambient laboratory illumination
10-20 uE/m2/s (Ambient laboratory levels)
16 h light/ 8 h darkness
300ml
200 ml/replicate
48 hour minimum
1-5 days; 24-h range in age
10
minimum, 2 for effluent tests, minimum, 4 for receiving water tests
Newly hatched Artemia nauplii (less than 24 h old)
Feed 40 nauplii per larvae twice daily, morning and night
cleaning not required
None, unless DO concentration falls below 4.0 mg/L,
then aerate all chambers.
Uncontaminated lum filtered natural seawater or
hypersaline brine prepared from natural seawater
Effluent: Minimum of 5 and a control; 0, 6.25,12.5,25,50,100%
Copper Ref. Tox.; 0, 94, 127, 169, 225, 300, 350 ppb
Receiving waters: 100% receiving water and a control.
Effluents: > 0.5 Receiving waters: None or > 0.5
Acute: 96 h; Chronic: 7 d
90% or greater survival in controls
2L per renewal
Effluents: Survival (e.g. LC50)
Receiving waters: Survival (Significant Difference from control)
                                    40

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2.8 TOPSMELT LARVAL SURVIVAL TEST (ACUTE)
For Atherinops affmis
Test Type
Salinity
Temperature
Light quality
Light intensity
Photoperiod
Test Chamber type/size
Test solution volume
Renewal of test solutions
Age of test organism
No. Larvae/test chamber
No. of replicate chambers/concentration
Source of food
Feeding regime
Cleaning
Aeration
Dilution water
Test Concentrations
Dilution factor
Test Duration
Test acceptability criteria
Sample volume required
Endpoint measured
static-renewal
15-34 ppt (ฑ 2 ppt of the selected test salinity)
21 ฑ 1 ฐC
ambient laboratory illumination
10-20 uE/m /s (Ambient laboratory levels)
16 h light / 8 h darkness
400ml
200 ml / replicate
48 hour minimum
9-15 days post-hatch
10
minimum, 2 for effluent tests, minimum, 4 for receiving water tests
Newly hatched Artemia nauplii
Feed 40 nauplii per larvae twice daily, morning and night
cleaning not required
None, unless D.O. concentration falls below 4.0 mg/L,
then aerate chambers. Rate should be less than 100 bubbles/min.
Uncontaminated lum filtered natural seawater or
hypersaline brine prepared from natural seawater
Effluent: Minimum of 5 and a control; 0, 6.25,12.5,25,50,100%
Copper Ref. Tox.; 0, 56, 100, 180, 320 ppb
Receiving waters: 100% receiving water and a control.
Effluents: > 0.5 Receiving waters: None or > 0.5
Acute: 96 h, Chronic: 7d
90% or > survival in controls
2L per day
Effluents: Survival (LC50)
Receiving waters: Survival (Significant Difference from control)
                                    41

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3.0 PROCEDURES FOR EQUIPMENT
3.1 PROTOCOL FOR AUTOCLAVE
•   The autoclave is a device used for sterilizing objects by exposing them to steam at above
    atmospheric pressure (and thus at a temperature above the normal boiling point of water)1.

•   The Autoclave is found in the Rm 127, down the hall from the Bioassay Lab.
•   Turn water valve on (orange valve located on the wall to the left of the autoclave). On position
    is vertical, while the off position is horizontal.
    1.  Switch main power on by pulling the lever down (lever located on the wall to the right and
       behind the autoclave).
    2.  Place glassware and  stoppers in metal tray inside autoclave.
    3.  Close autoclave door and turn handle to the right until tightened (locking mechanism will move
       into place).
    4.  Set Sterilizing dial to 20 minutes (Located on right hand side of autoclave).
    5.  Set Exhaust dial to 10 minutes (Located below sterilizing dial).
    6.  Turn red on/off switch to regular (switch located on autoclave below door).
    7.  Turn power switch to the "on" position.
    8.  Buzzer will indicate  when cycle is finished*.
    9.  Check that chamber pressure gauges are at zero before opening autoclave door.
    10. Open autoclave door slowly to release steam.
    11. Glassware may be HOT!! Use protective gloves when removing glassware.
    12. Leave autoclave door slightly ajar.
    13. Turn all switches back to the off positions.
    14. Turn light off.
* As of July 2004, the sterilization timer is broken. Please take note of sterilization time in order to
manually switch off.
1 http://www.webster-dictionary.net/definition/Autoclave
                                              42

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3.2 CALIBRATION AND USE OF THE ORION 720A ISE METER/AMMONIA PROBE

      I.  STORAGE OF AMMONIA PROBE

           A.  Between measurements, keep tip immersed in a 10~3 or 10 ppm standard with ISA added.
              For low-level measurements, keep tip in pH 4 buffer between measurements.

           B.  For overnight or week-long storage, place electrode tip in a 0.1 M or 1000 ppm standard
              w/o ISA.

           C.  For storage over a week, disassemble completely and rinse the inner body, outer body,
              and bottom cap with D.I. water. Dry and reassemble electrode without filling solution or
              membrane.

     II.  PROBE CALIBRATION

            A.  Calibration must be performed every time meter is used.

            B.  Plug in meter.

            C.  Look on back of meter to see which input the electrode is plugged in to. Make sure this is
                the number on the prompt line. If not, press the 2nd key, and then channel repeatedly
                until selected input is displayed.

            D.  Using the 0.1M NH4+ standard, prepare concentrations that bracket the expected sample
                range and differ in concentration by a factor often.

                     Example: if sample concentration is estimated to range from 0-50 ppm, make up 3
                     calibration solutions such as 0.1 ppm, 10 ppm, 100 ppm. These bracket the range
                     and differ by a factor often from each other. Since the 0.1 M NFL,+ standard is
                     equivalent to 1700 ppm, use CiVi=c2v2 to solve for dilutions;

                     For the O.lppm  solution: (1700 ppm)(vO=(0.1 ppm)(1000 mL)
                     (vi)= .058 mL or 58 uL of 1700 ppm in 999.94 mL of deionized water

                     For the 10 ppm  solution: (1700 ppm)(vi)=(10 ppm)(100 mL)
                     (vi)= 0.588 mL or 588 uL of 1700 ppm in 99.41 mL of deionized water

            E.  Measure 25 mL of the more dilute standard into a 30 mL beaker.

            F.  Add 0.5 mL ISA and stir thoroughly.

            G.  Select concentration mode by pressing mode until "CON" is displayed. Then press 1st -
                Calibration, when asked enter number of standards to be measured and press 2 or 3
                depending on how many standards  are being used, then yes.

            H.  Rinse electrode with deionized water then blot dry.

            I.   Place electrode in beaker and stir moderately.
                                             43

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PROTOCOL FOR CALIBRATING THE ORION 720AISE METER AND ORION AMMONIA PROBE Cont'd

             J.  When "READY ENTER VALUE" is displayed on prompt line, enter value of standard
                and press yes.
             K.  Example: For the 0.1 ppm dilution, type 0.100 on keypad and press yes.

             L.  The meter automatically switches to  standard 2. Rinse electrode and add 0.5 mL of ISA
                to the second standard.

             M. Place electrode in beaker and stir moderately.

             N.  When "READY ENTER VALUE" is displayed, once again enter value of 2nd standard
                and press yes.

             O.  Repeat steps 8-10 for the 3rd standard.

             P.  The electrode slope is then calculated and displayed. Slope should be within the range of
                -54 to -60.

    III.  MEASUREMENTS

             A.  After calibration meter will automatically proceed to "MEASURE" mode.

             B.  Place electrode into sample, when "RDY" is displayed and meter beeps, record sample
                results.

    IV.  NFL.+PROBE TROUBLESHOOTING

             1.  Membrane life can be anywhere from one week to several months. If there are any dark
                spots or discoloration on the membrane it needs to be changed. Follow instructions on
                page 4 of the Orion Ammonia electrode instruction manual.

             2.  Obtaining the slope (slope= Ain mV/ tenfold A in concentration)  provides the best means
                for checking electrode operation, (page 6 of electrode instruction manual)

                    1.  Place 100 mL of DI water in a 150 mL beaker.
                    2.  Add 2 mL of IS A and stir.
                    3.  Set the function switch to mV mode.
                    4.  Rinse electrode with deionized water and place in solution.
                    5.  Pipet ImL of 0.1 M NH4 into beaker and record mV's when reading is stable.
                    6.  Next, pipet 10 mL of 0.1 M NH4 solution into the same beaker. Stir thoroughly
                       and measure mV reading when stable.
                    7.  The difference between the first and second reading should be between -54 to -
                       60 mV/decade.

             3.  If electrode slope is low during operation, check electrode inner glass body, (page 24 of
                electrode instruction manual)

                    1.  This is done by first soaking the inner glass body in filling solution for at least
                       two hours, if it has been dry.
                                              44

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PROTOCOL FOR CALIBRATING THE ORION 720AISE METER AND ORION AMMONIA PROBE Cont'd

                    2.  Next, rinse inner-body with deionized water and immerse in 200 mL of pH 7
                       buffer w/ 0.1 M NaCl added. Assure that reference element is covered, stir and
                       record stable mV reading.
                    3.  Rinse in DI water and immerse in 200 mL of pH 4.0 buffer with 0.1M NaCl
                       added.
                    4.  Watch the change in meter readings carefully. The reading should change 100
                       mV in less than 30 seconds after immersion.
                    5.  After 3-4 minutes the reading should stabilize, the difference between the pH 7
                       and pH 4 should be greater than 150 mV.

     V.  METER TROUBLESHOOTING

            A.  The set up and self-test should be performed on the meter. Easy to follow instructions
                are on page 6 of the Orion manual for the 720A meter.

            B.  Next, run the checkout procedure on page 10 of the same book mentioned in the
                previous step. If you receive any error codes, go to the back of the manual for further
                troubleshooting tips.
                                              45

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3.3 CALIBRATION AND USE OF THE ACCUMET PH METER

Calibration should be performed at least once daily and about every hour (once measurements begin) for
more accurate results since the electrode's slope and zero potential may change overtime.

Note: Meter should be on Channel A for pH readings.  It should be on Channel A already, but if it is not,
press "Channel" until it reads "A" only.

    I.  PROBE CALIBRATION

           A.  Press "Standardize" and select "2" to clear existing standards.

           B.  Press "Standardize" again and select "1" to add the first standard.

           C.  Obtain fresh yellow pH 7.0 buffer solution and pour about 15 mL into 20 mL scintillation
               vial and add a small magnetic stir bar.

           D.  When the Buffer Value screen appears type in "7.0" and press Enter.

           E.  The Prepare Buffer/Standard screen will appear.

           F.  Remove electrode from pH 4.0 or 7.0 storing solution, rinse with deionized water, and blot
               dry.

           G.  Place pH electrode inside 7.0 buffer solution with magnetic stir plate on low.

           H.  Press Enter.

           I.   When accurate reading of buffer value is displayed, press the enter key to manually accept
               the reading.

           J.   Repeat these steps for the  second standard (pH 10.0).

           K.  When accurate reading of buffer value is displayed, press the enter key to manually accept
               the reading.

           L.  Press "Slope/Efficiency" button and ensure efficiency is 100 ฑ 2%.

           M.  If efficiency is poor, recalibrate with fresh standards.

   II.  MEASUREMENTS

           A.  Rinse electrode with deionized water.

           B.  Blot dry electrode with a KimWipe.
                                               46

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PROTOCOL FOR CALIBRATION AND USE OF THE ACCUMET pH METER Cont'd

           C.  Immerse electrode in sample, stirring gently (i.e. with magnetic stirrer).

           D.  Wait for pH reading to stabilize, and record value. The electrode also provides the
               temperature, if needed.

           E.  Rinse electrode with deionized water between samples.

           F.  Store rinsed electrode in pH 4.0 or 7.0 storage solution when finished.
                                                47

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3.4 CALIBRATION AND USE OF THE ORION (MODEL 840) DISSOLVED OXYGEN
PROBE

Calibration should be performed at least once daily and about every hour (once measurements begin) for
more accurate results.

    I.  BEFORE USE

          A. Be sure sponge in calibration sleeve is saturated with distilled or deionized water.

          B. If probe has been disconnected from instrument or silver anode has been cleaned, it must
              be reconnected and allowed to polarize for 20 to 50 minutes before use.

   II.  CALIBRATION

          A. Turn meter on by depressing "On/Off button.

          B. Depress and hold down "Mode" button until display cursor is at "Cal". As long as the
              Mode key is depressed, the display will cycle.  *Be sure calibration sleeve is completely
              covering probe and the probe is lying flat on lab counter during calibration steps.

          C. Depress quickly and release the Mode key. The word "SAL" will appear on display and
              then the salinity will appear. Adjust as necessary with up and down arrows.

          D. After correct salinity is entered, quickly depress the mode key again and three dashes  (—)
              should appear on the display.

          E. After a few moments, the slope of the electrode/membrane will be displayed. It should
              read between  0.7 and 1.2.  If it does not, see Troubleshooting below.

  III.  SAMPLE MEASUREMENTS

          A. Remove calibration sleeve. You are now ready to make D.O. measurements.

          B. Depress Mode key to choose mg/L or %.

          C. Immerse probe in sample, making sure the stainless steel thermistor is submerged.

          D. Stir slowly so that flow rate past the membrane is approximately 15 cm/sec.

          E. Take reading when the value on the display is stable. Also record temperature from
              display window next to D.O. value.

          F. Rinse in deionized water and return to calibration sleeve when finished.
                                              48

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CALIBRATION AND USE OF THE ORION (MODEL 840) DISSOLVED OXYGEN PROBE Cont'd

  IV.  TROUBLESHOOTING

       A.  Slope out of range or an error message "El" indicates electrolyte may need replacement,
           electrode cap is old, or electrodes need cleaning.

       B.  To replace electrolyte and cap, first disconnect probe from instrument.

       C.  Unscrew and discard old membrane cap.

       D.  Rinse electrode assembly with distilled water.

       E.  Moisten inside of new membrane with a few drops of electrolyte from the Probe Service Kit
           that should be on the counter adjacent to the meter.

       F.  Completely fill membrane cap with electrolyte.

       G.  Holding probe at a slant, with the flat surfaced vent on top, insert electrode assembly vertically
           into the new membrane cap and tighten cap quickly. Excess electrolyte will be expelled
           through vent.  If air bubbles are in the membrane cap, repeat procedure.

       H.  Plug probe back into instrument and allow approximately 20 minutes for repolarization.

       I.   Calibrate as before. If the slope still does not fall within range still, the electrode may need
           cleaning. Refer to "Cleaning the Electrode" section of Orion 840 Instruction Manual.
                                               49

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3.5 MEASUREING AMMONIA WITH THE HACK DR/2400 SPECTROPHOTOMETER


    I.  GETTING STARTED

       A. Turn power on with the blue power on/off key on the far left side of the spectrophotometer.

       B. The main menu will appear, if screen is hard to read turn on the backlight with the button that
          has a light bulb symbol on it.

       C. To select an operator, go to Instrument Setup and press Operator ID. Either select your
          initials or enter new.

       D. At the main menu select either Hach Programs or Favorite Programs, which contains
          frequently used programs.

   II.  MEASURING NITROGEN AS AMMONIA (SALICYLATE METHOD, 385N OR 8155)

       A. Select program 385N and press Start.

       B. Fill a round 10 mL sample cell to the 10 mL mark with deionized water (this is the blank).

       C. Fill another round 10 ml sample cell to the 10 mL mark with sample.

       D. Add the contents of one Ammonia Salicylate powder pillow to each cell. Stopper and shake to
          dissolve the powder.

       E. Touch the timer icon. Touch OK to start a 3 minute reaction period.

       F. When the timer beeps,  add one Ammonia Cyanurate reagent powder pillow to each cell.
          Stopper and shake to dissolve  reagent.

       G. Touch the timer icon. Touch OK to start a 15 minute reaction period. A green color will
          develop if ammonia nitrogen is present.

       H. When the timer beeps,  wipe blank with a Kimwipe to remove fingerprints and place in cell
          holder.

       I.  Touch Zero, display will show 0.00 mg/L.

       J.  Wipe the sample with a Kimwipe and place into holder.

       K. Touch Read. Results will appear in mg/L - NH3-N (unionized). To read in NH3 or NH/ go to
          Options. Select Chemical Form and select desired form.

       L. When finished touch Return and sample value will be converted.
                                             50

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PROTOCOL FOR MEASUREING AMMONIA WITH THE HACK DR/2400 SPECTROPHOTOMETER Cont'd

  III.  PREPARING AN AMMONIA NITROGEN STANDARD

       A. Prepare a 0.20 mg/L ammonia nitrogen standard solution.

       B. Dilute 2.00 Ml Ammonia Nitrogen Standard Solution (10 mg/L) to 100 mL with deionized
          water.

       C. To adjust the calibration curve using the reading obtained with the 0.20 mg/L standard
          solution, touch Options on the program menu.

       D. Touch Standard Adjust. Touch On. Touch Adjust to accept the displayed concentration.
                                             51

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3.6 BARNSTEAD E PURE WATER PURIFICATION SYSTEM

The Barnstead E-Pure water purification system is used to produce deionized (reagent) water with
a resistivity of as high as 18.2 megohms/cm and TOC content of less than 10 ppb.  The unit is
located on the wall near the fume hood in Rm 244.  The following steps should be taken to obtain
E-Pure water:

          A. Open orange-colored water valve every morning.

          B. Turn pump on and monitor water resistivity.

          C. When water has reached desired resistivity, open draw off valve to get water. Close
             draw off valve.

          D. Leave pump on.

          E. At end of day, turn pump off & close water valve.

          F. Cartridges are replaced on an as needed basis by Ignacio Rivera.
                                          52

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3.7 CALIBRATION AND USE OF THE ORION APLUS (105A+) BASIC
CONDUCTIVITY METER

Choose standards that bracket expected sample values. Brackish water and seawater range from 1-100
milli-Siemens. The meter will shut off automatically after 20 minutes of non-use. To turn this feature off,
depress the mode button while turning the meter on. A low-pitched beep should indicate that auto shutoff
has been disabled. This feature will be reactivated when the meter is shut off and turned on again.

  III.  PROBE CALIBRATION

           A.  To turn on the meter, press the "on" button.

           B.  Disable the temperature compensation by depressing the "setup" button. Change the
               number located at the bottom of the screen to 0.0 by pressing the "down arrow (T)"
               button.

           C.  Press the "mode" button to return to measurement screen. Press "cal" button to initiate
               calibration. The last cell constant used will appear on display.

           D.  Immerse conductivity cell in the standard. Agitate solution slightly to remove air bubbles
               from probe.

           E.  Enter the cell constant printed on the cell cable (1.00), the decimal point can be moved by
               pressing the up or down arrows. Press "yes" to accept cell constant value.

           F.  Meter will return to measurement mode, compare the displayed value with the standard at
               its specified temperature value (see tables included with manual).

           G.   If the correct standard value is not displayed, calculate the cell constant adjustment factor
               using the following formula:

                  Q = Standard value / Displayed Value

           H.  Multiply the initial cell constant (1.00) by Q.  This is the new cell constant.

           I.   Repeat steps C-G.

           J.   If Displayed Value is still different from the Standard Value, calculate Q again (step G)
               and multiply the derived Q by the previous cell constant.

