United States             Office of Water         EPA821-B-01-005
 Environmental Protection        (4303)             September 2001
 Agency
FinalRePrt:
Interlaboratory Variability Study of
EPA Short-term Chronic and
Acute Whole Effluent Toxicity Test
Methods,
Vol. 2: Appendix

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LIST OF APPENDICES

Appendix A: WET Study Plan

Appendix B: Participant Laboratory Standard Operating Procedures
      Ceriodaphnia Acute Test Standard Operating Procedure
      Ceriodaphnia Survival and Reproduction Test Standard Operating Procedure
      Fathead Minnow Acute Test Standard Operating Procedure
      Fathead Minnow Larval Survival and Growth Test Standard Operating Procedure
      Selenastrum Growth Test Standard Operating Procedure
      Mysid Survival, Growth, and Fecundity Test Standard Operating Procedure
      Sheepshead Minnow Acute Test Standard Operating Procedure
      Sheepsheed minnow Larval Survival and Growth Test Standard Operating Procedure
      Inland Silverside Acute Test Standard Operating Procedure
      Inland Silverside Larval Survival and Growth Test Standard Operating Procedure

Appendix C: List of Referee and Participant Laboratories

Appendix D: Preliminary Testing Results

Appendix E: Analysis of Percent Minimum Significant Differences

Appendix F: Method Performance Including Referee Laboratory Data

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      STUDY PLAN FOR DETERMINING INTERLABORATORY
     VARIABILITY OF THE EPA SHORT-TERM CHRONIC AND
      ACUTE WHOLE EFFLUENT TOXICITY TEST METHODS


                               TABLE OF CONTENTS

SECTION 1: INTRODUCTION AND BACKGROUND	  A-3

SECTION 2: OBJECTIVES 	  A-6

SECTION 3: STUDY MANAGEMENT	  A-8

SECTION 4: TECHNICAL APPROACH	  A-8
      4.1    Phase 1 - Laboratory Procurement	  A-8
             4.1.1   Identification of Potential Laboratories 	  A-9
             4.1.2   Selection of Referee Laboratories	  A-9
             4.1.3   Selection of Participant Laboratories 	  A-9
             4.1.4   Prequalification Requirements  	 A-10
             4.1.5   Prequalification Information Turnaround Requirements 	 A-13
      4.2    Phase 2 - Preliminary Testing  	 A-13
             4.2.1   Part 1- Characterization of Physical, Chemical, and Toxicological Properties of
                         Real-World Matrix Types  	 A-14
             4.2.2   Part 2 - Determination of the Toxicity of Spiked Reference Toxicants in the
                         Sample Matrix	 A-14
             4.2.3   Part 3 - Holding Time Testing  	 A-14
             4.2.4   Part 4 - Definitive Testing	 A-15
      4.3    Phase 3 - Sample Preparation, Packaging, and Distribution 	 A-15
             4.3.1   Description of Test Samples	 A-15
             4.3.2   Collection of Real-World Samples	 A-15
             4.3.3   Preparation of Test Samples	 A-16
             4.3.4   Packaging and Distribution of Test Samples  	 A-17
             4.3.5   Sample Tracking	 A-17
      4.4    Phase 4 - Interlaboratory Testing	 A-18
             4.4.1   Study Initiation	 A-18
             4.4.2   Preliminary Study Schedule 	 A-18
             4.4.3   General Testing Requirements  	 A-21
             4.4.4   Method-Specific Requirements	 A-23

SECTION 5:   DATA REPORTING AND EVALUATION	 A-36
                                        A-l

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                                      LIST OF TABLES

TABLE       TITLE                                                          PAGE

1.  WET Methods Included in the WET Interlaboratory Variability Study	  A-4
2.  Twelve Acute and Short-Term Chronic WET Methods	  A-5
3.  Four Phases of the WET Interlaboratory Variability Study	  A-6
4.  General Responsibilities of Parties Contributing to the WET Interlaboratory Variability Study . . .  A-8
5.  Preliminary Schedule for WET Interlaboratory Study	 A-20
6.  Fathead Minnow, Pimephales promelas, Acute Test	 A-24
7.  Fathead Minnow, Pimephales promelas, Larval Survival and Growth Test 	 A-25
8.  Cladoceran, Ceriodaphnia dubia, Acute Test	 A-26
9.  Cladoceran, Ceriodaphnia dubia, Survival and Reproduction Test	 A-27
10. Green Alga, Selenastrum capricornutum, Growth Test	 A-28
11. Inland Silverside,Me/'d/'a beryllina , Acute Test 	 A-29
12. Inland Silverside,Me/'J/'a beryllina, Larval Survival and Growth Test 	 A-30
13. Mysid, Holmesimysis costata, Acute Test  	 A-31
14. Red Macroalga, Champiaparvula, Reproduction Test  	 A-32
15. Sheepshead Minnow, Cyprinodon variegatus, Acute Test	 A-3 3
16. Sheepshead Minnow, Cyprinodon variegatus, Larval Survival And Growth Test  	 A-34
17. Mysid Shrimp, Mysidopsis bahia, Survival, Growth, and Fecundity Test	 A-35
18. Data Reporting Format	 A-36
                                             A-2