           K.  Repeat steps C-G until the Standard Value and Displayed Value are the same.

           Example:

           Initial Cell Constant = 1.00
           1st Standard Value = 9.288 (@ 21.4 ฐC)
           1st Displayed Value = 9.01
           Q = 9.288/9.01 = 1.0308 = new cell constant
                                               53

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PROTOCOL FOR CALIBRATION AND USE OF THE ORION APLUS (105A+) BASIC CONDUCTIVITY METER
Cont'd

           2nd Standard Value = 9.233 (@ 21.2 ฐC)
           2nd Displayed Value = 9.29
           Q = 9.233/9.29 = 0.9938
           Multiply 1.0308 (last cell constant) X 0.9938 (newest cell constant) = 1.0244
           3rd Standard Value = 9.215 (@ 21.1 ฐC)
           3rd Displayed Value  = 9.21
           Because the Standard and Displayed Values are the same, the instrument is calibrated.

  IV.  MEASUREMENTS

           A.  Rinse conductivity cell with deionized water.

           B.  Blot cell with a Kimwipe.

           C.  Immerse cell in  sample and agitate gently to remove air bubbles.

           D.  Press the "mode" button to move between conductivity and salinity.

           E.  Allow reading to stabilize.

           F.  For storage overnight or longer, conductivity cell should be clean and dry. While in use,
               the cell can remain in deionized or seawater.

   V.  TROUBLESHOOTING

           A.  Check battery, 9V battery  required and calibrate after battery change.

           B.  Run a self-test.

                    1.  Disconnect the conductivity cell  (probe).

                    2.  Press  and hold the "yes" button while pressing the "on/off button to turn meter
                        on.

                    3.  This will cause the meter to perform an electronic hardware diagnostics test.

                    4.  After test "7", a "0" will appear on display.

                    5.  Press  each key on meter, each key must be pressed within four seconds of the
                        previous key.

                    6.  After test "7", the meter's display will read "test 8" and turn off.

                    7.  An operator assistance code will be displayed if any errors are found.

                    8.  See troubleshooting guide in manual for further instruction.
                                               54

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3.8 CALIBRATION AND USE OF THE ORION (MODEL 830A) PORTABLE
DISSOLVED OXYGEN PROBE

Calibration should be performed at least once daily.

    I.  BEFORE USE

          A. Be sure sponge in calibration sleeve is saturated with deionized water.

          B. If the meter has been  off for longer than 72 hours, it will need to re-polarize for 60
              minutes.

          C. Probe storage: for short-term storage (overnight or between measurements), probe should
              remain plugged into meter and kept in the moist sleeve. For long-term storage, probe
              should be disconnected from meter, membrane cap should be removed, and probe should
              be stored cleaned and dry.

   II.  CALIBRATION

          A. Turn meter on by pressing the "power" button.

          B. To change salinity, simultaneously depress the "cal" and "power" button. Pressing the
              "cal" button allows you to scroll through configuration options. After changing salinity to
              the value of your sample using the up and down arrows, press "meas" key to return to
              measure mode.

          C. Press the "cal" button to enter the calibration mode. Press the "cal" button a second time to
              begin calibration. Be  sure calibration sleeve is completely covering probe and the probe is
              lying flat on the lab counter during calibration steps.

          D. A range value will appear. If the slope is out of the required range of 60-120%, "error" is
              displayed.

          E. To abort calibration, press the "meas" button at any time.

          F. The  display screen features a "Stat face" that resembles a smiley face.  It provides
              information on the electrode condition. If a sad face appears, calibration may be needed.

  III.  SAMPLE MEASUREMENTS

          A. Remove calibration sleeve.

          B. To change the  measurement mode (ie.  mg/L or % saturation), simultaneously press the
              "cal" and "power" button. Pressing the "cal" button allows you to scroll through
              configuration options. After choosing appropriate measurement, press  "meas" key to return
              to measure mode.
                                              55

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CALIBRATION AND USE OF THE ORION (MODEL 830A) PORTABLE D.O. METER Cont'd

           C. Immerse probe in sample, making sure the stainless steel thermistor is submerged.

           D. Take measurement when the value on the display is stable, if Auto-read is on (indicated by
              an "A" on the right hand side of the screen), the "A" will stop flashing when the reading is
              stable. The temperature can also be recorded from the display window below the D.O.
              value.

           E. Rinse probe in deionized water and return to calibration sleeve when finished.


  IV.  TROUBLESHOOTING & MAINTENANCE

           A. The display screen features a "Stat face" that resembles a smiley face. It provides
              information on the electrode condition (slope, response time, etc.). Deterioration of
              electrode condition is shown first by a straight face and then by the frowning of "Stat
              face." An improvement can only take place after calibration.

           B. The display screen also features what looks like a bulb with flashing lines coming from it.
              This symbol is an indication of electrode response time. Response time can become
              sluggish due to aging, lack of maintenance, or membrane tearing and fouling.

           C. To replace electrolyte and membrane cap, first disconnect probe from instrument.

           D. Unscrew and discard old membrane cap.

           E. Fill the new membrane cap halfway with electrolyte solution (Polarographic DO Probe
              Electrolyte 080514).

           F. Holding probe at a slant, insert electrode assembly vertically into the new membrane cap
              and tighten cap quickly.  Excess electrolyte will be expelled through vent.  If air bubbles
              are in the membrane cap, repeat procedure.

           G. Plug probe back into instrument and allow approximately 25 minutes for repolarization.

           H. Calibrate. If the slope still does not fall within the required range, the electrode may need
              cleaning. Use  polishing paper to clean probe then repeat steps E-H.

           I.  Please consult manual for further troubleshooting and definitions of error messages.
                                               56

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3.9 PERCIVAL SCIENTIFIC 136LL INCUBATOR

    I.  LIGHTING

          A. To enter the Lights Menu, press the "LIGHTS" key. To navigate through the menu, use the
             up and down arrow keys.

          B. When the display reads "Light 1," press "ENTER." Switch the setting to "ON" or "OFF"
             using the arrow keys. Press "ENTER" to accept the setting.

          C. Use the arrow keys to scroll until the display reads "Light 2." Switch the setting to "ON"
             or "OFF" using the arrow keys. Press "ENTER" to accept the setting.

          D. To exit the Lights Menu, press the "LIGHTS" key.

   II.  TEMPERATURE MENU

          A. To enter the Temperature Menu, press the "TEMP/ALARM" key. To navigate through the
             menu, use the up and down arrow keys.

          B. To set the temperature manually,
                   1.  Press "ENTER" when the display reads "Manual Temp Set Pt."
                  2.  The temperature reading will begin to flash. Use the up and down arrows
                      to change the set point to the desired temperature.
                  3.  Press "ENTER" to accept value.

          C. To set the temperature high safety setting,
                   1.  Press "ENTER" when the display reads "Safety High Alarm."
                  2.  The temperature will begin to flash. Use the up and down arrows to
                      change the display to the desired temperature, which is recommended to
                      be 3ฐC above the highest programmed temperature. When the temperature
                      gets higher than this value, a safety alarm will be triggered and the
                      incubator will shut down its control functions.
                  3.  Press "ENTER" to accept value.

          D. To set the temperature low safety setting,
                   1.  Press "ENTER" when the display reads "Safety Low Alarm."
                  2.  The temperature will begin to flash. Use the up and down arrows to
                      change the display to the desired temperature, which is recommended to
                      be 3ฐC below the lowest programmed temperature. When the temperature
                      gets lower than this value, a safety alarm will be triggered and the
                      incubator will shut down its control functions.
                  3.  Press "ENTER" to accept value.

          E. To exit the Temperature Menu, press the "TEMP/ALARM" key.
                                           57

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PERCIVAL SCIENTIFIC 136LL INCUBATOR Cont'd
  III.  96 STEP PROGRAM SETUP

          A.  Press the "PROG" key.

          B.  Use the up and down arrow keys to select "Enter/Edit 96 Step" and press "ENTER."

          C.  If a program has not been entered, a message will be given that there are no steps in the
              profile. If a program has been entered, press the "PROG" key, select "Delete All Steps,"
              and press "ENTER."

          D.  To add the first step,
                   1.  Press the "PROG" key, select "Add Step," and press "ENTER."
                   2.  Press the "TIME" key. Use the up and down arrows to change the time.
                   3.  Press the "TEMP/ALARM" key. Use the up and down arrows to change the
                      temperature.
                   4.  Press the "LIGHTS" key. Use the up and down arrows to change the lighting so
                      that "1" represents on and "0" represents off.
                   5.  Press "ENTER" and verify that no settings are flashing.

          E.  To add the second step,
                   1.  Press the "PROG" key, select "Add Step," and press "ENTER."
                   2.  Follow steps D2-5.

  IV.  RUN 96 STEP PROGRAM

          A.  Press the "PROG" key

          B.  Use the up and down arrow keys to select "Run 96 Step" and press "ENTER."

   V.  SAMPLE 96 STEP PROGRAM

          Below is a sample 96 Step Program for a 16 hour light: 8 hour dark cycle.
                      Step 1 9:00 am
                            15.0 C
LT11
                      Step 2  1:00 am
                             15.0 C
LT:00
                                           58

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3.10 CALIBRATION AND USE OF THE OAKTON PH 11 METER

Calibration should be performed at least once daily for more accurate results.

  VI.  PROBE CALIBRATION

           N.  Make sure that the MODE on the meter is set to measure pH, as indicated in the upper right
               corner of the display.

           O.  Remove the probe from the electrode storage bottle, rinse with deionized water, and shake
               dry.

           P.  Place the electrode in pH 4.0 buffer solution and stir.

           Q.  Press CAL/MEAS.  The CAL indicator will be shown.

           R.  When the measured pH value is stable, press the HOLD/ENTER key to confirm
               calibration.

           S.  Rinse the electrode with deionized water and shake dry. Place the electrode in pH 7.0
               buffer solution and stir.

           T.  Repeat steps D and E.

           U.  Rinse the electrode with deionized water and shake dry. Place the electrode in pH 10.0
               buffer solution and stir.

           V.  Repeat steps D and E

           W.  When finished calibrating, the meter will automatically return to Measurement mode.  If
               this does not occur, press CAL/MEAS to return manually.

           X.  In Measurement mode, perform a calibration check using pH 7.0 buffer solution. If the
               measured value is not within the required range, change buffer solutions and repeat
               calibration.

  VII.  MEASUREMENTS

           G.  Rinse the electrode with deionized water and shake dry.

           H.  Immerse the electrode in sample and stir gently.

           I.   Wait for the pH reading to stabilize and record the value. The electrode will also provide a
               temperature reading, if needed.

           J.   Rinse the electrode with deionized water between samples.

           K.  Store rinsed electrode in electrode storage bottle when finished.
                                               59

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4.0 STANDARD OPERATING PROCEDURES- MISCELLANEOUS

4.1 GLASSWARE AND PLASTICWARE CLEANING

I.     NEW PLASTICWARE

      Rinse new plasticware with sample dilution water before use.

II.    NEW GLASSWARE

      New glassware must be soaked overnight in 10% acid, then rinse well in deionized water
      and seawater.

III.   NON-DISPOSABLE SAMPLE CONTAINERS, TEST VESSELS, PUMPS, TANKS
      AND OTHER EQUIPMENT

      Any equipment coming in contact with samples must be washed to remove surface
      contaminants as described below:

    1.   Rinse with tap water several times.
    2.   Soak in tap water and 10% Liquinox or other detergent for at least 15 minutes, then scrub
        with brush.
    3.   Rinse in tap water several times.
    4.   Rinse in 10% Nitric (HNO3) or hydrochloric (HC1) acid to remove scales, metals, and
        bases. 10% =10 mL concentrated acid + 90 mL deionized water.
    5.   Rinse several times in deionized water.
    6.   If organic toxicant used, rinse once with pesticide grade acetone  in fume hood.
    7.   Rinse three times with deionized water.

IV.   SEDIMENT-WATER INTERFACE TUBES

   A. AFTER USE

     1.  Soak in 10%  Citranox or RB S for 24 hours.
     2.  Scrub screen  surface and tube gently with brush and rinse 2-3 times  in tap water.
     3.  Dip screen tubes in 10% nitric acid for 5-10 seconds.
     4.  Rinse thoroughly in deionized water.

   B. PRIOR TO NEXT USE

     1.  Soak in seawater for 24 hours.
     2.  Rinse 3 times in deionized water.
                                         60

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PROTOCOL FOR CLEANING GLASSWARE/PLASTICWARE (Cont'd)

V.    CARBOYS

   B. SEMI-ANNUALLY

          1.  Soak in -2% nitric acid solution for approximately one week, check that pH of acid
             solution is below 2.00.
          2.  Rinse 3 times with deionized water.
          3.  When refilling with Scripps water, rinse the inside, nozzle and outside of the
             carboy 2-3 times with seawater before refilling. Avoid touching the metal nozzle of
             the hose inside the carboy.

VI.   SEDIMENT CORE TUBES

      A. AFTER USE

          1.  Soak in 10% Citranox or RBS for 24 hours.
          2.  Scrub tube gently  with brush and rinse 2-3 times in tap water.
          3.  Dip core tubes in 10% nitric acid for 5-10 seconds.
          4.  Rinse thoroughly in deionized water.

      B. PRIOR TO NEXT USE

          1.  Soak in seawater for 24 hours.
          2.  Rinse 2-3  times in deionized water.

VII.   EMBRYO/LARVAL IN-SITU DRUMS

    A. AFTER USE

      1.  Remove plastic  screws from ends.
      2.  Soak in 10% Citranox or RBS for 24 hours.
      3.  Scrub screens very gently with brush and rinse 2-3 times in tap water.
      4.  Dip drums in  10% nitric acid for 5-10  seconds.
      5.  Rinse thoroughly in deionized water.

    B. PRIOR TO NEXT USE

      1.  Soak in seawater for 24 hours.
      2.  Rinse 2-3 times  in deionized water.

VIII. DINOFLAGELLATE FLASKS

   A. AFTER USE

      1. Soak in 10% Citranox or RBS for 24 hours.
                                         61

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PROTOCOL FOR CLEANING GLASSWARE/PLASTICWARE (Cont'd)

       2.  Scrub with brush and rinse 2-3 times in tap water.
       3.  Place in 10% nitric acid for 5-10 seconds.
       4.  Rinse thoroughly (2-5 times) in deionized water.

   B. PRIOR TO NEXT USE

   1.  Sterilize in autoclave (see protocol for using autoclave1)

IMPORTANT: All glassware must be soaked overnight in dilution water prior to use in each test.
                                           62

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4.2 RECEIVING AND HOLDING TEST ORGANISMS

This protocol is intended for receiving and holding of mysid shrimp (Americamysis bahid),
topsmelt larvae (Atherinops a/finis), and inland silversides (Menidia beryllina):

   1.  Upon arrival, check temperature before placing into aquarium/holding tank (6 L or 22 L).
       Test organisms should not be subjected to changes of more than 3 ฐC in water temperature
       or 3 ppt salinity in any 12-hour period.

   2.  In order to acclimate animals, place shipping bag in clean aquarium/holding tank for at
       least 60 minutes. After initial water quality measurements are taken, the top of the bag
       should be propped open and water should be gently aerated. A small amount of food may
       be added if the animals do not appear stressed.

   3.  After temperature in the shipping bag has approached appropriate holding temperature
       (depending on test method), remove the shipping bag and add filtered seawater to the
       holding tank.

   4.  Mysids: gently siphon mysids into holding tank using a wide-bore pipette and tygon
       tubing. As mysid:water ratio in the shipping bag decreases, siphon out the excess water
       into a clean beaker. When all of the mysids have been transferred, rinse the bag with
       filtered sea water and check for mysids that may have stuck to the sides of the bag, also
       check the excess water that was siphoned off into the clean beaker. Loading rate for mysids
       should not exceed 20 mysids per liter.

       Fish Larvae: Carefully siphon off extra water from the travel bag in order to concentrate
       fish larvae. Gently pour larvae into clean holding tank. Be sure not to transfer any fish that
       died during shipment. When bag level gets low,  individually pipette larvae into holding
       tanks using a wide-bore pipette. Loading rate for fish should not exceed 0.4  g fish per liter.

   5.  Gently aerate each holding tank with a small airstone.

   6.  Animals should be fed newly hatched Artemia nauplii liberally.

   7.  Check temperature  frequently to make sure it is maintained at appropriate holding
       temperature + 2 ฐC.  If temperature is not maintained in  range, organisms should be held
       an additional day prior to testing. Organisms should be acclimated for at least 2 days prior
       to testing.

   8.  Ensure that the photoperiod to be used during testing is being used during acclimation.

   9.  Renew holding water every other day or renew one half of the water every day. This
       depends on the amount of fecal  matter and density of animals in the holding tank. All  fecal
       matter, dead, etc. should be siphoned daily.
                                           63

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PROCEDURE FOR RECEIVING/HOLDING TEST ORGANISMS (Americamysis bahia, Atherinops affmis,
and Menidia beryllina) Cont'd

    10. If the organisms need to acclimate to the testing salinity, mix filtered sea water with the
       appropriate amount of deionized water to obtain the desired salinity (do not adjust salinity
       more than 3ppt in a 12-hr period) during water changes.

    11. The following should be recorded during the holding period:

           a.  Condition of the organisms upon arrival and every day thereafter.
           b.  Temperature in holding tanks
           c.  Frequency of water change and siphoning
           d.  Dissolved oxygen level in  holding tanks
           e.  Frequency and approximate quantity of feeding
           f.  General appearance of water (cloudy, clear, etc.) and organisms (active,  dead, etc.)

    12. Before disposal, any surviving test organisms are killed, generally by concentrating into a
       container and freezing. Under no  circumstances are test organisms ever released to the
       wild or used more than once for testing.
                                            64

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4.3 MAINTAINING DINOFLAGELLATE CULTURES

FACILITY:            SPAWAR SYSTEMS CENTER
                       BIOASSAY LABORATORY (RM 116)
                       CODE 71750
                       53475 STROTHE RD.
                       BLDG.  111
                       SAN DIEGO, CA 92152
I.      OBJECTIVE: To maintain organism health and propagation, dinoflagellate cultures need to be
       split about every two weeks to so that they do not become too dense. Species being maintained
       include; Lingulodinium polyedrum, Ceratacorys horrida, Pyrocystis noctiluca, Gonyaulax
       grindleyii, Pyrocycstis lunula, and Pyrocystis fusiformis.

II.     NECESSARY MATERIALS AND SUPPLIES

           Beakers- 1 Liter, Class A, borosilicate glass-  3 or 4 for media
           Pipets, automatic - adjustable, to cover a range of 0.01 to 5 ml and pipette tips
           Watch glasses - for covering 1 L beakers
           Colored labeling tape
           Stock solutions A, B and C1
           Erlenmeyer flasks  - 5-10 acid washed and autoclaved for split cultures
           Glass microscope slides - for examining dinoflagellate species
           Light biological Microscope
           Microwave
           Foam stoppers - to stopper flasks with culture

III.    METHODS

**Be aware of cross contamination! Wear gloves at all times, not allowing anything that will come in
contact with the inside of the flasks to touch countertops - use different pipette tips for each culture, etc.

   A. ONE DAY PRIOR TO  CULUTRE SPLIT

       1.   Sterilize all flasks  and stoppers in the autoclave2.

       2.   Filter 2 to 3 (depending on how many cultures you will split) liters of seawater (collected from
           the cold room in bldg. Ill) with 0.22 (im filter paper.

       3.   To each 1 Liter glass beaker add:
           •   Stock A-15 mL
           •   Stock B - 1  mL
           •   Stock C - 0.5 mL

       4.   Add 1 L of filtered seawater to each beaker.
                                             65

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PROTOCOL FOR MAINTAINING DINOFLAGELLATE CULTURES OF SEVERAL SPECIES Cont 'd

       5.  Heat in microwave for 25 minutes. Include a small beaker filled with deionized water for
           possible evaporation.
       6.  Allow media to cool to room temperature (about 20 ฐC) overnight.

    B. DAY OF CULTURE SPLIT

       1.  Remove a 25 mL aliquot from each beaker of media and take pH, salinity and temperature.
           Salinity should be approximately 34 %o, pH from 8.0-8.1 and temperature range should be 19
           ฐCฑ2ฐC.

       2.  Record data on the dinoflagellate log.

       3.  Choosing dinoflagellate stocks to split

           a.  Turn off the lights and return to the incubator.
           b.  Swirl each culture and move brightest (most dense) cultures to the front row.
           c.  Remove all flasks in front row, making sure that one of each six cultures is selected.
           d.  Depending on the culture density remove .020 mL to 1 mL and view under the microscope
              to determine viability, presence of motility (for some species) and density.

       4.  Label all new flasks with current date and species name and strain (if applicable).

       5.  Rinse each sterilized flask with approximately 50 mL of medium.

       6.  Add approximately 50 mL into flask (to cushion entry of dinoflagellates).

       7.  For high density cultures (i.e. L. polyedrum) add 100 mL of culture in new flask then bring up
           to 250 mL line with media.

       8.  For low-density cultures (P.  noctiluca and C. horrida) add 125 mL of culture in new flask and
           then bring up to the 250 mL line with media.

       9.  Additionally, bring source flask up to the 250 mL mark with  new media.

       10. Always assure that incubator is functioning at 19 ฐC when replacing cultures.
    1 Please refer to "PROTOCOL FOR PREPARATION OF ENRICHED SEAWATER MEDIUM" located on: C:\Documents
    and Settings\zacharia\Desktop\Laboratory 116\Protocols and logs

    2 Please refer to "PROTOCOL FOR AUTOCLAVE" located on: C:\Documents and Settings\zacharia\Desktop\Laboratory
    116\Protocols and logs
                                               66

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4.4 PREPARATION OF ENRICHED SEAWATER MEDIUM (ESM)1
FACILITY:             SPAWAR SYSTEMS CENTER
                        BIOASSAY LABORATORY (RM 116)
                        CODE 71750
                        53475 STROTHE RD.
                        BLDG.  111
                        SAN DIEGO, CA 92152
I.      OBJECTIVE:  This method allows one to prepare stock solutions that serve as growth medium for
       algae, diatoms and dinoflagellates.

II      NECESSARY MATERIALS AND SUPPLIES

           Polycarbonate bottles - (3) 1 Liter
           Deionized water
           Graduated cylinders - Class A, borosilicate glass or non-toxic plastic labware, 5 0-1000ml for
           making test solutions
           Colored labeling tape
           Pipets, automatic - adjustable, to cover a range of 0.01 to 5 ml and pipette tips
           Calculator
           Wash bottles - for topping off graduated cylinders
           Analytical toploading scale - for measuring chemicals

III     METHODS

       A.  MICRONUTRIENT STOCK SOLUTION (A)

             1.   For Cultures:

             To a 1 L  polycarbonate bottle, add 1 L deionized water and the following chemicals
             in the order listed:

             FeCl3 • 6H2O - 0.072 g
             MnCl2-4H2O-0.144g
             ZnSO4 • 7H2O - 0.045 g
             CuSO4 • 5H2O - 0.157 mg (see below)
             CoCl2 • 6H2O - 0.404 mg (see below)
             H3BO3-1.140g
             Na2EDTA- l.Og

Note: Analytical scales will not accurately measure some chemicals required in amounts below 1 mg. To obtain these amounts of
chemical accurately, see example below;
    For CuSO4 • 5H2O - 0.157 mg, measure 0.157 g of chemical, and add to volumetric flask (100 mL). Fill flask to line and
    invert several times. Use a 100 |iL aliquot to obtain correct amount of chemical. If using a different volumetric flask follow
    example calculation:
    for CoCl2 • 6H2O - 0.404 mg and a 200 mL flask; 0.404 mg / 100 |iL x 1 ml /1000 [iL = 4.04mg / 1 mL and if we are using
    trying to create 200 mL of this solution, we need 4.04mg/lmL x200= 808 mg of chemical in 200 mL of deionized water, use
    100 |iL of this solution for the correct amount of chemical. Check calculation: 808mg/200mL x 0.100mL= 0.404 mg.
                                              67

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PROTOCOL FOR PREPARATION OF ENRICHED SEAWATER MEDIUM (ESM) Cont'd

            2.  For Bioassays:

             Follow the same procedure, but omit copper (CuSO4 • 5H2O) and add only .05 g Na2EDTA
             instead of 1.0 g.

       B. MACRONUTRIENT STOCK SOLUTION (B)

            1.  For Diatoms (i.e. Skeletonema costatum):

                    To a 1 L polycarbonate bottle, add 1 L deionized water and the
                     following chemicals in the order listed:

                 •  K3PO4-3.0g
                 •  NaNO3 - 50.0 g
                 •  NaSiO3 • 9H2O - 20.0 g

            2.  For Dinoflagellates:

                    Do not add any NaSiO3 • 9H2O.


       C. VITAMIN STOCK SOLUTION (C)

          To a 1 L polycarbonate bottle, add 1 L deionized water and the following chemicals in the
          order listed:

          •  Thiamine hydrochloride - 500 mg
          •  Biotin- 0.1 mg
          •  B12-1.0mg


To renew dinoflagellate cultures, stock solutions are added to a sterile container containing natural
seawater that has been filtered through a 0.22 |j,m membrane filter in the following proportions:

       Stock A: 15 mL / L of medium
       Stock B: 1 mL / L of medium
       Stock C: 0.5 mL / L of medium

Adjust to pH 8.0  ฑ 0.1 with NaOH or HC1.

Store excess medium in the dark at approximately 4 ฐC until use.
 modified from 1995 ASTM vol. 11.05, sectionE 1218, p.581-2
                                           68

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4.5 HATCHING BRINE SHRIMP AND THEIR USE AS TEST ORGANISM FOOD

TESTING FACILITY:  SPAWAR SYSTEMS CENTER
                       BIOASSAY LABORATORY (RM 116)
                       CODE 71750
                       53475 STROTHE RD.
                       BLDG. 111
                       SAN DIEGO, CA 92152

I.      OBJECTIVE: Brine shrimp (Artemia spp) are the preferred and most convenient food for Mysids
       (Americamysis bahia) and Topsmelt (Atherinops affinis) for whole effluent toxicity testing and
       holding/acclimation.

II      NECESSARY MATERIALS AND SUPPLIES

           Separatory Funnels - (2), 2-Liter capacity
           Air pump
           Plastic tubing - to provide aeration in separatory funnels
           Glass Pasteur pipettes
           Flashlight
           Dark Material - to aid in collection of brine shrimp
           Brine Shrimp (Artemia) cysts

       Note: EPA suggests use of Brazilian or Colombian brine shrimp cysts. These can be
       purchased from Aquarium Products, 180L Penrod Ct, Glen Burnie, MD 21061. Other
       suppliers are on p. 28 of EPA/600/R-95/136.

Ill     METHODS

      1.   Add 1 L of seawater to a 2-L separatory funnel, or equivalent.

      2.   Add 10 mL or 1-2 grams of Artemia cysts to the separatory funnel and aerate for 24 hours at 27
         ฐC.  Actual hatching time will vary with temperature and strain.

      3.   After 24 hours, remove the air supply from the separatory funnel. Cover funnel with a dark cloth
         or paper towel while directing the beam of a flashlight through the bottom of the funnel for 5-10
         minutes. Artemia are phototactic, and will concentrate at the bottom of the funnel. Do not leave
         concentrated nauplii at bottom for more than 10 minutes without aeration, or they will die.

      4.   Drain the nauplii into a funnel fitted with a <150 jam Nitex or stainless steel screen, and gently
         rinse with seawater.

      5.   Gently spray nauplii into a beaker and fill until desired concentration is reached.

      6.   Approximately 40-50 nauplii per feeding per test organism is targeted for most tests. In order to
         feed 10 organisms, this requires 200 \i\ of a suspension with a density of 2000 nauplii/ml. This
         concentration can be achieved by dilution or concentration of nauplii following cell counts under
         a light microscope.  For test protocols using 5 organisms per beaker, 100 jol of the suspension
         would be used.
                                             69

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4.6 HYPERSALINE BRINE AND ARTIFICIAL SEA SALT USE

    I  OBJECTIVE

       Since many effluents entering marine and estuarine systems have little measurable salinity, salinity
       adjustment may be necessary for tests with marine/estuarine organisms. It is important to maintain
       an essentially constant salinity across all treatments. Two methods are available to adjust salinity -
       artificial sea salts and hypersaline brine. Some test methods (e.g. embryo tests, QwikLite) may
       require use of HSB due to toxicity associated with artificial salting.

   II.  MAKING HYPERSALINE BRINE:

       A.     Collect seawater on an incoming tide and pour through a 10 jam filter.

       B.     Store 4 L of filtered seawater in a carboy that has a bottom valve.

       C.     Freeze for approximately 6 hours at -10 ฐC to -20 ฐC.

       D.     Remove hypersaline water from container, leaving behind ice (primarily freshwater).

       E.     Check salinity and pH, adjust if necessary (salinity should never exceed 100 ppt).

       F.     Filter through a 1 jam filter.

       G.     Cap, label, date and store in the dark at 4 ฐC.

  III.  DILUTIONS WITH HYPERSALINE BRINE (SEE BRINE DILUTION WORKSHEET):

       Several dilutions of effluent are needed in a defmitve test. The highest test concentration
       will include a combination of effluent and hypersaline brine. The concentration of the
       highest test concentration will depend on how much brine is required. If the target salinity
       is 34 ppt, diluting to make different test concentrations must be done  with dilution water
       that is also 34ppt. Use the following equation to determine the volume of brine to be added
       to effluent.
       VB = VE x (34 - SE) / (SB - 34), where

       VB = volume of brine to be added in ml
       VE = volume of effluent to be added in ml
       SE = salinity of the effluent (ppt)
       SB = salinity of the brine (ppt)
+ Dilution water (34 ppt)
x^ ^s
6.25%
tl
12.5%
x^ ^s

25%
                                             70

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PROTOCOL FOR HYPERSALINE BRINE, BRINE CONTROLS AND ARTIFICIAL SALT Cont'd

       For example, if the brine is 68 ppt (SB) and the effluent is 2 ppt (SE), to 1 L effluent,
       you would add 1000 ml x (34-2) / (68-34) = 941.18mL brine to make a 34 ppt effluent
       solution (51.5% effluent).

       Serial dilution with dilution water (baseline water) can then be used to achieve other
       effluent concentrations (i.e. 6.25%,  12.5%, 25%, 50%).

       *Check pH of all solutions, and adjust appropriately by adding dropwise, dilute hydrochloric acid
       or sodium hydroxide.

   IV.  BRINE CONTROLS

       Brine controls should contain the quantity of brine used in the highest effluent concentration.  First,
       D.I. water should be adjusted to the salinity of effluent using dilution water (34 ppt) therefore the
       same amount of brine can be added to the control as the effluent. The amount of reagent water (D.I.
       water + dilution water) (VE) added  to the brine controls can be determined by the following
       equation:

       VE = VB x (SB-34) / (34-SE)
   V.  ARTIFICIAL SALT

      A.  For every Ippt increase in salinity desired, add Ig/L of artificial salt (Crystal Sea Marine Mix)
          directly to effluent. Underestimation is good practice, so that the sample does not get over-
          salted.

      B.  Dissolve salt by use of magnetic stirrers.

      C.  If undissolved salt remains, decant effluent into another flask, leaving behind the particulates.

      D.  The pH of the effluent may be a little different from natural seawater, but it is generally not
          adjusted.
                                               71

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4.7 REFERENCE TOXICANT TEST DILUTIONS
  I.  OBJECTIVE

       Reference toxicant tests provide an indication of the sensitivity of the test
       organisms and the performance of the testing laboratory. A dilution factor of 0.5 or greater is
       generally used. Below are concentrations generally used for various tests used by the Bioassay Lab
       and general procedures for preparing test dilutions.
Species
Atherinops qffinis
Menidia
beryllina
Americamysis
bahia
Mytilus edulis

Crassostrea gigas
Strongylocentrotus
purpuratus
Dendraster
excentricus
Ceratocorys
horrida
Eohaustorius
estuarius
Rhepoxynius
abronius
Toxicant
CuS04
CuS04
CuS04
CuS04

CuS04
CuS04
CuS04
CuS04
CdCl2
CdCl2
Test
Endpoint
Survival:
LC50
Survival :
LC50
Survival:
LC50
Normal shell
development
EC50
Normal shell
development
EC50
Normal larval
development
EC 50
Normal larval
development
EC 50
Photon
emission:
EC50
Survival/
reburial
Survival/
reburial
Concentrations
(ug/L, ppb)
0, 50, 100, 200, 400
0, 50, 100, 200, 400
0, 25, 50, 100, 200,
400
0,4.1,5.9,8.4, 12,
17.2, 24
0,4.1,5.9,8.4, 12,
17.2, 24
0, 5.8, 8.4, 12, 17.2,
24,35
0,4.1,8.4, 12, 17.2,
24,35
0, 15.6,31.3,62.5,
125,250


Test
Duration
96 h
96 h
96 h
48 h

48 h
72 h
72 h
24 h
96 h
96 h
  II  MAKING REFERENCE TOXICANT STOCK SOLUTIONS

       A 1 ppt Cu solution is made on an annual basis (or as needed) and stored in the KM 116
       refrigerator. The solution is made as follows (other stock solutions are made similarly, with the
       appropriate weight of solid material substituted for the ones shown here):

           A.  Obtain reagent grade CuSO4ป5H2O crystals from chemical storage area in RM 116.
           B.  Weigh out 0.982g CuSO4ป5H2O on Sartorius balance in Rm 115.
           C.  Add to 250 ml E-pure (deionized) water in clean polycarbonate bottle.
                                              72

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PROTOCOL FOR REFERENCE TOXICANT DILUTIONS (Cont'd)

           D. Label bottle with estimated concentration (e.g. 1 ppt), date, and analyst initials.
           E. Have solution analyzed by STGFAA.
           F. Label the bottle with the measured concentration and date, and record on log sheet.

 III.  MAKING SUB-STOCK FOR USE IN DILUTIONS

       A.  The day of the test, make up 200 mL of 1 ppm (or other relevant concentration) Cu stock
           solution by pipetting 0.2 mL of the Ippt stock in 199.8 mL filtered seawater. This volume is
           adequate for most types of testing.

       B.  Store at testing temperature until use.

 IV.   MAKING TEST DILUTIONS

       A.  Construct a table or use log sheet with pre-constructed table similar to the one below
Coll
Col 2
Col 3
Col 4
Col 5
Concentration
(ppb)
Control
15.63
31.25
62.5
125
250
mL 1 ppm Cu
stock in seawater
0
0.78
1.56
3.13
6.25
12.5
mL filtered
seawater
(diluent)
48
47.22
46.44
44.87
41.75
35.5
mL
dinoflagellate
culture
2
2
2
2
2
2
Total mL
50
50
50
50
50
50
              Where,

              Column 1 is determined by the specific test endpoint and species.
              Column 2 is determined by the equation: CYVi= C2V2

              Example:   (Ippm =1000 ppb) x(Vi) = (15.625 ppb) x (50 ml),  Vi= 0.78ml

              where: Ci= concentration of stock solution (Ippm)
                     Vi= unknown volume of stock to be added
                     C2= desired concentration in dilution flask (e.g. 15.625 ppb)
                     V2= volume of dilution flask (e.g. 50 ml)

              Column 3 is determined by subtracting ml of Cu stock and ml of culture added.

              Example:  Col 3 = Col 5- (Col 4 + Col 2)

              Column 4 is determined by concentration of culture needed for specific test (e.g.
              QwikLite).

              Column 5 is determined by how many replicates are in each treatment. Excess should be
              made so water chemistry can easily be measured.
                                              73

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       Example: if each treatment has 3 replicates each containing 20ml total, at least 110 ml of
       solution should be made. 3 replicates x 20 ml= 60 ml + 50 ml for water quality
       measurements.
B.  After dilutions have been made, cover with parafilm.

C.  Wait 1-2 hours for equilibration before exposure to test organisms.
                                        74

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4.8 ACQUISITION, REDUCTION, AND REPORTING OF DATA


  I.  MANUAL DATA REDUCTION

     A. Precisely measure and record all readings and output.

     B. Calculate final results using select suitable formulas and programs, (e.g. unionized
        ammonia or proper statistical tests).

     C. Manually enter at least one sample calculation onto data sheet or notebook.

     D. Double check recorded data when transferred into forms or spreadsheets.

     E. Compare raw data entries with summaries and results to assure accurate initial data entry.

   All raw data must be retained as a part of the study records. These records must be identified
   with the following information: date; sample ID; analyst or operator; species identification,
   and instrument operating conditions (if applicable). Raw data is stored in a filing system
   maintained in the Bioassay Laboratory.


 II.  COMPUTER DATA REDUCTION

   A.  Ascertain that all data used in final calculations are entered accurately: mortality, number
       normal, number alive, water quality reporting, etc.

   B.  Record appropriate and accurate information concerning sample identification, date;
       sample ID; analyst or operator; species identification, and instrument operating conditions
       (if applicable).

   C.  Identify analysis in the "Test ID Log" notebook and assign a test number that can be cross-
       referenced.

   D.  Calculate results using appropriate computer software and analyses.

   E.  Manually enter at least one sample calculation onto data sheet or notebook.

   F.  Properly interpret the computer output.

   G.  Attach all relevant test material (raw data, summaries, analyses, and reports) and place in
       appropriate binder or filing system.
                                            75

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4.9 RECORDING AND HANDLING DATA


I.  OBJECTIVE: To provide guidelines on recording data in notebooks, forms and any other
   media to ensure legibility, accuracy, validity and clarity.

II  GUIDELINES

       A.     All entries should be made legible.

       B.     All entries should be made with a black or blue ballpoint pen.

       C.     Use initials or name to indicate the originator of entries.

       D.     All blank cells with no data should contain a short slash or horizontal dash.

       E.     Abbreviations should not be used unless they are for chemical names (i.e. NaCl for
              Sodium Chloride).

       F.     Cross out errors with a single line and note initials.

Ill NOTETAKING

       A.     Notes should be recorded in a laboratory notebook.  Include date, person(s)
              responsible, project name, and signatures.

       B.     A generic note page can be found on the laptop in room 116 at: C:\Documents and
              Settings\zacharia\Desktop\Laboratory 116 titled "Notepage".

       C.     Fill in the top of the page where there is space for the date (month day and year)
              and the notetaker's name.

       D.     Record any observations such as experimental procedure, equipment, materials and
              calculations.

       E.     Attach note page  with all other relevant test data and file into a binder or folder
              with corresponding project.


IV. RECORDING DATA

   Standard units are used among organizations to ensure consistency. When appropriate, the
   mean and standard deviation should be reported.

   pH           pH units
   Salinity       ppt  or %o
   Temperature  ฐ C


                                            76

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   Dissolved Oxygenmg/L
   Ammonia     % unionized NH3
   Sulfide       H2S mg/L

V DATA MANAGEMENT

       A.     The analyst shall internally review data by checking for completeness and
              accuracy. The analyst will verify:

                   1.  Analyses are within the calibration curve range
                   2.  QC samples meet acceptance criteria
                   3.  Data meets quality  objectives
                   4.  Calculations are performed correctly

       B.     When entering data into an electronic format, analyst shall use a hardcopy to
              compare with entries or use a double entry technique.