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SECTION 1: INTRODUCTION AND BACKGROUND

The Clean Water Act (CWA) requires the U.S. Environmental Protection Agency (EPA) to promulgate
guidelines establishing test procedures for data gathering and compliance monitoring under the National
Pollutant Discharge Elimination System (NPDES). Test procedures are specified at 40 CFR Part 136. On
October 16, 1995, EPA promulgated a final rule approving the use of seventeen whole effluent toxicity
(WET) test methods to protect aquatic life in NPDES compliance monitoring (60 FR 53529). Whole
effluent toxicity is defined as the aggregate toxic effect of an effluent or receiving water measured
directly as an organism response in a toxicity test.  The Agency-approved WET test methods are listed in
40 CFR 136.3, Table IA. These WET test procedures employ a suite of standardized freshwater, marine
and estuarine plants, invertebrates, and vertebrates to measure acute and short-term chronic toxicity. The
EPA-approved WET methods resulted from many years of development and testing by EPA, States,
municipalities, academia, and the regulated community. As part of a settlement agreement to resolve a
judicial challenge to the WET methods rule, EPA will conduct the WET Interlaboratory Variability Study
(hereinafter referred to as the "WET Study").

Twelve of the seventeen promulgated WET methods will be evaluated in the WET study. These include
five acute and seven short-term chronic WET methods.  The study will be implemented in three rounds.
Freshwater tests will be conducted in Round 1, and marine tests will be conducted in Rounds 2 and 3.  The
WET methods and the round in which they will be performed in the WET Study are listed in Table 1.
Table 2 identifies the test duration and test endpoints for the five acute and seven short-term chronic
methods included in the WET Study.

The WET Study was designed to quantify the interlaboratory variability of the 12 WET test methods.
This will be accomplished through (at a minimum) the determination of the coefficient of variation (CV)
for the LC50 and IC25 endpoints and the range of values for the NOEC endpoints for each method in the
study. Other measurements of method variability such as ASTM's h and k statistics also may be used to
quantify interlaboratory variability. The study  was designed to provide data on the rate at which
participating laboratories successfully complete tests initiated (test completion rate) and the rate at which
the tests indicate the presence of toxicity when measuring non-toxic samples (false positive rate).

The general design of the WET Study is as follows:

      A total of 12 WET methods (5 freshwater methods and 7 marine methods) will be conducted (See
       Tables 1 and 2).

      A minimum of 9 and a maximum of 20 participant laboratories (that meet prequalification
       requirements) will be selected to perform each WET test method.  This will  constitute the "base"
       study design. Additional laboratories (above 20) may participate on a more limited basis as part
       of an "extended" study design (see Section 4.1.3).

      Referee laboratories will conduct WET tests for each method during preliminary testing and
       simultaneously with participant laboratories  during interlaboratory testing. Preliminary testing
       will document sample characteristics and consistency, and referee laboratory results during
       interlaboratory testing will provide further information on sample consistency and may be pooled
       with participant laboratory data in the evaluation of interlaboratory method variability.

      For each method, laboratories participating in the base study design will conduct WET tests with
       four blind test samples. A "test sample" is a single bulk sample preparation (i.e, matrix, recipe)
       that is divided and distributed by the referee laboratory to participant laboratories for the conduct
       of a given test.  Aliquots of the bulk sample will be shipped to the participant laboratories as

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        whole volume (volume necessary to conduct the test) or ampules (to mix and dilute to required
        volume) for test initiation and test renewals (if necessary).

        Laboratories that are participating in the extended study design will conduct WET tests with two
        or three blind test samples received as ampules.

        Test samples received by participant laboratories will include some combination of the following
        test sample types:  reference toxicants, industrial and/or municipal wastewater effluents, ambient
        receiving water, and method "blanks"( i.e., moderately hard reagent water prepared as explained
        in the test method manuals).