       C.     Data entered should be backed up as frequently as needed.

       D.     Any data that is analyzed with  a computer program such as Toxcalc 5.0 or
              Microsoft Excel should be verified with randomly chosen hand calculations.
              Provide calculations on original sheet and initial.
VI HANDLING SUSPECT AND ERRONEOUS DATA

       A.    If suspect data is identified during review, it should be examined further.

       B.    Document investigation of suspect data.

       C.    If necessary, report erroneous data to laboratory director for corrective action.

Please refer to the document titled "Corrective Action" which can be found at: C:\Documents and
Settings\zacharia\Desktop\Laboratory 116\Protocols and logs\QA Manual Documents
                                           77

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4.10 STATISTICAL ANALYSIS OF DATA

I. OBJECTIVE: To statistically analyze data for determination of NOEC/LOECs via hypothesis
tests and/or LCSOs using point estimation techniques.

II. METHODS

ACUTE TOXICITY ANALYSIS

       A. Fill out data tables using data sheets specific to test method.

       B. Enter data into ToxCalc 5.0 - found in the Windows XP Programs Menu.

       C. ToxCalc automatically runs Shapiro-Wilk's test for normality and Bartlett's test for
             equality of variance. If data is not normally distributed, perform an arc-sin square
             root transformation.

       D. Run Hypothesis test function in ToxCalc

             Determining which Hypothesis test to use:

             1. Equal # of reps & data is normal - Dunnet's Multiple Comparison test

             2. Equal reps & data in non-normal - Steel's Many -One Rank test (only if there
             are at least 4 replicates per treatment).

             3. Unequal reps & data is normal - T-test with a Bonferroni Adjustment

             4. Unequal replicates & data is non-normal - Wilcoxon Rank Sum test

       E. Results of a hypothesis test are expressed in terms of the No-Observed-Effect
             Concentration (NOEC) and the Lowest-Observed-Effect Concentration (LOEC).

       F. Use the Maximum likelihood probit for the point estimation. If data do not fit this
             method, use the Trimmed Spearman-Karber method.  Linear Interpolation is used if
             neither of the former methods can be performed based on the test data. Results of
             the point estimate techniques are expressed as EC, 1C, or LC values.

       G. Verify that all data was entered into database correctly and save.

       H. Print spreadsheets with data output and graphs for future reference.
                                           78

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CHRONIC TOXICITY ANALYSIS

SURVIVAL ENDPOINT

   A. Fill out data tables using data sheets specific to test method.

   B. Calculate proportion surviving each day.

   C. Toxcalc will automatically run Shapiro-Wilk's test for normality and Bartlett's test for
       equality of variance on data. If data is not normally distributed, perform an arc-sin square
       root transformation.

   D. Run Hypothesis test function in ToxCalc

       Determining which Hypothesis test to use:

       1. Equal # of reps & data is normal - Dunnet's Multiple Comparison test

       2. Equal reps & data in non-normal - Steel's Many -One Rank test (only if
             there are at least 4 replicates per treatment).

       3. Unequal reps & data is normal - T-test with a Bonferroni Adjustment

       4. Unequal reps & data is non-normal - Wilcoxon Rank Sum test

   E. Results of a hypothesis test are expressed in terms of the No-Observed-Effect Concentration
       (NOEC) and the Lowest-Observed-Effect Concentration (LOEC).

   F. Use the Maximum likelihood probit for the point estimation. If data do not fit this method,
       use the Trimmed Spearman-Karber method.  Linear Interpolation is used if neither of the
       former methods can be performed based on the test data. Results of the point estimate
       techniques are expressed  as EC, 1C, or LC values.

   G. Verify that all data was entered into database correctly and save.

   H. Print spreadsheets with data output and graphs for future reference.

GROWTH ENDPOINT

   A. Follow the same procedures as used from survival data.

   B.  Concentrations with 100% mortality and concentrations with significant mortality are not
       included in growth analysis.

   C.  Graph the mean growth values for each concentration with ranges.

   D.  Summarize any other information indicating toxicity.


                                           79

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   4.11 HAZARDOUS MATERIAL STORAGE, DISPOSAL AND SAFETY INFORMATION

 I. OBJECTIVE: This document provides guidelines for the lifecycle management of hazardous
    materials (HM) and hazardous wastes at the Space and Naval Warfare Systems Center San
    Diego (SSC-SD).

    What is a hazardous material?
    Any material that, because of its quantity, concentration, physical or chemical characteristics,
    poses a present or potential health hazard to human health and safety or to the environment1.

    How to identify a hazardous material:
    Look on the label of the original container; if the material is hazardous it will usually indicate
    this. If there are any uncertainties refer to the Material Safety Data Sheet (MSDS) located in
    room 116.

II. PURCHASING HAZARDOUS MATERIALS

      A. Please refer to the Hazardous Materials Information notebook located in room 116 for
         specific purchasing instructions.

      B. When any new chemicals are received (either hazardous or non-hazardous) notify the
         Safety office at X3-3 873.

Ill  STORAGE AND MANAGEMENT OF HAZARDOUS MATERIALS

      A. LABELING OF HAZARDOUS MATERIAL
         All hazardous material must be labeled with:

         Original Containers
         1.  Chemical name(s) or common name(s)
         2.  Manufacturer's name and address
         3.  Chemical hazards (flammable, corrosive, etc.)
         4.  HSMS barcode label (contact Safety office if missing)

         Secondary Containers
         1.  Chemical name or common name
         2.  Manufacturer's name and address
         3.  Chemical hazards (flammable, corrosive, etc.)
         4.  Date that the container was filled
         5.  Name of the owner of the HM

      B. Periodically inspect HM to ensure that there aren't any leaks and labels are intact.

      C. Give useable HM that is no longer needed to the HazMin Center, Bldg. 116, for reissue.
                                             80

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   In addition, all hazardous materials must have a MSDS on site where they are stored (electronic
   access is acceptable). MSDS's in room 116 are kept in a black notebook located near the fume
   hood in the southeast corner of the room. Please refer to the hazardous materials information
   notebook located in room 116 for MSDS explanations.
IV.  HANDLING HAZARDOUS MATERIALS

      A.  CATEGORIES OF HAZARDS

          1.  Flammables
             These are substances that have a flash point (The lowest temperature at which the
             vapor of a combustible liquid can be made to ignite momentarily in air) below 100 ฐF.
                 a.  most liquids volatile, generating flammable vapors
                 b.  vapors are irritating or toxic
                 c.  skin or eye contact can be irritating

                 examples: hexane, benzene, methanol, acetone, most paints, propane

          2.  Halogenated Solvents
             Solvents containing a halogen such as chlorine, fluorine, bromine, or iodine
                 a.  non-flammable
                 b.  volatile
                 c.  vapors are irritating or toxic
                 d.  skin or eye contact can cause burns

                 examples: trichloromethane, carbon tetrafluroide
                 Organic solvent management - concentrated wastes (essentially pure or as water
                 mixtures that have flashpoints below 140 ฐF) must not be discharged into the
                 sewer, these include alcohols, ketones, and solvents immiscible with water.

          3.  Corrosives
             Acidic or caustic materials that can cause irreversible alterations to human skin tissue
                 a.  Generally liquid, but may be granular or powdered solids
                 b.  skin or eye contact can cause severe burns
                 c.  skin contact with hydrofluoric acid may be fatal
                 d.  vapors irritating to eyes, skin and mucous membranes and are generally toxic

                 examples: nitric acid, acetic acid, ammonia, potassium hydroxide

          4.  Toxics
             Lethal dose higher than limits designated by OSHA
                 a.  May be solid, liquid or gas
                 b.  May exhibit flammability or corrosivity

                 examples: heavy metals (cadmium, lead, mercury), toluene, oils and greases,
                 adhesives, detergents, paints

                                               81

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5.  Compressed Gases
   Gases stored at pressures above one atmosphere (1 atm)
       a.  May be toxic (chlorine, ammonia)
       b.  May be flammable (propane, oxygen)
       c.  May be inert (nitrogen, argon, helium)
       d.  May be cryogenic (liquid nitrogen)

6.  Oxidizers
   Materials that readily contribute oxygen to a reaction or combustion
       a.  Unstable and reactive
       b.  May be flammable
       c.  Are corrosive
       d.  May be explosive
       e.  skin or eye contact can cause burns
       f.  fumes from reaction, decomposition or fire may be toxic and cause irritation to
          skin and eyes.

       examples: hydrogen peroxide, benzoyl peroxide, t-butyl peracetate

7.  Water Reactives
   Materials that react violently when exposed to  water
       a.  Can react with moisture in air
       b.  Can react with oxygen containing liquids such as alcohols and ketones
       c.  Difficult to extinguish if ignited
       d.  May be explosive

       examples: lithium, sodium, sodium hydroxide, magnesium nitride, calcium carbide,
       nitric acid

8.  Pyrophorics
   Materials that spontaneously combust with exposure to air
       a.  May be liquid or powder
       b.  May have violent reactions
       c.  Skin contact may cause burns
       d.  Fumes from  fire or reaction may be irritating or toxic
       e.  Extinguished fire may re-ignite
   examples: diethyl zinc, trimethyl aluminum, elemental phosphorus

9.  Explosives
   Materials that release a tremendous amount of energy in the form of heat, light, and
   expanding pressure in a very short period of time
       a.  May be sensitive to shock or heat
       b.  Many are flammable
       c.  Explosions can produce projectiles or pressure shock waves

       examples: TNT, picric acid, nitroglycerine, ammonium nitrate


                                     82

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     B.  PROTECTION FROM HAZARDS

            1.  Eliminate the possibility of exposure through material substitution
            2.  Use engineering controls, such as fume hoods, to eliminate exposure
            3.  Use personal protective equipment (gloves, goggles and aprons)
V.  HAZARDOUS WASTE DISPOSAL

       Hazardous waste is any discarded, excess or spilled material that is solid, liquid or gas and
       meets the definition of a hazardous material1. It is either Characteristic (toxic, reactive,
       ignitable or corrosive) or Listed (appears on a specific EPA or state list).

       The following are prohibited sewer discharges:

      -   Flammable or explosive substances - flashpoint < 140 ฐF
      -   Corrosives - pH <5.0 or > 12.5
         Hazardous Wastes
      -   Trucked pollutants (from offsite)
         Substances that may obstruct flow (solid or viscous)
         Odorous wastes
         Uncontaminated ground, storm and surface water
      -   Sludge
      -   Heated wastestreams >150 ฐF
         Radioactive wastes
      -   Greases and oils (that will cause interference or pass through treatment system to ocean)

      A batch discharge request may be made to discharge small and large quantities of wastewater
      by contacting Brett Radsliff (code 20384) at X3-1437.

      A.  DETERMINE IF WASTE IS HAZARDOUS

              1.  If waste needs to be analyzed, contact Mary Anne Flanagan at X3-6363.

      B.  DISPOSAL

              1.  Schedule a pick up by  calling X3-7464  as soon as possible after generation.
                   a.  Complete all paperwork prior to pickup
                      - HW Disposal Request Form (Required for all HW)
                      - HW Profile Sheet (not req'd for  materials in original containers)
                      -CopyofMSDS's
                   b.  Forms  available in Hazardous Materials Information notebook or are
                      accessible on line at:
                      https://iweb.spawar.navy.mil/services/sti/publications/inst/forms/

              2.  If you need to accumulate HW

                                             83

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                    a.  Contact Mary Anne Flanagan at X3-6363, so she can visit work site and
                       determine which type of accumulation you require.
                    b.  She will complete an accumulation area designation form.
                    c.  Two methods of HW accumulation;
                       i. Satellite Accumulation (accumulate for no longer than 9 months, 55
                          gallon limit, under control of designated operator)
                       ii. Standard Hazardous Waste Accumulation (accumulate for no longer
                          than 45 days, no volume restriction, under the control of code's
                          representative)
                    d.  Both methods require analyst to fill out a HW label and affix to containers
                    e.  When ready to turn in HW, call X3-7464 and complete all paperwork prior
                       to pickup:
                       i. HW Disposal Request Form (Required for all HW)
                       ii. HW Profile Sheet (not required for materials in original containers)
                      iii. Copy ofMSDS's
                    f.  Forms available in Hazardous Materials Information notebook or are
                       accessible on line at:
                       i. https://iweb.spawar.navy.mil/services/sti/publications/inst/forms/
                    g.  If Disposal Containers are needed contact Louie Don or Rudie at X3-7464.

               3.  OTHER HW DISPOSAL METHODS

                    h.  Fluorescent light tubes - call the HW Office, X3-7464, and they will pick
                       them up.
                    i.  Printer toner cartridges recycling - Bldg. 116 A-33 Wing 6, Ground Floor.
                       Each cartridge must be in original box and re-sealed.
                    j.  Batteries - from pagers, phones, flashlights, clocks, etc. are collected in a
                       bucket located in Bldg. Ill, 2nd floor, northeast corner.
                    k.  Glass - If broken, dispose of in a broken glass container, to obtain more
                       glass disposal boxes, call Joel Baumbaugh at X3-5030.

VI.  HAZARDOUS MATERIALS SPILLS
      A.  If material spill is unknown, a large spill, a danger to personnel, or too large to contain or
          clean up
             1.  evacuate the area
             2.  report to X9-911 (Federal Fire Department) and X3-5024

      B.  If spill is safe and type of spill is known
             1.  Contain the spill using proper protective equipment and spill kit material
             2.  Identify hazards through use of the MSDS
             3.  Absorb the spill with appropriate spill pads or absorbent material
             4.  Bag and  Tag spill and material used
             5.  Call X3-5024 for disposal coordination
   Report all spills to the Safety and Environmental Office X3-5024.
   1 Protocol  derived from  SSC-SD Document 4110.1
                                              84

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4.12 COUNTING SPERM WITH A HEMOCYTOMETER

Preparation

    1.  Mix sperm by agitating the tube with a vortexer. Add about 0.025 ml of sperm to a 100 ml beaker
       containing 50 ml of 15 ฐC dilution water and stir with a Pasteur pipette. Cover and keep at 15 ฐC,
       use within 1.5 hours.

    2.  Slowly withdraw a subsample of semen (i.e. 0.5 ml), dispense it into a 1% glacial acetic acid
       solution (killing solution) in an Erlenmeyer flask (i.e. 5 ml of 10% acetic acid in 45 ml of filtered
       seawater). Rinse residual semen from pipet several times by filling and emptying into flask.  Cover
       flask with parafilm and mix thoroughly by repeated inversion. *(0.5 ml of semen in 50 ml of 1%
       acetic acid is a 100-fold dilution (50 / .05 = 100)).

    3.  Transfer well-mixed acetic acid/sperm samples to a hemocytometer and wait 15
       minutes to settle before counting.

Cell Counting

(Adapted from: http://www.ruf.rice.edu/~bioslabs/methods/microscopy/cellcounting.html)

A device used for cell counting is called a counting chamber. The most widely used type  of chamber is
called a hemocytometer, since it was originally designed for performing  blood cell counts.
To prepare the counting chamber the mirror-like polished surface is carefully cleaned with lens paper. The
coverslip is also cleaned. Coverslips for counting chambers are specially made and are thicker than those
for conventional microscopy, since they must be heavy enough to overcome the surface tension of a drop of
liquid. The coverslip is placed over the counting surface prior to putting on the cell suspension. The
suspension is introduced into one of the V-shaped wells with a pasteur or other type of pipet. The area
under the coverslip fills by capillary action. Enough liquid should be introduced so that the mirrored
surface is just covered. The charged counting chamber is then placed on the microscope stage and the
counting grid is brought into focus at low power.
                                               85

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PROTOCOL FOR COUNTING SPERM WITH A HEMOCYTOMETER cont'd
                      I ••••••••••!••••• I
                      (•••••••••••••••••I-
                           •••••••••••••••I
                      Illllllllllllllllllll
                      •	••••illinium
                      Illllllllllllllllllll
                      Illllllllllllllllllll
                      •	• •••illinium
                           •••••••••••••••i
One entire grid on standard hemocytometers with Neubauer rulings can be seen at 40x (4x objective). The
main divisions separate the grid into 9 large squares (like a tic-tac-toe grid). Each square has a surface area
of one square mm, and the depth of the chamber is 0.1 mm. Thus the entire counting grid lies under a
volume of 0.9 mm-cubed.

Cell suspensions should be dilute enough so that the cells do not overlap each other on the grid, and should
be uniformly distributed. To perform the count, determine the magnification needed to recognize the
desired cell type. Now systematically count the cells in selected squares so that the total count is  100 cells
or so (number of cells needed for a statistically significant count). For large cells this may mean counting
the  four large corner squares and the middle one. For a dense suspension of small cells you may wish to
count the cells in the four 1/25 sq. mm corners plus the middle square in the central square. Always decide
on a specific counting pattern to avoid bias. For cells that overlap a ruling, count a cell as "in" if it overlaps
the  top or right ruling, and "out" if it overlaps the bottom or left ruling.

To get the final count in cells/ml, first divide the total count by 0.1 (chamber depth) then divide the result
by the total surface area counted. For example if you counted 125 cells in  each of the four large corner
squares plus the middle, divide 125 by 0.1, then divide the result by 5 mm-squared, which is the total area
counted (each large  square is  1 mm-squared). 125/ 0.1 = 1250.
                                                86

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PROTOCOL FOR COUNTING SPERM WITH A HEMOCYTOMETER cont'd

1250/5 = 250 cells/mm-cubed. There are 1000 mm-cubed per ml, so you calculate 250,000 cells/ml.
Sometimes you will need to dilute a cell suspension to get the cell density low enough for counting. In that
case you will need to multiply your final count by the dilution factor.

Using the equation on the egg and sperm count sheet, determine concentration of sperm. Using either table
5 in EPA manual or C1V1=C2V2, determine volume of concentrated sperm stock needed to make a 500:1
solution.
                                              87

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4.13 COUNTING MUSSEL/OYSTER LARVAE USING AN INVERTED MICROSCOPE
TESTING FACILITY:  SPAWAR SYSTEMS CENTER
                       BIOASSAY LABORATORY (RM 116)
                       CODE 71750
                       53475 STROTHE RD.
                       BLDG.  111
                       SAN DIEGO, CA 92152
I.      OBJECTIVE: This method allows for estimation of the chronic toxicity of effluent and receiving
       waters to the embryos and larvae of bivalve mollusks. After larvae have been exposed to test
       solutions for the specified amount of time they should be preserved in 1.0 mL of 37%
       (concentrated) buffered formalin so that each sample has a final formalin concentration of 4%.
       Larvae should ideally be examined within one week of preservation.

II      NECESSARY MATERIALS AND SUPPLIES

          Inverted microscope - for inspecting gametes and counting embryos and larvae (Located in
          room 152, Bldg.  Ill)
          Counter, two unit, 0-999 - for recording counts of embryos and larvae
          Data record sheets

III     METHODS

          A.  Turn on microscope and adjust lamp to reasonable brightness.

          B.  Ensure that the magnification is set to 40X (4x objective and lOx oculars).

          C.  Carefully unscrew cap and place vial on the center of the mechanical stage. If vial has
              been shaken at all, contents must be allowed to settle before counting.

          D.  Using the mechanical stage, begin at the upper left corner of one end of the vial and rotate
              the stage so that the field of view moves downward  as you count all larvae, scoring them as
              normal or abnormal. When you've come to the bottom edge of the vial, focus on an
              embryo or particle that lies  at the edge of the field of view and move stage so that the
              particle has moved from one end to the other (i.e. right to left), bringing in view only
              larvae that have not been counted. Count that field of view, and repeat this procedure until
              the entire vial has been counted.