        Replicate (i.e., duplicate) test samples will be included among the four blind test samples
        distributed to participant laboratories for each test method.
Table 1.  WET Methods Included in the WET Interlaboratory Variability Study	

Round 1 - Freshwater Tests
(1) Fathead Minnow, Pimephales promelas, Acute Test1
(2) Method 1000.0: Fathead Minnow, Pimephales promelas, Larval Survival and Growth Test2
(3) Cladoceran, Ceriodaphnia dubia, Acute Test1
(4) Method 1002.0: Cladoceran, Ceriodaphnia dubia, Survival and Reproduction Test2
(5) Method 1003.0: Green Alga, Selenastrum capricornutum, Growth Test2

Round 2 - Marine Tests
(1) Inland Silverside, Menidia beryllina, Acute Test1
(2) Method 1006.0: Inland Silverside, Menidia beryllina, Larval Survival and Growth Test3
(3) Mysid, Holmesimysis costata, Acute Test1
(4) Method 1009.0: Red Macroalga, Champiaparvula, Reproduction Test3

Round 3 - Marine Tests
(1) Sheepshead Minnow, Cyprinodon variegatus, Acute Test1
(2) Method 1004.0: Sheepshead Minnow, Cyprinodon variegatus, Larval Survival and Growth Test3
(3) Method 1007.0: Mysid, Mysidopsis bahia, Survival, Growth, and Fecundity Test3

'USEPA, 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
2USEPA, Short-Term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Water to Freshwater Organisms,
Third Edition, EPA-600-4-91-002, July  1994
3USEPA, Short-Term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Water to Marine and Estuarine
Organisms, Second Edition, EPA-600-4-91-003, July 1994
NOTE: EPA will conduct the WET Interlaboratory Variability Study using the specific test protocols promulgated at 40 CFR
Part 136, including, as appropriate, reference to EPA guidance entitled "Clarifications Regarding Flexibility in 40 CFR Part 136
Whole Effluent Toxicity (WET) Test Methods" dated April 10, 1996 from Tudor T. Davies, EPA Office of Science and
Technology to EPA Water Management Division Directors and EPA Environmental Services Division Directors. Additional
corrections to the method manuals are included in the following document: USEPA, Errata for Effluent and Receiving Water
Toxicity Test Manuals: Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms; Short-Term
Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms; and Short-Term
Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Marine and Estuarine Organisms, EPA-
600/R-98/182, January 1999.
                                                  A-4

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Table 2.  Twelve Acute and Short-Term Chronic WET Methods.
Round
1
1
1
1
1
2
2
2
2
3
3
3
EPA Methods for the WET Interlahoratory
Variability Study
Fathead Minnow, Pimephales promelas, Acute
Test
Method 1000.0: Fathead Minnow, Pimephales
promelas, Larval Survival & Growth Test
Cladoceran, Ceriodaphnia dubia, Acute Test
Method 1002.0: Cladoceran, Ceriodaphnia
dubia, Survival & Reproduction Test
Method 1003.0: Green Alga, Selenastrum
capricornutum, Growth Test
Inland Silverside, Menidia beryillina, Acute
Test
Method 1006.0: Inland Silverside, Menidia
beryillina, Larval Survival and Growth Test
Mysid, Holmesimysis costata, Acute Test2
Method 1009.0: Red Macroalga, Champia
parvula, Reproduction (cystocarp production)
Test
Sheepshead Minnow, Cyprinodon variegatus,
Acute Test
Method 1004.0: Sheepshead Minnow,
Cyprinodon variegatus - Larval Survival &
Growth Test
Method 1007.0: Mysid, Mysidopsis bahia,
Survival, Growth, and Fecundity Test
Acute Tests
Survival
LC50
X

X


X

X

X


Test
Duration
(Hours)
96

48


96

96

96


Short-Term Chronic Tests
Survival
LC50
NOEC

X

X


X



X
X
Growth
ICB
NOEC

X


X

X



X
X
Reprod
IC25
NOEC



X




X


X
Test
Duration
(Days)

7

81
4

7

7-93

7
7
    The C. dubia test acceptability criteria states that the test is complete when 60% of controls have 3 broods (approximately 7 days); for
    purposes of this study, all tests will continue for 8 days and each laboratory must carefully distinguish and carefully record the number of
    broods (see Section 4.4.3 and 4.4.4 of this study plan).
    The EP A-approved acute test with Holmesimysis costata will be performed using the acute test procedures for Mysidopsis bahia and test
    conditions optimized for H. costata.
    C. parvula are exposed to test substance for two days, followed by a 5-7 day recovery period in control water.
The remainder of this study plan describes the design of the WET Study. In the performance of each
WET method, participating laboratories shall follow the specific instructions that EPA (or EPA's
authorized representative) provides to perform the testing in accordance with their routine laboratory
practices using the applicable test methods from the WET final rule. Additionally, EPA will provide all
laboratories interested in the referee or participant laboratory role with detailed statements of work
(SOWs) that articulate the specific tasks, instructions, deliverables, and turnaround requirements
associated with each task. EPA may modify this study plan, the SOWs, or any specific instructions prior
to or during the performance of the WET Study.
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SECTION 2: OBJECTIVES
The primary objectives of the WET Study are to (1) generate data to characterize the interlaboratory
variability of the 12 WET methods targeted in the study, (2) obtain data on the rate at which participating
laboratories successfully completed WET tests initiated, and (3) generate data on the rate at which WET
tests indicate "toxicity" is present when measuring non-toxic samples.