          E.  Use the fine focus to view larvae that may be at different depths near the bottom of the
              vial. This is particularly important around the edges of the vial, where objects can appear
              distorted. It is important to count all larvae in the vials, so take time ensuring that this is
              done correctly.

IV     DISTINGUISHING BETWEEN NORMAL AND ABNORMAL LARVAE

          A.  Larvae that were live before preservation with completely developed D-hinged shells
              should be marked as normal. Larvae that appear slightly deformed, but have achieved the

-------
PROTOCOL FOR ASSESSING MUSSEL/OYSTER LARVAE USING AN INVERTED MICROSCOPE
(Mytilus galloprovincialis or Crassostrea gigas) Cont'd

              D-hinge stage should still be counted as normal unless they are not clearly D-shaped.  If
              the shells are empty, they are considered dead and this should be noted as abnormal.

           B. Mortality will be assessed by comparing the number of normal and abnormal larvae in the
              test vials with a set of initial embryo vials that were preserved at the beginning of the test.
                                              89

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5.0 LOGS AND DATA SHEETS




5.1 ECHINODERM EMBRYO-LARVAL DEVELOPMENT TEST - WATER QUALITY DATA




                  WATER QUALITY DATA - 96 Hour Echinoderm Embryo Development Test
Marine Chronic Bioassay
Project:
Sample ID:
Test No.:
Concentration
%











Salinity
(PPt)
0








24









48









Technician Initials: WQ Readings:
Dilutions made by:
Animal Source/Date Received:
Comments: 0 hrs:


72








96








Test Species:
Start Date/Time:
End Date/Time:
Temperature
PC)
0








24








48








0 24 48 72 96











72








96








Water Quality Measurements
S. purpuratus



Dissolved Oxygen
(mg/L)
0








24








48








72








96









PH
(pH units)
0








24








48








72








96











24hrs:
48 hrs:
72 hrs:
QC Check:



Final Review:



                                                90

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5.2 BIVALVE EMBRYO-LARVAL DEVELOPMENT TEST - WATER QUALITY DATA




                    WATER QUALITY DATA - 48 Hour Bivalve Embryo Development Test
Marine Chronic Bioassay
Project:
Sample ID:
Test No.:
Concentration
(%)
Lab Control
Brine Control
6.25
12.5
25
50




Salinity
(PPt)
0








24








Technician Initials: WQ Readings:
Dilutions made by:
48







Temperature
(ฐC)
0







24







0 24 48






48







Water Quality Measurements
Test Species:
Start Date/Time:
End Date/Time:
Dissolved Oxygen
(mg/L)
0








24







48










PH
(pH units)
0







24







48








Animal Source/Date Received:
Comments: 0 hrs:
24 hrs:
48 hrs:
QC Check:





Final Review:
                                                91

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5.3 EMBRYO-LARVAL DEVELOPMENT TEST CALCULATIONS
Embryo-Larval Development Test - SPAWNING CHECKLIST & CALCULATIONS
Batch ID:	Spawn/Test Date:	Test Species	
Analyst:	
Task
Spawning Inducement Initiated
Spawning Begins
Females/Males Isolated in Incubator
Fertilization Initiated
Fertilzation Terminated/eggs rinsed
Embryo Counts
Embryo addition to vials
Time







Embryo Counts:
Embryo Stock #1:
Embryo Stock #2:
Embryo Stock #3:
Mean =
Mean =
Mean =
uL* 1000uL/mL =
uL* 1000uL/mL =
uL* 1000uL/mL =
Adjust selected embryo stock to 2000 embryos/ml.  Confirm density:
Selected Stock:    ,    ,     Mean =    /    uL * lOOOuL/mL =
Add 100 |il of 2000 embryo/ml stock to obtain 20 embryos/ml in test vials.
Notes:
  cells/mL
  cells/mL
  cells/mL
cells/mL
                                          92

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5.4 EMBRYO-LARVAE DEVELOPMENT TEST RESULTS RAW DATA SHEET
Embryo L
Project:
Sample ID:
Test No.:
.arval Bioassay



Random #
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
QC Check:
Number Counted





































Test Species:
Start Date:
End Date:
Number Normal



































Final Review:
96-Hour Development



Technician Initials






































                                 93

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Embryo L
Project:
Sample ID:
.arval Bioassay


Random #
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
QC Check:
Number Normal





































Test Species:
Start Date:
End Date:
Number Abnormal



































Technician Initials



































Final Review:
48-hour Development




5.5 DINOFLAGELLATE PMT COUNT SHEET FOR COPPER REFERENCE TOXICANT
TEST
                                 94

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       Qwiklite (Sealite) Data for Ceratocorys horrida - Copper Reference Toxicant Test
TEST ID:
Backround noise
Mean:
Control -
Mean:
Cone.
Cone.
Mean:
Cone.
Oppb
25 ppb
50 ppb
                               Cone.
Rep
1
2
3
4
PMT count




Rep
1
2
3
4
5
PMT count





                                        Mean:
                                        Cone.
Rep
1
2
3
4
5
PMT count





Mean:
                                        Mean:
                                        Cone.
                                        Mean:
Rep
1
2
3
4
5
PMT count





5.
Rep
1
2
3
4
5
M&bNOFJ
PMT count






LAGELLAfE PMT COUNT SHEET
                                                         DATE:
                                              TOXCALC TEST ID:'
100 ppb
Rep
1
2
3
4
5
PMT count





                                         200 ppb
Rep
1
2
3
4
5
PMT count





                                         400 ppb
Rep
1
2
3
4
5
PMT count





                                            95

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                     Qwiklite (Sealite) Data for Ceratocorys horrida
                                                        DATE:
  TEST ID:
  Backround noise
Rep
1
2
3
4
PMT count




  Mean:
  Control -
Rep
1
2
3
4
5
PMT count





  Mean:
  Cone/Sample ID:
Rep
1
2
3
4
5
PMT count





  Mean:
  Cone/Sample ID:
Rep
1
2
3
4
5
PMT count





  Mean:
  Cone/Sample ID:
Rep
1
2
3
4
5
PMT count





  Mean:
U.C TEST ID:
Cone/Sample ID:
Rep
1
2
3
4
Mean:
PMT count





Cone/Sample ID:
Rep
1
2
3
4
5
Mean:
PMT count






Cone/Sample ID:
Rep
1
2
3
4
5
Mean:
PMT count






Cone/Sample ID:
Rep
1
2
3
4
5
Mean:
PMT count






Cone/Sample ID:
Rep
1
2
3
4
5
PMT count





Mean:
DINOFLAGELLATE PMT COUNT ANALYSIS
                                             96

-------
SPAWAR - C. horrida 24 hour exposure test
Date
Cone. (%)
0





15.625





31.25





62.5





125





250




PMT Counts



































Mean PMT Counts





#DIV/0!




#DIV/0!





#DIV/0!





#DIV/0!





#DIV/0!





#DIV/0!
SD





#DIV/0!




#DIV/0!





#DIV/0!





#DIV/0!





#DIV/0!





#DIV/0!
CV (%)





#DIV/0!




#DIV/0!





#DIV/0!





#DIV/0!





#DIV/0!





#DIV/0!
% control





100




#DIV/0!





#DIV/0!





#DIV/0!





#DIV/0!





#DIV/0!
Normalized
SD





#DIV/0!




#DIV/0!





#DIV/0!





#DIV/0!





#DIV/0!





#DIV/0!
                                                 97

-------
5.7 NEANTHES 28 DAY WATER CHEMISTRY DATA SHEET
28-Day Marine Sediment Bioassay
Static-Renewal Conditions
Client:
Sample ID:
Test Day
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28


Salinity
(PPt)





























Temperature
(ฐC)





























Dissolved
Oxygen (mg/L)





























PH
(units)





























Water Quality Measurements
Test Species:
Start Date/Time:
End Date/Time:
Fed





























Water
Change





























Technician
Initials
































Comments





























QC Check: Final Review:
                                   98

-------
5.8 NEANTHES SURVIVAL DATA SHEET
     Marine Sediment Bioassay
        Client:
     Project ID:
              Organism Survival

  Test Species: N. arenaceodentata
Start Date/Time: 	
 End Date/Time:
Sample ID















Initial No.















No.
Recovered















Pan
Weight
(mg)















Pan + Org.
Weight
(mg)
WET















Pan + Org.
Weight
(mg)
DRY















Technician
Initials















                 QC Check:
                                                   Final Review:
                                         99

-------

-------
5.9 AMPHIPOD 10 DAY WATER CHEMISTRY DATA SHEET
   10-Day Marine Sediment Bioassay
   Static Conditions
                   Water Quality Measurements
   Client:

   Sample ID:
 Test Species:  E. estuarius

Start Date/Time: 	

 End Date/Time:

Test Day
0
1
2
3
4
5
6
7
8
9
10
Salinity
(PPt)











Temperature
(ฐC)











Dissolved
Oxygen (mg/L)











PH
(units)











Technician
Initials











Comments











    QC Check:
   Final Review:
                                                      101

-------
5.10 AMPHIPOD SURVIVAL DATA SHEET
  Marine Sediment Bioassay
Organism Survival
Client:
Project ID:



Sample ID















Initial No.















Test Species:
Start Date/Time:
End Date/Time:
No.
Recovered















Technician
Initials















E. estuarius



                  QC Check:
                                      Final Review:
                                 102

-------
5.11 ACUTE FISH/MYSID SURVIVAL SHEET
        ACUTE FISH / MYSID SURVIVAL AND WATER QUALITY
                            DATA
Marine Acute Bioassay
Static-Renewal Conditions
Project:
Sample ID:
Test No.:
Concentration
PPb
Lab Control



50



100



200



400



800







Initial Counts
QC'd by:



Re
P
A
B
C
D
A
B
C
D
A
B
C
D
A
B
C
D
A
B
C
D
A
B
C
D
A
B
C
D
Number of Live
Organisms
0
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5





24





























48




























72




























96




























Test Species:
Start Date/Time:
End Date/Time:
Salinity
(PPซ)
0




























24




























48
1
f


1
f


1
f


1
f


1
f


'
f


'
f



Animal Source/Date Received:
72




























96































Water Qi
STes
Counts:
Readings:
Dilutions made by:
Temperature
0




























24




























Age at Initiation:
48
1
f


1
f


1
f


1
f


1
f


'
f


'
f


72




























96




























Dissolved Oxygen
mg/L)
0




























24





























48
1
f


1
f


1
f


1
f


1
f


'
f


'
f



Comments: i = initial reading in fresh test solution, f = fina reading in test chamber priorto renewal
QC Check:

Organisms fed priorto initiation, circle one ( y / n)
Tests aerated? Circle one ( y / n) if yes, sample ID(s): Duration:
Aeration source:
Fin
72




























96




























AM:
PM:
al Review:
jality Measurements
t Organism Survival
Tech Initials
0



24



48



72



96





0




























24




























PH
units)
48
1
f


1
f


1
f


1
f


1
f


'
f


'
f


72




























96





























Feeding Times
0


24


48


72


96



                             - 103 -

-------
5.12 DINOFLAGELLATE MAINTENANCE LOG
                 Dinoflagellate Maintenance Log
Date











































Media
ID











































Salinity
(ppt)











































pH











































Temp
(ฐC)











































Date of
next
media
split











































Date of Culture Being Split
(check mark indicates that culture has
been split at that date)

Q L. polyedrum
Q C. horrida
Q P. noctiluca
Q G. gridleyii
Q P. lunula
Q P. fusiformis
Q L. polyedrum
Q C. horrida
Q P. noctiluca
Q G. gridleyii
Q P. lunula
Q P. fusiformis
Q L. polyedrum
Q C. horrida
Q P. noctiluca
Q G. gridleyii
Q P. lunula
Q P. fusiformis
Q L. polyedrum
Q C. horrida
Q P. noctiluca
Q G. gridleyii
Q P. lunula
Q P. fusiformis
Q L. polyedrum
Q C. horrida
Q P. noctiluca
Q G. gridleyii
Q P. lunula
Q P. fusiformis
Q L. polyedrum
Q C. horrida
Q P. noctiluca
Q G. gridleyii
Q P. lunula
Q P. fusiformis
Q L. polyedrum
Q C. horrida
Q P. noctiluca
Q G. gridleyii
Q P. lunula
Q P. fusiformis
Normal
Temp, and
Light
Regime?
(18ฐC-12h
light/12h dark)


a Yes

a No



a Yes

a No



a Yes

a No



a Yes

a No



a Yes

a No



a Yes

a No



a Yes

a No


                             - 104-

-------
5.13 Brine Dilution Worksheet
 Marine Chronic Bioassay

 Project:      	

 Sample ID:   	
                        Brine Dilution Worksheet
 Salinity of Effluent

 Salinity of Brine

 Target Salinity

 Test Dilution Volume
 Salinity Adjustment Factor:
 (TS-SE)/(SB-TS) =
   TS = target salinity
   SE = salinity of effluent
   SB = salinity of brine
                              Effluent
   QC Check:
                 Analyst:

                Test Date:

               Test Type:



        Date of Brine used:

 Alkalinity of Brine Control:



Brine Control
                             Dl Volume
              Brine Control
                                                           0.0
                          Total Brine Volume Required (ml): I
                 0.0
             Final Review:
mg/Las CaCOS
Concentration
%
Control
6.25
12.5
25
50

Effluent
Volume
(ml)
NA
12.5
25.0
50.0
100.0

Salinity
Adjustment
Factor
NA





Brine
Volume
(ml)
NA





Dilute
to:
(ml)
200
200
200
200
200
200
                                                                        200
                                           - 105-

-------
                         Brine Dilution Worksheet Summary
STEP 1: Calculate the Effluent Salinity Adjustment Factor
Salinity Adjustment Factor:
           TS-SE      TS = target salinity
           SB - TS      SE = salinity of effluent
                       SB = salinity of brine
                                  Ex:   34-10
                                       71 -34
                                 24    =  0.65
                                 37
Concentration %
     Control
      6.25
      12.5
       25
       50
      61*
Effluent
Volume
  (mL)
   NA
  31.3
  62.5
  125
  250
  303
        Salinity
       Adjustment
           NA
          0.65
          0.65
          0.65
          0.65
          0.65
 Brine
Volume
 (mL)
  NA
  20.3
  40.6
  81.2
 162.5
  197
Dilute to
  to:
  (mL)
  500
  500
  500
  500
  500
  500
                Dl Volume
  Brine Control
214
            0.92
  197
  500
STEP 2: Calculate Effluent Volumes
Multiply 500 by each cone, in decimal form
Ex: 500x0.0625 = 31.3

STEP 3: Calculate Brine Volumes
Multiply the effluent volumes by the
salinity adjustment
Ex: 31.3x0.65 = 20.3

STEP 4: Determine the Highest
Obtainable Test Concentration *
Divide 500 by 1 + the salinity adjustment
Ex: 500/1.65 = 303
Then divide 303 by 500 to determine a %
303/500 = 61%
STEP 5: Calculate the Brine Control Salinity Adjustment Factor

Brine Control Calculation:       TS - 0        Ex:     34 - 0   =  0.92
                           SB-TS
                               71 -34
STEP 6: Calculate the Dl Volume Required for the Brine Control
Divide the highest brine volume from above by the brine control salinity adjustment factor
                      Ex:
                             197
                             0.92
                     =  214
                                              - 106-

-------
6.0 REFERENCES

ASTM 2000. Annual Book of ASTM Standards. Vol. 11.05. American Society for Testing and
Materials. 2000.

EPA 1994. Methods for Assessing the Toxicity of Sediment-associated Contaminants with
Estuarine and Marine Amphipods. EPA 600/R-94/025. June 1994

EPA 1995. Short-Term Methods for Estimating the Chronic Toxicity of Effluents and Receiving
Waters to Marine and Estuarine Organisms. First edition. EPA/600/R-95/136. August 1995.

EPA 1993. Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to
Freshwater and Marine Organisms. Fourth Edition. EPA/600/4-90/027F. August 1993.

EPA 2002. Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to
Freshwater and Marine Organisms. Fifth Edition. EPA/82l/R-02/012. October 2002.

Tidepool Scientific 2002. Toxcalc User's Guide. Version 5.0. Tidepool Scientific Software.
McKinleyville, CA, USA.
                                       - 107-

-------
             SFWAR
              Systems Center
                  PACIFIC
            Bioassay Laboratory
         Quality Assurance Manual
                 Version V
                May 11, 2011
Approvals:
Laboratory Director
Gunther Rosen
                                  Date:
Code 71750 Branch Head
D. Bart Chadwick
                                  Date:
                  53475 Strothe Road
                 Bldg. Ill Room 116
                San Diego, CA 92152-5000
              (619)553-0886  • (619)553-2766

-------
                        Table of Contents

Section                                                        Page

INTRODUCTION	4

1.0 LABORATORY ORGANIZATION & PERSONNEL RESPONSIBILITIES	5

  1.1 LABORATORY ORGANIZATION	5
  1.2 PERSONNEL RESPONSIBILITIES	5
  1.3 EXPERTISE AND PROFICIENCY	6

2.0 FACILITIES AND EQUIPMENT	8

  2.1 FACILITIES	8
  2.2 EQUIPMENT	11

3.0 QUALITY ASSURANCE OBJECTIVES	12

  3.1 QA/QC AND TOXICITY DEFINITIONS*	12
  3.2 DATA ACCURACY, PRECISION, COMPLETENESS, REPRESENTATIVENESS,
  AND COMPARABILITY	16

4.0 SAMPLE AND TEST ORGANISM HANDLING	18

  4.1 RECEIVING SAMPLES	18
  4.2 HOLDING SAMPLES	19
  4.3 SAMPLE HANDLING AND CHAIN OF CUSTODY	19
  4.4 ORGANISM AND SAMPLE LOG	22
  4.5 SAMPLE COLLECTION RECORD LOG	23
  4.6 RECEIVING/HOLDING TEST ORGANISMS	24

5.0 HAZARDOUS MATERIAL (HM) STORAGE, DISPOSAL AND SAFETY
CONSIDERATIONS	25

  5.1 PURCHASING HAZARDOUS MATERIALS	25
  5.2 STORAGE AND MANAGEMENT OF HAZARDOUS MATERIALS	25
  5.3 HANDLING HAZARDOUS MATERIALS (SAFETY)	25
  5.4 HAZARDOUS WASTE DISPOSAL	25
  5.5 HAZARDOUS MATERIALS SPILLS	26

6.0 CALIBRATION, USE, AND TROUBLESHOOTING OF
INSTRUMENTATION	27

  6.1 CALIBRATION OF BASIC LABORATORY INSTRUMENTATION	27
  6.2 LABORATORY STANDARDS	28

7.0 CLEANING GLASSWARE/PLASTICWARE	29

8.0 QUALITY CONTROL SAMPLES	30

  8.1 NEGATIVE CONTROLS	30
  8.2 REFERENCE TOXICANT TESTS	30

9.0 PREVENTIVE MAINTENANCE PROCEDURES FOR LABORATORY
EQUIPMENT AND CHEMICALS	31
  9.1 PREVENTIVE MAINTENANCE FOR EQUIPMENT	31
  9.2 PREVENTIVE MAINTENANCE FOR CHEMICALS	35

-------
10.0 ACQUISITION, REDUCTION AND REPORTING OF DATA	36

  10.1 ACQUISITION	36
  10.2 DATA REDUCTION AND REPORTING	36

11.0 REPLICATION AND TEST SENSITIVITY	37

12.0 CORRECTIVE ACTION	38
  12.1 DETERMINING THE PROBLEM	38
  12.2 RESOLUTION	39

13.0 AUDITS AND QUALITY ASSURANCE REPORTS	40

  13.1 INTERNAL AUDITS	40
  13.2 EXTERNAL AUDITS	40
  13.3 QUALITY ASSURANCE REPORTS	40

REFERENCES	41

-------
INTRODUCTION

Space and Naval Warfare Systems Center Pacific (SSC Pacific) is responsible for
development of the technology to collect, transmit, process, display and, most critically,
manage information essential to U.S. Navy operations.  The mission of the
Environmental Sciences and Applied Systems Branch (Code 71750) at SSC Pacific is to
provide cost effective technology for Navy environmental compliance and restoration
through ecological risk assessment and restoration research, sediment characterization
and management technology development, and environmental sensor and instrument
development. The use of both standardized and innovative bioassays for evaluating
effluents, receiving water, sediments, and other environmental samples have been a
critical component to research within the branch for a number of years. The potential for
toxicity data generated by the Bioassay Laboratory to be used in modification of Navy
discharge permits as well as uses towards other regulatory issues led to the effort to
obtain certification by the state of California's Environmental Laboratory Accreditation
Program (ELAP) and by the State of Washington Department of Ecology.

Code 71750 consists of approximately 40 personnel, and is made up of biologists,
chemists, oceanographers, and engineers, two-thirds of which have advanced degrees
with a general emphasis in environmental science.  Typically, a small number of the staff
are directly involved in studies requiring toxicity testing, and the Bioassay Laboratory
itself is generally run by two to three people, the laboratory director and one to two
analysts, due to the relatively small scale of the projects being conducted.  The Bioassay
Laboratory at SSC-Pacific is not a production laboratory, yet is dedicated to producing
results of the highest quality.

This manual presents the Bioassay Laboratory's quality assurance plan.  It includes
laboratory procedures with emphasis on Quality Assurance/Quality Control (QA/QC)
requirements based on EPA guidelines for aquatic bioassays,  specifically whole effluent
toxicity (WET) testing as intended for compliance with National Pollution Discharge
Elimination System (NPDES) permits.  This manual is intended for Bioassay Lab staff
and any other relevant parties interested in understanding the laboratory's approach to
quality assurance.

-------
1.0 LABORATORY ORGANIZATION & PERSONNEL RESPONSIBILITIES

1.1 LABORATORY ORGANIZATION

TESTING FACILITY: SSC PACIFIC
                      BIO ASS AY LABORATORY (RM 116), CODE 71750
                      53475 STROTHE RD.
                      BLDG. 111
                      SAN DIEGO, CA 92152

Space and Naval Warfare Systems Center Pacific (SSC-Pacific) is responsible for
development of the technology to  collect, transmit, process, display and, most critically,
manage information essential to U.S. Navy operations.  The mission of the
Environmental Sciences and Applied  Systems Branch (Code 71750) at  SSC-Pacific is to
provide cost effective technology for Navy environmental compliance and restoration
through ecological risk assessment and restoration research, sediment characterization
and management technology development, and environmental sensor and instrument
development.  The use of both standardized and innovative bioassays for evaluating
effluents, receiving water, sediments,  and other environmental samples  have been a
critical component to research within  the branch for a number of years.  The potential for
use of toxicity data generated by the Bioassay Laboratory to modify Navy discharge
permits and make other  regulatory changes led to application for certification by the state
of California's Environmental Laboratory Accreditation Program  (ELAP) and by the
State of Washington Department of Ecology.

Code 71750 consists of  approximately 40 personnel, and is made up of  biologists,
chemists, oceanographers, and engineers, two-thirds of which have advanced degrees
with a general emphasis in environmental science.  Typically, a small number of the staff
are directly involved in  studies requiring toxicity testing. The Bioassay Laboratory is
typically operated by two to three  people, and includes the laboratory director and one or
two analysts.  When needed, assistance in the lab is provided by other branch members.
The Bioassay  Laboratory at SSC-Pacific is not a production laboratory,  and projects are
generally manageable without additional support.

1.2 PERSONNEL RESPONSIBILITIES

The Bioassay  Lab staff is responsible for conducting acute and/or chronic toxicity testing
as well as sample handling, and laboratory equipment calibration and maintenance. The
work performed by this  group consists of acquisition, management, analysis,
interpretation  and presentation of toxicological data. The group utilizes  several tools to
manage, analyze, and present data (Toxcalc 5.0, SigmaPlot, SigmaStat,  Microsoft Excel,
Microsoft Word, and Microsoft PowerPoint). These analyses are reported to the
appropriate principal investigator or to the project sponsor.

There is considerable overlap with respect to individual responsibilities  within the
toxicology group.  There are two primary positions, however, including the

-------
laboratory/technical director and one or two analysts. Additional support is provided by
other members of Code 71750 where needed.  The roles of these positions are briefly
described below:

         A) Laboratory/Technical Director

         Responsibilities of this position are overseeing laboratory operations,
         establishing quality assurance and quality control (QA/QC) policies and
         enforcing them, conducting toxicity testing and data analysis, verifying the
         quality of the data and taking corrective action when needed, interfacing with
         other scientists and project sponsors, attending project-related meetings, report
         writing, and presentation of project results. For this position, an advanced
         degree in the biological sciences and several years of related experience is
         preferred.

         B) Analyst

         An analyst is responsible for sample handling, preparation, and disposal, test
         organism maintenance, carrying out toxicity testing, data analysis, record
         keeping, calibration and troubleshooting of instruments, inventory, and general
         implementation of QA/QC policy. This position requires at least a bachelor's
         degree in biological or related sciences and at least one year of related
         experience.

         C) Additional Branch Support

         There are several other scientists in the branch with extensive capabilities that
         assist in one way or another with the functioning of the Bioassay Lab (e.g.
         sampling or sample handling, chemical analysis of reference toxicant stock
         solutions, assistance with larger scale testing, etc.).
1.3 EXPERTISE AND PROFICIENCY

To ensure a high level of professionalism, the staff is expected to be at the forefront of
scientific research in their respective field. All scientists in code 71750 have a minimum
of a bachelor's degree, while approximately 67% have advanced degrees (e.g. M.S.,
Ph.D.).  A number of resources for professional development are also available at SSC-
Pacific. Employees may access SSC-Pacific's Marine Environmental Support Office
(MESO) in Bldg. Ill for a large collection of technical reports and scientific journals.
SSC-Pacific also has a technical library located at Topside in building 81 and the research
library at Scripps Institution of Oceanography is nearby.  Subscriptions to journals in the
areas of toxicology and marine biology/chemistry, memberships to professional
associations, internet links to scientific journals, are other avenues for increasing
technical knowledge.  Employees  are also encouraged to attend and present at seminars
and departmental and division  meetings to extend communication and promote

-------
interdepartmental organization.  Career development via additional training/certification
programs is encouraged. General training in laboratory safety and hazardous materials
handling, storage and disposal are provided to the staff via seminars and training
conducted at SSC-Pacific.
                                      Division Head
                                        Code 717
                             Advanced Systems & Applied Sciences
                                     Martin Machniak

Branch Head
Code 71 750
Environmental Sciences & Applied Systems
D. Bart Chadwhick

_____^^ 	 L 	 	 J 	 	 1 	
Scientist, YF-II
(Oceanographer)
Charles Katz

Scientist, YD-II
(Analytical Chemist)
Ignacio Rivera

Scientist, YD-II
(Bioassay Laboratory Director)
Gunther Rosen

Scientist, YD-II
(Chemist)
Ernest Arias
Biologist
(Analyst)
Marienne A Colvin

Scientist, DP-MI
(Analyst/Chemist)
Joel Guerrero
Organizational chart for the Bioassay Lab and relevant additional staff at SSC-Pacific.

-------
2.0 FACILITIES AND EQUIPMENT

2.1 FACILITIES

The Bioassay Laboratory is located on the first floor of Bldg.  111 at the Bayside location
of SSC-Pacific's Point Loma campus, just north of the Submarine Base.  Bldg. Ill is a
combination of office, laboratory, and storage space with a net working space of 35,662
sq. ft. The first floor is primarily dedicated to laboratories.

The main Bioassay Lab is located in Room 116, but a number of other labs are also
utilized. A temperature controlled lab space is located across the hall in Room 124.
Flow-through experiments can be conducted in the Rm 124 (also known as the "cold
room") as it is plumbed to receive clean seawater from north San Diego Bay. The cold
room also receives clean compressed air and has fluorescent lighting wired to a timer.
Additional equipment, storage, and sample processing space can be found in the
following  locations (see map of Bldg. 111 on following page): 115 (counter space,
balances), 127 (autoclave), 244 (E-pure water), and 246 (centrifuge).

Bldg. Ill  was built in 1982 and was originally designed to facilitate naval research in
marine sciences.  All labs, therefore, were plumbed to receive natural seawater, deionized
water, and compressed air. Natural seawater is pumped from Pier 160, located near the
mouth of San Diego Bay, and passes through two sand filters before it is stored in a
settling tank on the top of the building.  From there, it is distributed to individual
laboratories. Reagent water is provided by a water purification system that includes
carbon filtration, water softener,  reversed osmosis cartridge prefilters, and a UV-sterilizer
to produce deionized water with a resistivity of > 15 megohms/cm.  Where required,
reagent water with a resistivity of > 18 megohms/cm is available from the Barnstead E-
pure System located in Rm 244.  Compressed  air is dehydrated before distribution to labs,
and is subsequently filtered in the Bioassay Lab  and cold room with a 5-|im in-line filter
to remove potential particulates,  oil, or residual moisture.

Adjacent to Bldg. Ill, there are three research piers bordering north San Diego Bay. Pier
169 is home to the RV/ECOS, the branch's 40-foot survey boat that is used to collect
many of the samples used for bioassays. The vessel is part of the branch's Marine
Environmental Survey Capability (MESC), which is houses an elaborate flow through
system used to obtain a variety of water quality parameters in real-time.  A 22-foot
whaler is also available for use, and is launched  from the boat ramp located near Pier 160.
Pier  160 is a concrete pier that supports various other SSC-Pacific research vessels (e.g.
Acoustic Explorer, USS Dolphin).  Pier 159 is primarily dedicated to the Navy's Marine
Mammal Program.

-------
Schematics of Bldg. Ill
First floor (top figure), second floor (bottom figure). Blue shaded rooms represent those
housing equipment or space used by the bioassay laboratory.
        M             ^	'
        ซ?!   i4s      147  j
          L „     I     F"l
120
119
118
tr
117

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Lab
115
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-------
                  ฃtf,  r' f <*  • *4f
                  EkLit^    ..^ - -•  -
     •B
  Aerial photo of SSC-Pacific Bay side.
Aerial photo of piers at SSC-Pacific Bayside.
            10

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2.2 EQUIPMENT




A list of major equipment used by the Bioassay Lab is below:
Description
Incubators (2, temp/light
controlled)
Incubator (temp/light
controlled)
Light Microscope
Inverted Microscope
Spectrophotometer
UV/VIS Spectrophotometer
Ion Selective Electrode
(Ammonia)
Conductivity Meter
Dissolved Oxygen Meter
pH Meter
Drying Oven
Ultracentrifuge
Ice Maker
E-Pure Water Purification
System
Analytical Balance
Analytical Balance
Fume Hood
Microtox Toxicity
Analyzer
Fluorometer
Dinoflagellate Toxicity
Analyzer
Microwave
Light Tables (2)
Magnetic stirrers/hot plates
(10)
Make/Model
Percival Scientific, Model 1-3 5LL VL
Percival Scientific, Model 136LL
Olympus/CH-2
Olympus
HACH/DR2400
ThermoSpectronic/Genesys 10UV
Orion/720A
Orion/1 05+
Orion/840
Accumet/50
Yamato Gravity Convection Oven,
Model DX-600
Beckman/L8-80M
Scotsman
Barnstead 18 megohm-cm
Mettler PE22 top-loading, 0.1 g
Sartorius, 0.1 mg
Labconco
Microtox/2055
Turner/112
QwikLite
Daewoo
Porta-Trace/1618
Corning
Location
(Rm.)
116
116
116
116
116
114
116
116
116
116
116
246
246
244
115
115
All labs
123
116
152
116
116
116
                                      11

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Refrigerator
Whirlpool Estate TT18TKXSQ
116
3.0 QUALITY ASSURANCE OBJECTIVES

The purpose of the SSC-Pacific Bioassay Laboratory Quality Assurance Program is to
ensure that the lab provides high-quality data for principal investigators, project sponsors,
and other clients.  The laboratory aspires to adhere to the following objectives:

   •   Data should be accurate in terms of agreement with reference "true" values (for
       water quality parameters only)

   •   Data should agree among individual measurements made under similar conditions

   •   Data should be complete in terms of the amount of valid data achieved vs.
       planned

   •   Data should be comparable to prior relevant data for evaluation and testing
       purposes

   •   Data should be representative of the overall population of database of parameter
       measurements

   •   Data should be reproducible under similar conditions at any site

These objectives are achieved by ensuring that all staff members participate in the
QA/QC program, which covers all phases of the data generation, including strict
compliance with SOPs for sample handling, equipment calibration and proper use, record
keeping, and data handling.

3.1 QA/QC AND TOXICITYDEFINITIONS*

Accuracy- The degree of agreement of an analytical result with the true (reference)
value.  Accuracy is affected by both random and systematic errors, but is sometimes used
improperly to denote only systematic error (see "Bias" below). Because "true" values
don't necessarily apply to toxicity testing, this term is not typically applied.

Batch- A set of consecutive determinations (analyses) made without interruption; a
"run".  Results are usually calculated from the same calibration curve or factor.

Bias- That part of inaccuracy of analytical results caused by systematic error.

Blank- An analysis made by the same procedure as a sample, but intended not to contain
the analyte.
                                       12

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Calibration- Standardization of a measurement or instrument by use of another standard
or instrument to adjust any variance in accuracy.  The concentrations of the calibration
standards should bracket the expected concentration of the test materials.

Calibration Curve- The graphical relationship between the known values, such as
concentrations, of a series of calibration standards and their instrument response.

Calibration Standard- A substance or reference material used to calibrate an instrument.

Certified Reference Material (CRM)- A reference material one or more of whose
property values are certified by a technically valid procedure, accompanied by or
traceable to a certificate or other documentation which is issued by a certifying body.

Chain of Custody (COC) Form- Record that documents the possession of the samples
from the time of collection to receipt in the laboratory. The record generally includes:
number and types of containers; mode of collection; collector; time of collection;
preservation (if any); and requested analyses.

Coefficient of Variation (CV)- A standard statistical measure  of the relative variation of
a distribution or set of data, defined as the standard deviation divided by the mean. It is
also called the relative standard deviation  (RSD). The CV can be used as a measure of the
precision within and between laboratories, or among replicates for each treatment
concentration (EPA, 2000).

Control Chart- A cumulative summary chart of results from QA tests with reference
materials (e.g. reference toxicants). The results of a given QA test are compared to the
control chart mean value and acceptance limits (typically 95%  confidence limits, i.e.
mean + 2 standard deviations) or warning limits (typically 99% confidence limits, i.e.
mean + 3 standard deviations).

Corrective Action- The action taken to eliminate the causes of an existing
nonconformity, defect, or other undesirable situation in order to prevent recurrence.

Data Quality Objectives (DQOs)- A statement of the overall level of uncertainty that a
decision maker is willing to accept in results derived from environmental data.  This is
qualitatively distinct from quality measurements such as precision, bias, and detection
limit.

Data Validation- The process of evaluating the available data  against the project DQOs
to determine to what degree the objectives were met.

Detection Limit- The lowest concentration or amount of the target analyte that can be
identified, measured, and reported with confidence that the analyte concentration is not a
false positive value.
                                        13

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Effect Concentration (EC)- A point estimate of the toxicant concentration that would
cause an observable adverse effect (e.g., death, immobilization, or serious incapacitation)
in a given percent of the test organisms, calculated from a continuous model (e.g., Probit
Model). EC25 is a point estimate of the toxicant concentration that would cause an
observable adverse effect in 25 percent of the organisms (EPA, 2000).

False negative- A determination that a material is nontoxic when it is in fact toxic.

False positive- A determination of toxicity when the material is in fact nontoxic.

Holding Times- The maximum times that samples may be held prior to analysis and still
be considered valid or not compromised.

Hypothesis Testing- A statistical technique (e.g. Dunnett's test) for determining whether
a tested concentration is statistically different from the control. Endpoints determined
from hypothesis testing are NOEC and LOEC. The two hypotheses commonly tested in
WET are: Null Hypothesis (Ho)- The effluent is not toxic. Alternate hypothesis (Ha)- The
effluent is toxic (EPA, 2000).

Inhibition Concentration (1C)- A point estimate of the toxicant concentration that
would cause a given percent reduction in a non-lethal biological measurement (e.g.,
reproduction or growth), calculated from a continuous model (e.g., Interpolation
Method). IC25 is a point estimate of the toxicant concentration that would cause a 25-
percent reduction in a non-lethal biological measurement (EPA, 2000).

LC50 (lethal concentration, 50 percent)- The toxicant or effluent concentration that
would cause death in 50% of the test organisms (EPA, 2000). The concentration is
calculated from the data set using statistical or graphical models. The lower the LC50, the
more toxic the chemical or effluent sample. Other LC values, e.g. the LC90 or LC5 may
also be calculated to determine concentrations causing more or less mortality to the
population. Note: The LC value must always be associated with the duration of exposure.
Thus a 48-h LC50, 96-h LC50, etc. is calculated.

LOEC (Lowest-observed-effect-concentration)- The lowest concentration of an
effluent or toxicant that results in adverse effects on the test organisms (i.e., where the
values for the observed endpoints are statistically different from the control) (EPA,
2000).

Negative Control- A negative control is a part of an experiment where the experimental
conditions are identical to the regular experiment except the substance being tested in not
present.

NOEC (No-observed-effect-concentration)- The highest concentration of an effluent or
toxicant that causes no observable  adverse effects  on the test organisms (i.e., the highest
concentration of toxicant at which  the values for the observed responses are not
statistically different from the control) (EPA,  2000).
                                        14

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NPDES (National Pollutant Discharge Elimination System)- Created under the Clean
Water Act. The permitting system under which point source discharges are regulated to
eliminate or minimize the discharge of toxicants into surface waters. States frequently
oversee their own programs which must comply with (i.e. be equally or more stringent)
the national permit program.

Precision- 1) A qualitative term used to denote the scatter of results. Precision is said to
improve as the scatter among results becomes smaller.  Precision is usually measured as
standard deviation (SD), coefficient of variation (CV), or relative percent difference
(RPD). 2) A measure of the reproducibility within a data set. Precision can be measured
both within a laboratory and between laboratories using the same test method and
toxicant (EPA, 2000).

Quality Assurance (QA)- An integrated system of activities involving planning, quality
control, quality assessment, reporting and quality improvement to ensure that a product or
service meets defined standards of quality with a stated level of confidence.

Quality Control (QC)- The overall system of technical activities whose purpose is to
measure and control the quality of a product or service so that it meets the need of users.

Reagent Water- Water suitable for use in making up critical reagents or for use in
sensitive analytical procedures.

Reference Toxicant Test- Reference toxicants are routinely tested to demonstrate the
continuing ability of the laboratory  to successfully perform the tests and to evaluate the
overall health and sensitivity of the test organisms over time. The coefficient of variation
(CV) for the test LCSOs (acute tests) or IC25s (chronic tests) provides a measure of test
repeatability or precision; the lower the CV value, the less variable the results and the
lower the frequency of false positive and false negative results. Individual reference test
results are compared to control charts to determine acceptability.

Replicate- Each of several experimental units that are tested simultaneously using the
same experimental conditions (ASTM, 2002).

Standard Curve- A plot of concentration of known analyte standards versus the
instrument response to the analyte.

Toxicity Test- A procedure to determine the  toxicity of a chemical or an effluent using
living organisms. A toxicity test measures the degree of effect of a specific chemical or
effluent on exposed test organisms (EPA, 2000).

*Unless otherwise noted, definitions were taken from EPA SW-846 Revision 1, July
1992 and Washington Department of Ecology Model Quality Assurance Model
http ://www. epa. gov/epaoswer/hazwaste/test/pdfs/chap 1 .pdf.
                                        15

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3.2 DATA ACCURACY, PRECISION, COMPLETENESS,
REPRESENTATIVENESS, AND COMPARABILITY

    1)  Precision

   Toxicity test precision is determined by comparison of a) the variation among
   laboratory replicates of individual samples, and b) reference toxicant tests.
   Depending on the study objectives and the test method, replication will vary.
   Typically, three to five replicates are analyzed for each sample/test concentration.
   Standard deviations or CVs can be compared with results from other studies to
   estimate specific test precision. Reference toxicant tests are useful because they can
   both verify the technical quality of the testing facility as well as the sensitivity of the
   test organisms. Because toxicity of reference toxicants is assumed to be constant,
   their use is a good indicator of precision.  Resulting LCSOs are plotted on control
   charts indicating current and past performance. If test results fall within 2 standard
   deviations of the running mean, the test is generally considered acceptable.
   Calculated CVs of the LC50 should also not exceed the 75th percentile of CVs
   reported nationally as reported in EPA (2000). See Section 8.2 for more information
   on the use of reference toxicants.

   2)  Accuracy

   Because there is no "true" or "correct" response against which to compare toxicity
   test results, accuracy cannot be determined. Therefore, data quality objectives to test
   accuracy of toxicity tests are not available. Water quality measurements, however,
   are assessed for accuracy by comparing measured values of standards against known
   values.  If they differ by more than 10% for dissolved oxygen, pH, or salinity, or by
   more than 30% for ammonia, corrective action will be taken.

   3)  Completeness

   It is anticipated that all samples received will be tested.  There are several factors that
   may affect the  successful completion of testing including: a) acceptable negative
   control  response; b) acceptable reference  toxicant (positive control) tests; c)
   acceptable test condition variability; and d) test organism availability. For these
   reasons, it is important that enough sample volume be collected in case retesting is
   necessary. A test failure rate of approximately 20% is estimated, but with retesting a
   completeness rate of 95% is expected. Sample holding time may become an issue in
   the case of retesting, and needs to be considered by the project planner.

   4)  Representativeness

   A number of factors  determine the degree to which toxicity tests represent actual
   effects of typical effluent discharges. These include sampling design, sample
                                        16

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handling, test species and endpoint used, and exposure time. Careful consideration of
how samples and what kinds of samples (e.g. grab vs. composite) are collected,
therefore, is important.

5)  Comparability

The use of standardized testing allows for comparability among laboratories. The use
of negative controls and reference toxicant tests can be used to compare test results
with other studies.  Splits of samples can also be used to compare the Bioassay Lab's
performance with other laboratories.  Comparability should take into account
variables such as species, test conditions (e.g. pH, temperature, salinity, dissolved
oxygen), dilution factor, test endpoint, reference toxicant, and dilution water
characteristics.
                                     17

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4.0 SAMPLE AND TEST ORGANISM HANDLING

4.1 RECEIVING SAMPLES

1) Upon arrival, samples should be handled with the utmost care. Samples should be
received in a well-ventilated area in case leakage has occurred during shipping.

2) A chain of custody form should be completed and a copy given to the person who
delivered the sample. The chain of custody form should contain the following
information:

   •   Sample ID

   •   Sample description/quantity

   •   Date received

   •   Date collected

   •   Sample collector

   •   Location of delivery

   •   Analyses requirements for each sample

   •   Date and signatures of the delivery person and receiver

3) The sample should be logged in and given an identification number. The number
given should be the next consecutive number on the list for SSC-Pacific Sample IDs. The
following information should also be recorded in the logbook:

   •   Time/date that the sample was collected.

   •   Time/date that the sample was received.

   •   Temperature of sample during collection and upon arrival at lab.

   •   Company or organization the sample came from.

   •   Type of analyses that will be performed on the sample.

   •   Description of sample (volume, type [seawater, freshwater, sediment], preserved
       or frozen, compounds known or suspected to  contain).

   •   Initials of the person who received the sample.
                                      18

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   •   Location sample was stored upon arrival.

4) Dilution or laboratory water such as Scripps Institution of Oceanography (SIO)
seawater is collected at the Scripps pier in La Jolla, California by a laboratory technician.
Dilution or laboratory water is logged into the "Seawater Collection Log" upon arrival
and is stored in a clean carboy or a suitable HOPE container for a maximum of 14 days.
4.2 HOLDING SAMPLES

If samples are not immediately prepared for testing, they are stored at a target
temperature of 4 ฐC in the cold room (Bldg.l 11, Rm. 124) until used. Temperature of the
cold room is logged daily during sample holding and should at no time fall outside the
range of 0-6 ฐC (USEPA 2002). Every effort should be made to initiate testing with
effluent sample on the arrival day, and effluent sample-holding time should not exceed
36h. Sediment samples must be tested within 14 days.
4.3 SAMPLE HANDLING AND CHAIN OF CUSTODY

   I.   OBJECTIVE: Methods for tracing and transfer of samples ensure the integrity
          from time of collection to sample disposal. Custody of samples is defined as
          either having physical possession, being in a person's view after taking
          possession, security from tampering or holding in a place restricted to
          authorized personnel.

   II  METHODS

   A.  Transferring Custody

          1.  Records shall be kept in permanent ink on a chain of custody form for
             receiving samples.

          2.  Chain of custody forms should always travel with test organisms or
             samples.

          3.  Upon arrival of samples, examine containers to detect any damage or
             tampering.

          4.  If containers are damaged, it should be noted on the chain of custody
             form.

          5.  Note the date and time on the form and sign.

   B.  Subdividing Samples
                                       19

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          If samples need to be sub-divided and sent to other laboratories, this should be
          noted on the original chain of custody form and a new chain of custody form
          should be made.

   C.  Sample Disposal

          Indicate disposal of samples, which terminates the chain of custody.

Copies of chain of custody forms shall be kept in the laboratory or with all corresponding
       project data.
                                       20

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      SfVUfflR
      Systems Center
         PACIFIC

   ENVIRONMENTAL SCIENCES AND
APPLIED SYSTEMS BRANCH, CODE 71750
       53605 HULL STREET
      San Diego, CA 92152-5000
CHAIN OF CUSTODY RECORD
                                                 Date
                                                 Page
of
Project Title / Project Number:
Remarks / Air
Bill:


Sampler(s): (Signature)
Tel:
Fax:
E-mail:
Special Instructions:
Field Sample
Identification








Sampling
Temp. (ฐC)








Sampling
Date








Relinquished by: (Signature)
Relinquished by: (Signature)
Sampling
Time








Matrix








Project Leader:
Contact:
Contact Tel:
Requested Analyses



























Received by: (Signature)
Received by: (Signature)













































Date:
Date:













































Time:
Time:
                                                                 21

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4.4 ORGANISM ARRIVAL LOG
                                                       ORGANISM ARRIVAL LOG
Batch ID


















Date
Received


















Received
From


















Project Title


















Species


















Age when
shipped


















Number
Ordered


















Organism Condition
(e.g. number dead)


















Initial Water Quality
pH


















D.O.


















Temp.


















Salinity


















Storage
Location


















Analyst
Initials


















 Species
 A.a. - Atherinops affinis
 A.b.- Americamysis bahia
 C.g. - Crassostrea gigas
 C.h. - Ceratocorys horrida
 M.g. - Mytilus galloprovincialis
R.a.- Rhepoxinius abronius
S.p. - Strongylocentrotus purpuratus
E.e. - Eohaustorius esturaius
M.b. - Menidia beryllina
Other:
                                                                              22

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4.5 SAMPLE COLLECTION RECORD
                           SAMPLE COLLECTION RECORD
SSC Sample ID
(correspond w/Toxcalc ID)
























Sample Name
























Sampling Date
Begin
























End
























Sampling Time
Begin
























End
























Collection
Temp.
























Water Quality on Arrival
pH
























D.O.
























Temp.
























sal.
























Sample Type
(e.g. Grab/Com p.)
























Received By
(print name)
























Company or
Organization
























Storage
Location
























                                        23

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4.6 RECEIVING/HOLDING TEST ORGANISMS

With the exception of dinoflagellates, test organisms used by the Bioassay Lab are
generally not cultured on the premises; therefore, they are purchased and shipped or hand
delivered by outside vendors.  The fastest method of shipment is always used to prevent
unnecessary stress on the test organisms. Analysts are trained in the proper procedures
for receiving and maintaining test organisms. Notes are recorded for all stages, from
arrival in the lab to termination and disposal of unused organisms.  See "Receiving  and
Holding Test Organisms" SOP for details. General considerations with respect to
successful maintenance of test organisms are:

          •  Minimum shipping time
          •  Shipped in aerated containers
          •  Immediate assessment of water quality and organism health upon arrival
          •  Preparation and maintenance of high quality food supply
          •  Daily feeding
          •  Acclimation to test conditions at safe rate for species
          •  Regular water changes
          •  Water quality regularly monitored
          •  Organisms are not overcrowded in holding tanks
          •  Minimization of disturbances to prevent stress

Before disposal, any surviving test organisms are humanely killed, generally by
concentrating into a container and freezing.  Under no circumstances are test organisms
ever released to the wild or used more than once for testing.
                                       24

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5.0 HAZARDOUS MATERIAL (HM) STORAGE, DISPOSAL AND SAFETY
CONSIDERATIONS

A hazardous material (HM) is any material that, because of its quantity, concentration,
physical or chemical characteristics, poses a present or potential health hazard to human
health and safety or to the environment1.  Staff members receive an initial hazardous
materials training course as well as annual refresher trainer from SSC-Pacific. It is the
responsibility of the laboratory director and analyst that all hazardous materials are
acquired, handled, stored, and disposed of according to policy detailed in SSC- Pacific
Document 4110.1.  A detailed description of the policy is available in the "Hazardous
Material Storage, Disposal and General Information" SOP. A summary is provided
below:
5.1 PURCHASING HAZARDOUS MATERIALS

       Purchasing instructions are provided in a handbook located in Rm 116. Upon
       receipt of new chemicals, the Safety Office is notified (x33873).

5.2 STORAGE AND MANAGEMENT OF HAZARDOUS MATERIALS

       Prior to storage, all HM needs to be labeled with name, manufacturer, hazard,
       barcode label, and owner, whether in original or secondary containers. HM is
       inspected regularly to ensure absence of leakage and that labels are intact.  A
       notebook in Rm 116 contains Material Safety Data Sheets (MSDS) for each
       chemical stored in the lab.

5.3 HANDLING HAZARDOUS MATERIALS (SAFETY)

       Hazardous materials are grouped into different categories including: flammables,
       halogenated solvents, corrosives, toxics, compressed gases, oxidizers, water
       reactives, pyrophorics, and explosives. All staff members are trained in the
       characteristics of these materials, and the safety considerations associated with
       working with these materials. Engineering controls such as fume hoods are used
       to eliminate exposure. Personal protective equipment such as gloves, goggles,
       respirators, and aprons are also used where necessary. Safety equipment
       including first aid kits, fire extinguishers, and eye wash stations are located in
       each lab.  Emergency showers are also located in designated areas of the building.

5.4 HAZARDOUS WASTE DISPOSAL

       Hazardous waste is any discarded, excess or spilled material that is solid, liquid or
       gas and meets the definition of a hazardous material1. It is either Characteristic
       (toxic, reactive, ignitable or corrosive) or Listed (appears on  a specific EPA or
       state list).  The staff is trained in which wastes are permitted sewer discharges,
       and which are not. Questionable waste can be analyzed by contacting the Safety
                                       25

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       and Environmental Office (x36363).  Otherwise, proper labels and paperwork are
       filled out (HW Disposal Request Form, HW Profile Sheet, copy of MSDS) and
       the code's hazardous waste coordinator is notified so that it can be removed
       during the next scheduled pick up date. Forms are available in Hazardous
       Materials Information notebook or are accessible on line at:
       https://iweb.spawar.navy.mil/services/sti/publications/inst/forms/

5.5 HAZARDOUS MATERIALS SPILLS

       All staff is trained in how to respond to FDVI spills. Larger spills deemed a
       potential danger to personnel result in area evacuation and a call to the fire
       department  (9-911).  Smaller, known spills are cleaned up using proper protective
       equipment and spill kit materials stored in each lab. All spills are reported to the
       Safety and Environmental Office (x35024).
                                       26

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6.0 CALIBRATION, USE, AND TROUBLESHOOTING OF
INSTRUMENTATION

Calibration refers to the standardization of a measurement or instrument by use of
another standard or instrument to adjust any variance in accuracy.  Proper calibration of
equipment prior to sample measurement is the responsibility of the analyst. Calibration
procedures for laboratory instrumentation are covered in the standard operating
procedures (SOPs) manual.

6.1 CALIBRATION OF BASIC LABORATORY INSTRUMENTATION

A) Spectrophotometers

The HACK spectrophotometers are put through a "self-test" each time they are turned on
and generally do not require calibration.  However, chemical standards certified by
HACK are used to verify accuracy prior to measurement of samples. If necessary, a
standard curve is constructed to make corrections to samples measurements.

B) Pipettes

Pipettes are routinely checked for accuracy,  and calibrated only if necessary. Checking
calibration is performed by repetitively weighing aliquots of distilled water at room
temperature. After weight is converted to volume, the value is compared against
permitted values outlined in the equipment manual.  If outside the permitted range,
pipettes are recalibrated with the enclosed service tool and rechecked.

C) Balances

Balances are calibrated by outside specialists on an annual basis.  If a balance has been
moved or if standard Class-S calibration weights indicate there is reason to suspect that
the balance is not producing accurate measurements (e.g. values vary by more than 2% of
calibration weight values), this is noted and alternate balances that are calibrated
correctly are used until calibration can be performed.

D) Ion electrodes

Ammonia, conductivity/salinity, pH, and dissolved oxygen electrodes are calibrated  each
time they are used, and recalibrated as necessary during sample measurements depending
on the specific method.  Refer to the SOP for each piece of equipment for specifics on
their calibration.

E) Thermometers

Thermometers are compared against an NIST certified thermometer on an annual basis.
If temperature readings on thermometers vary by more than 0.5ฐC, a correction factor
may be applied to the thermometer or the thermometer will no longer be used.
                                       27

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F) Temperature of cold room, incubators, freezers, lab

Temperature in the main laboratory, cold room, and both incubators are continuously
monitored with max/min thermometers and/or HOBO temperature data loggers. For
storage of samples, cold room temperature is maintained at 4(ฑ 2)ฐC, while freezers are
held at -20(ฑ 10)ฐC.  Temperature in incubators is dependent on the test being conducted.
If temperature is outside the range, the thermostat is adjusted accordingly and the
deviation recorded if samples or test organisms are suspected of having been affected.

G) Fume Hood

The fume hoods in all labs are measured annually for adequate air-flow by an industrial
hygienist provided by the Safety Office (POC: Gary Douglas, x35026).
6.2 LABORATORY STANDARDS

Analytical standards used for calibration and preparation of quality control samples shall
be traceable to standard reference materials. Reference toxicant stock solutions are
created from reagent grade chemicals, which are analyzed in-house by stabilized
temperature graphite furnace atomic absorption (STGFAA) spectroscopy by direct
injection.  The standard reference material (SRM) CASS4 (coastal seawater) from the
National Research Council of Canada is used to quantify the recovery of any
preconcentration, and SRM 1643d (trace metals in water) of the National Institute of
Standards & Technology is used to evaluate the precision and accuracy of the STGFAA
analysis. These measurements are done by injections in triplicate for each sample, with
relative  standard deviation in the absorbance measured of less than 10%. Accuracy better
than 15% is required for the SRM 1643d. An efficiency of + 10% (90% to 110%) in the
recovery of CASS4 is required in the case of samples that are preconcentrated following
the APDC/DDDC liquid/liquid procedure. The limit of detection is determined as three
times the standard deviation of the concentrations measured in blanks. Metal stock
solutions are prepared in E-Pure water and stored in the dark at 4ฐC in polycarbonate
bottles to minimize binding to wall surfaces.
                                       28

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7.0 CLEANING GLASSWARE/PLASTICWARE

In general, containers used to hold effluent samples are not reused because they could
carry over adsorbed toxicants from one test to another. Non-disposable sample
containers, test vessels, tanks, and other equipment that comes into contact with effluent
are washed according to EPA protocol (EPA 1993).  New plasticware or glassware not
previously used by the lab is first tested to ensure that no toxic effects are associated with
the container.  After an absence of toxicity has been established, new plasticware is
rinsed with dilution water prior to its first use, while all new glassware must be soaked in
10% acid and rinsed well  with deionized and dilution water prior to its first use. A brief
description of the cleaning procedure used for non-disposable containers after exposure
to effluent is provided below. More details are provided in the SOP Manual.

General cleaning procedures:

     1.   Rinse with tap water several times.
    2.   Soak in tap water and 10% Liquinox or other detergent for at least 15 minutes.
    3.   Rinse in tap water several times.
    4.   Rinse in 10% Nitric (HNOs) acid to remove scales, metals, and bases.
    5.   Rinse several times in deionized water.
    6.   Rinse once with pesticide grade acetone in fume hood.
    7.   Rinse three times with deionized water.
                                        29

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8.0 QUALITY CONTROL SAMPLES

One method for assessment of the quality of bioassay results is the evaluation of
performance for quality control (QC) samples.  QC samples used by the Bioassay
Laboratory include negative controls and reference toxicant tests (positive controls).

8.1 NEGATIVE CONTROLS

A negative control is a part of an experiment where the experimental conditions are
identical to the regular experiment except the substance being tested is not present.
Negative controls are suggestive  of test organism health and/or laboratory quality and are
used to assess if apparent effects  in experimental treatments are real. Performance of
negative controls is also used to determine test acceptability as dictated by individual test
methods.

Negative controls typically consist of dilution water (e.g. deionized water or filtered,
natural seawater). If an experiment calls for natural seawater, it is collected in a clean
carboy from the Scripps Institution of Oceanography pier in La Jolla, California.
Seawater is obtained a day or two before the test and discarded no later than 14 days after
collection.  Depending on the objectives of the test, there may be other types  of negative
controls including solvent controls, synthetic salt controls, or hypersaline brine (HSB)
controls. The analyst is responsible for determining which control(s) is/are relevant to a
test.

8.2 REFERENCE TOXICANT TESTS

Reference toxicant tests are a means of assessing test precision.  These tests are
conducted concurrently with effluent samples, and employ a known toxicant  known as a
reference toxicant. The reference toxicant is copper for most test species used at SSC-
SD. By exposing different batches of the test organism to the same concentrations of the
reference toxicant in the same dilution water, under identical testing conditions, the lab
can assess repeatability via comparison of LC50 or EC50 values for a given species.
Values are plotted on a control chart to monitor the lab's performance over time.  In
general, reference toxicant test results that fall within two standard deviations above or
below the running mean are an indication of acceptable performance. In addition to the
mean and standard deviation, the coefficient of variation (CV) may also be used to
demonstrate the lab's precision.  Actual tested concentrations in reference toxicant tests
are dependent on the test method due to differences in sensitivity among species and
endpoints.
                                        30

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9.0 PREVENTIVE MAINTENANCE PROCEDURES FOR LABORATORY
EQUIPMENT AND CHEMICALS

9.1 PREVENTIVE MAINTENANCE FOR EQUIPMENT

A preventive maintenance program for equipment increases laboratory efficiency,
reduces the potential for inferior quality test results, and prolongs the life of essential
laboratory tools.  Analysts are trained in the proper use of all laboratory equipment and
how to troubleshoot problems associated with their normal function. Equipment
operating manuals are stored in a drawer labeled "Equipment Manuals" in Rm. 116 for
easy reference. Standard Operating Procedures including troubleshooting guides are also
available for commonly used equipment. When repairs required are beyond the
capability  of the analyst, outside vendors are contacted for repair. A maintenance record
for equipment is kept in the lab. Information included in the maintenance record is
provided on the following page.

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    9.1 Preventive Maintenance for Equipment
     Equipment
     Description
              Storage
           Requirements
                 General
               Maintenance
 Frequency
Accumet
pH/i on/conductivity
meter model 50
Benchtop
Clean case with a mild detergent and damp
cloth. Never use solvent.
As needed
Accumet pH Probe
Between measurements: D.I. water
Overnight: pH 4.0 buffer
Long-term: cotton cap over electrode
Required when troubleshooting probe.
As needed
Orion Dissolved
Oxygen Meter model
840 and probe
Between measurements: put probe in
sleeve moistened with D.I. water
Overnight: turn meter off
Long-term: remove membrane and
replace when returned to service
Soak probe in silver anode cleaning solution,
refill electrolyte solution, and replace
membrane cap.
6 months or
As needed
Orion Portable
Dissolved Oxygen
Meter model 830A and
probe	
Between measurements: D.I. water
Overnight: turn meter off
Long-term: remove membrane and
replace when returned to service
Clean external surfaces with water and a mild
detergent. Never wipe the meter with a dry
cloth.
Batteries - alkaline AA.
As needed
Orion Conductivity /
Salinity Meter model
105A+
Between measurements: D.I. water or
seawater
Overnight or Long-term: clean and dry
Clean case and touchscreen with a damp cotton
cloth. Do not use strong solvents.
Batteries - 9 volt.
Always recalibrate after battery change.	
As needed
Orion 720A meter
Between measurements: lOppm
standard with ISA
Overnight: place electrode tip in a
lOOOppm standard without ISA
Long-term: disassemble completely and
rinse with D.I, water and dry	
Send to Orion Technical Service for repairs.
As needed
Orion Ammonia Probe
model 95-12
Between measurements: 0.1M ammonia
standard
Long-term: disassemble and place in
storage box	
Check membrane.
Refill internal filling solution.
As needed
                                                              33

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9.1 (cont.) Preventive Maintenance for Equipment
Equipment
Description
Hach DR 2400
Spectrophotometer
Storage
Requirements
Benchtop
General
Maintenance
Clean case and touchscreen with a damp cotton
cloth. Do not use strong solvents.
Batteries - 3 D-cell.
Send to HACH for recertification to maintain
accuracy in measurement.
Troubleshooting - www.hach.com for latest
information.
Frequency
As needed
                                                         34

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9.2 PREVENTIVE MAINTENANCE FOR CHEMICALS
Chemicals for laboratory functions, such as glassware cleaning (acid water baths) and
reference toxicant testing (metal stock solutions), need to be properly made, maintained,
and disposed of to ensure high quality test results. Analysts are trained in how to safely
and accurately prepare chemical solutions.  All containers holding chemicals are labeled
with the contents and the date prepared. The following chemicals are tracked on a log
sheet:
Reagent/
Solution
Nitric Acid bath -
10%
Concentrated
Nitric Acid
Copper (sulfate) -
1 ppt stock
Reagent Grade
CuSO4*5H2O
crystals
Zinc (sulfate) -
1 ppt stock
Reagent Grade
ZnSO4*7H2O
pH Buffers
0.1 N Ammonia
Standard
Ammonia ISA
Solution
Storage
20 L HOPE
container near
rear sink
Corrosives locker
500 mL
polycarbonate
container in
refrigerator at 4
ฐC
Original
bottle/Room
Temp
250 mL
polycarbonate
container in
refrigerator at 4
ฐC
Original
bottle/Room
Temp
Original
bottles/Room
Temp
Original
bottle/Room
Temp
Original
bottle/Room
Temp
                                       35

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10.0 ACQUISITION, REDUCTION AND REPORTING OF DATA
10.1 ACQUISITION

At the beginning of a test, a test identification number is assigned in the "Test ID" log
stored in Rm 116 for easy cross-referencing. The Test ID is used on all other applicable
data sheets. Raw data is recorded in non-erasable ink on computer-derived data sheets.
Notes are taken on computer-derived note sheets. At a minimum, all data and note sheets
require the date, Sample ID, Test ID, analyst or operator, and species/method
identification or water quality parameter. Standard units as defined below are always
used to ensure  consistency. Raw data and note sheets are stored in a notebook in the
Bioassay Laboratory. Copies are made for attachment in reports or other uses as
required. Details with respect to entry of data onto data sheets are provided in the SOP
Manual.
Parameter
PH
Salinity
Temperature
Dissolved Oxygen
Total Ammonia
Unionized Ammonia
Standard Unit
pH units
ppt or %o
ฐC
mg/L
mg/L
mg/L
10.2 DATA REDUCTION AND REPORTING

Data reduction is the process of transforming raw data into reportable material.
Mathematical manipulation and summary statistics are generated by means of computer
programs and laboratory equipment that perform these functions.

Once all measurements have been recorded on data sheets, data is manually entered into
spreadsheets or statistical programs.  All phases of data transfer are double checked.
When computer programs are used to derive calculations,  individual calculations are
performed by hand at random.  Summaries and results are compared with raw data entries
to assure accurate data entry. Any suspect data is reported.

Depending on the objectives of the study, toxicity  data are statistically analyzed using a
variety of tools, including ToxCalc 5.0, a software package designed specifically for
whole effluent toxicity test data. The staff is trained in the proper use of ToxCalc to
derive NOECs and LOECs using hypothesis testing and LC50/EC50 values using point
estimation techniques. The software is designed to be in accordance with EPA
recommend procedures for the analysis of whole effluent toxicity  data.  ToxCalc
summary sheets are saved on the computer in Rm  116 and printed out and placed into a
folder designated for the specific study for future reference.  The "Statistical Analysis of
Data" SOP provides a detailed description of data  analysis procedures for the staff to
follow.
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11.0 REPLICATION AND TEST SENSITIVITY

The sensitivity of toxicity tests will depend in part on the number of replicates per
concentration, the significance level selected, and the type of statistical analysis. If the
variability remains constant the sensitivity of the test will increase as the number of
replicates is increased. Minimum numbers of replicates are dictated by the individual
protocol. The actual numbers will depend on the objectives of the test and the statistical
method used for analysis of the data.  For example, 20 fish per concentration are
generally considered optimal for Probit analysis.  This typically equates to 4 replicates of
5 fish each. The Bioassay Laboratory meets minimum requirements for replication at all
times.  The actual number of replicates used is typically a function of costs and project
goals, and may be discussed with the  project manager/sponsor/client.
                                        37

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12.0 CORRECTIVE ACTION

12.1 DETERMINING THE PROBLEM

Since each situation is unique regarding resolution of erroneous data, the following
guidelines are used in a manner that best suits the existing circumstance. The analyst and
laboratory director generally work on this together. Documentation of the
troubleshooting techniques used is required to allow follow up on the situation.

       1.   Compare results from the reference toxicant test to control chart values to
           assess quality of test results. In general, if LC50 values fall within 2 standard
           deviations of the mean, they are acceptable.

       2.   Compare the control (e.g. negative, solvent, salt, brine control) response to
           test acceptability requirements as dictated by the SOP for the method.

       3.   Double-check all calculations (e.g. those for making reference toxicant stock
           solutions and sub-stock solutions, dilutions and test organism counts).

       4.   Verify quality of the reference toxicant solutions or samples  (e.g. were they
           correctly made, did they violate holding time, are water quality parameters
           within range tolerated by test organisms?).

       5.   Check all water quality parameters (pH, salinity, dissolved oxygen, ammonia,
           temperature).

       6.   If there are any other tests being run via an analogous procedure, then
           compare results. This may allow the analyst to exclude certain factors.

       7.   Assure properly treated/cleaned  glassware was used in  all phases of the test
           set up and testing.

       8.   Re-read all notes taken during testing to determine if there are any conditions
           in the laboratory that may conflict with proper testing procedures (light
           sources, temperature, debris in glassware, incorrect calibration of equipment,
           etc.).

       9.   Review scientific journals of relevance that may offer possible cause or
           resolution.

       10.  If the erroneous data is being observed during data analysis,  verify all entries
           and confirm all calculations by hand.
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12.2 RESOLUTION

If the source of error is determined, appropriate action to resolve issues (e.g. repeat test,
note and remove erroneous data, re-calibrate equipment, etc.) is taken. Detailed accounts
of the corrective action are made for future reference. Although some data may be
considered "unacceptable" by not meeting DQOs, the data may still be potentially
"useful" to the study.  Data should be evaluated for its usefulness on a case-by-case basis.

If the source of error is not determined, follow up testing or other measures may be
required. The approach may be discussed with the principal investigator or project
sponsor.
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13.0 AUDITS AND QUALITY ASSURANCE REPORTS

13.1 INTERNAL AUDITS

The laboratory director and analyst meet regularly to discuss status, required changes,
and proper execution of the Bioassay Lab's QA/QC program. Any deficiencies in the
program are documented and corrective action is taken towards remediation of the
problem(s). The following topics are typically covered in the review of the QA program
to ensure that:

          •  QA Manual and SOPs are up to date
          •  Equipment and Facilities are functioning properly
          •  Equipment is being calibrated correctly
          •  Reagents/standards/solvents are available and not expired
          •  Samples are being handled properly
          •  QC measures are being applied correctly
          •  QC records (e.g. control charts) indicate lab is in  control
          •  Data is being properly managed and reported
          •  Staff is receiving appropriate training

13.2 EXTERNAL A UDITS

To date, the Bioassay Lab has not been subjected to external audits. The laboratory,
however, agrees to comply and assist with future external audits that may be required.

13.3 QUALITY ASSURANCE REPORTS

The laboratory director and analyst meet on a regular basis to discuss results of internal
audits and evaluate the status of the quality assurance program.  A report documenting
issues associated with any of the below topics is compiled as needed, but on a quarterly
basis at a minimum, depending on the level of testing taking place in the lab. Reports are
available to the Bioassay Lab staff as well  as relevant principal investigators,
Environmental Sciences and Applied Systems (71750) Branch Head, and Advanced
Systems and Applied Sciences (717) Division Head. Areas covered in the quality
assurance report may include:

          •  Audit findings
          •  Certification status
          •  Problematic data and effectiveness of corrective action taken
          •  Modification/addition of SOPs
          •  Personnel and instrumentation changes
          •  Training courses held/attended
          •  Status of new methods evaluated/new lab capabilities
                                       40

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REFERENCES

   ASTM 2002. Standard Terminology Relating to Biological Effects and
   Environmental Fate. Standard E 943-00 in: Annual Book of Standards. Vol. 11.05
   Biological Effects and Environmental Fate; Biotechnology; Pesticides. ASTM
   International, West  Conshohocken, PA.

   USEPA 2000. Understanding and Accounting for Method Variability in Whole
   Effluent Toxicity Applications Under the National Pollutant Discharge Elimination
   System. June 2000.  EPA 833-R-00-003.

   USEPA 2002. Guidance for Developing  Quality  Systems for Environmental
   Programs. EPA QA/G-1. EPA 240/R-02/008. November 2002.

   SSC-SD 2004. The  Lifecycle Management of Hazardous Materials / Hazardous
   Waste at Space and Naval Warfare Systems Center San Diego. Space and Naval
   Warfare Systems Center San Diego (SSC-SD). Document 4110. 1 April 2004.

   Navy Regional Environmental Laboratory. 2003. Laboratory Quality Assurance
   Manual, Revision No. 4.6. Navy Regional Environmental Laboratory, Public Works
   Center Code 910, San Diego, CA. November 28, 2003.

   Washington State Dept of Ecology. 2002. Procedural Manual for the Environmental
   Laboratory Accreditation Program. Publication no. 02-03-055. November 2002.

   Washington State Dept of Ecology. 2005. Laboratory Guidance and Whole Effluent
   Toxicity Test Review Criteria.  Publication no. WQ-R-95-80. June 2005.
                                      41

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   I.      WATER SAMPLES

General Methodology

Water samples for copper analysis were collected in 30-mL acid-cleaned low-density
polyethylene bottles, which were acidified to pH <2 with quartz still-grade nitric acid (Q-HN03)
in a High Efficiency Particle Air (HEPA) class-100 all polypropylene working area. Copper
concentrations were measured with a Perkin-Elmer SCIEX ELAN DRC II inductively coupled
plasma with detection by mass spectrometry (ICP-MS; USEPA, 1994). If deemed necessary,
samples were diluted with 0.1 N Q-HN03 made up in high-purity (18 MO cm"1) water in order to
minimize matrix related interferences inherent to seawater. The samples were injected directly
into the ICP-MS via a Perkin-Elmer Autosampler 100.  Analytical standards were made with
Perkin-Elmer multi-element standard solution (PEMES-3) diluted in IN Q-HN03; which was
matrix matched to the salinity of the test samples. Standards were analyzed at the beginning
and end of the run. The analysis also included measurement of the Standard Reference
Material (SRM) 1643e from the National Institute of Standards & Technology (NIST), and
analytical blanks made up of IN Q-HN03 after every five samples. A coefficient of variation (CV)
of <5% for replicate measurements will be observed, as well as a recovery within 15% of SRM
1643e.

   II.     SEDIMENT SAMPLES

Sediment Digestion

Adapted from:
David Strom, Stuart L. Simpson, Graeme E. Batley, and Diane F. Jolley. 2011.  The influence of
   sediment particle size and organic carbon on toxicity of copper to benthic invertebrates in
   oxic/sub-oxic surface sediments. Environmental Toxicology and Chemistry 30(7): 1599-
   1610.
   •  Weigh a pre-labeled,  pre-dried 125 mL LDPE bottle with cap and record the  BOTTLE tare
      mass (g).
   •  Include at least six (6) blanks of a sample bottle with no sediment that will go thru all
      the treatments of a regular sample, and the three (3) Standard Reference Materials
      (SRMs), PACS-1, BCSS-1 and NIST 2709, each in triplicate (3X).
   •  Pour 0.20 ฑ 0.05 g dry sediment sample in the bottle. In case of using wet sediment,
      then pour 2.0 ฑ 0.05 g wet sediment.
   •  Weigh about 0.25 ฑ 0.05 g of each SRMs in each of three (3) separate  bottles.
   •  Set bottle with sediment in an oven at 60ฐC for at least 24 hours. Take the cap out from
      the bottle and set it down-side up by the bottle. NOTE: make sure the sediment is
      completely dry before weighing and proceeding to add any acid.
   •  Set bottle with dry sediment in a desiccator to cool down to room temperature.

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   •   Weigh and record the BOTTLE plus DRY SEDIMENT mass (g)
   •   Add 1.0 ml of concentrated trace metal grade (TMG) hydrochloric acid (HCI).
   •   Add 0.5 ml of concentrated TMG nitric acid (HN03).
   •   Allow the sample to digest at room temperature for 24 hours with loose cap.
   •   Warm up on hot plate in clean bench for 1 hour with loose cap. Hot plates are set to
       warm up to a temperature that does not melt the bottle
   •   Alternatively: Microwave 2 times for 20 minutes at 100 W with loose cap
   •   Add IN HN03 TMG to neck of bottle, about 130 g, and weigh and record BOTTLE plus
       DIGESTATE final mass (g)
   •   Allow particles to settle down and pipette volume required of digestate from the
       overlying water
   •   Alternatively: Filter thru 0.45 jam pore-size and pipette from filtered solution
Make up the appropriate dilution (e.g., 25, 50 or 100 ul of digestate to 15 ml or so) in an 15 ml
ICP-MS test tube with IN quartz-still grade HN03 (Q-HN03) for analysis using methodology
described above.

   III.    CLEAN ROOM TECHNIQUES

The clean room is located in Building 111 Room 242

   To turn on Epure System

   1.  Turn on the red valve located to the left of the Barnstead filter unit. The valve is on
       when it is parallel with the pipe.
   2.  Turn the switch located on the Barnstead unit on.
   3.  Allow digital read-out to reach 18.0 mega-ohm before using milli-Q (MQ) water.
   4.  Retrieve a lab coat and booties from adjacent room if there aren't any in the clean room
       already.
   5.  Place a bootie over one shoe at a time while stepping onto sticky pad at the entrance of
       room. Never let shoes touch the sticky pad without booties. This pad serves to remove
       any dust particles that have gotten on the booties.
   6.  Once inside the room, close the door, put on lab coat and affix all buttons.
   7.  Put on nylon gloves.
   8.  Put on one pair of plastic gloves without touching anything above the wrist area of the
       glove.
   9.  Put on a second pair of plastic gloves without touching anything above the wrist area on
       the glove. Consider the first pair of plastic gloves contaminated and do not touch the
       second pair anywhere above the wrist.
   10. Gloves can be tightened  over fingers by overlapping fingers. Hand should be kept in this
       position when not in use to avoid contamination. See figure 1.

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       Figure 1.
   11. Do not touch the walls or the inside of the hood glass with hands.
   12. Using the spicket, rinse gloves with MQ water, and then rinse the spicket itself.
   13. Rinse grating inside hood to remove any settled dust particles.
Bringing an item into the Clean room or Hood

   1.  When bringing new items into the clean room, place in container on the floor until use.
   2.  When bringing item into hood, rinse the outside entirely three to four times with MQ
       water from the spicket.
   3.  Next, rinse the inside entirely three to four times with MQ water from the spicket.
   4.  Water will be exiting into a bucket, be sure not to overflow.
   5.  Keep item near grating until rinsed thoroughly.
After use of clean room and hood

   1.  Rinse area down with spicket.
   2.  Use kimwipe to absorb water; do not repeat exposure of kimwipe surface after touching
       another.
Filtering Samples

          1.  Loosen all caps from samples
          2.  Bring in Container and label it (acid washed scint vial) to place filtered sample in.
          3.  You should have the following items; 5% nitric acid in small container, 2 MQ
              water containers, disposal container, cup with syringes, and filters.
          4.  Fill syringe with 5% nitric acid and place back into cup.
          5.  Empty acid into waste container from syringe
          6.  Semirinse with MQ water
          7.  Swirl  sample and take 5 ml through plastic tip on syringe

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          8.   Remove plastic tip from syringe within  sample allowing it to drain the tip into
              the original sample bottle, replace tip with a clean filter
          9.   Dispose of the first 5-10 drops
          10. Place remaining sample (about 5ml) in the labeled clean scint vial in hood.
          11. If there is any sample remaining, place into the disposal container
          12. Use additional MQ container to rinse syringe 2-3 times
          13. Clean with nitric acid by allowing it to sit in syringe.
          14. Rinse once with MQ water.
Begin with the lowest concentration and filter in order to the highest cone.

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