The WET Study will be conducted in four phases designed to accomplish the overall study objectives.
These phases, and the specific objectives  associated with each phase, are shown in Table 3.

Table 3.  Four Phases of the WET Interlaboratory Variability Study.	
 Phase
                          Objectives
  1 - Laboratory Procurement
Identify potential referee and participant laboratories to support the study
Prequalify and select referee laboratories for Phases 2, 3, and 4
Prequalify and select participant laboratories for Phase 4 of the study
 2 - Preliminary Testing
Determine the suitability of selected real-world sample matrices for use in the
study through characterization of physical, chemical, and lexicological properties
of the test sample
Determine the appropriate spiking concentrations for reference toxicant samples to
achieve the desired range of toxicity
Determine the persistence of toxicity in real-world test samples
Assess whether the desired range of sample toxicity will be maintained in test
samples following shipping and handling
 3 - Sample Preparation and
    Distribution
Prepare real-world and synthetic test samples for use by referee and participant
laboratories in Phase 4
Minimize variability between samples prepared for and distributed to each of the
Phase 4 laboratories
Distribute blind test samples to all qualified laboratories for initial use within 36
hours of individual sample shipment from the referee laboratories
 4 - Interlaboratory Testing
Obtain interlaboratory test data for each WET method using four real-world and
synthetic test samples to evaluate precision of the test methods, the rate at which
laboratories successfully completed tests initiated, and the rate at which the tests
indicate "toxicity" is present when measuring non-toxic samples	
Six data quality objectives (DQOs) have been identified as necessary to ensure that data produced will
meet the study objectives described above. These are:

(1)     All data produced in the study must be generated in accordance with the analytical and quality
        assurance/quality control (QA/QC) procedures defined in this study plan and the following
        documents:
               Short-Term Methods for Estimating the Chronic Toxicity of Effluents and Receiving
                Water to Marine and Estuarine Organisms, Second Edition, EPA-600-4-91-003, July
                1994; (hereinafter referred to as the "Marine Chronic Methods Manual").
               Short-Term Methods for Estimating the Chronic Toxicity of Effluents and Receiving
                Water to Freshwater Organisms, Third Edition, EPA-600-4-91-002, July 1994,
                (hereinafter referred to as the "Freshwater Chronic Methods Manual").
               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;
                (hereinafter referred to as the "Acute Methods Manual").
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               "Clarifications Regarding Flexibility in 40 CFR Part 136 Whole Effluent Toxicity (WET)
               Test Methods", memorandum from Tudor Davies, Office of Science and Technology,
               USEPA dated April 10, 1996.
              Errata for Effluent and Receiving Water Toxicity Test Manuals: Acute Toxicity of
               Effluents and Receiving Waters to Freshwater and Marine Organisms; Short-Term
               Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to
               Freshwater Organisms; and Short-Term Methods for Estimating the Chronic Toxicity of
               Effluents and Receiving Waters to Marine andEstuarine Organisms, EPA-600/R-98/182,
               January 1999.

       The first three documents are referred to collectively as the "methods manuals" throughout this
       document. The test requirements in Sections 4.4.3 and 4.4.4 of this study plan and the specific
       instructions provided by EPA will define the allowable flexibility in the WET methods included
       in this study. This study plan and the specific instructions will address items agreed to by EPA in
       the settlement that are currently not specified in the methods manuals.

(2)     All test results from controls must meet the required test acceptability criteria (i.e., survival,
       minimum growth, minimum offspring/reproduction, average dry weight) specified in the methods
       manuals and Section 4.4.4 of this study plan to be considered valid. The Ceriodaphnia dubia
       Survival and Reproduction Test (Method 1002.0) will be  conducted according to the method
       manuals as a three brood test, with careful notation of times of broods.  In addition, the test will
       be conducted for eight days, with survival and reproduction measurements continuing past the
       third brood (see Section 4.4.3 and 4.4.4 for further clarification).

(3)     Test parameters must meet the  range of chemical and physical test conditions (such as
       temperature, hardness, alkalinity, ammonia, conductivity, pH, salinity, etc.) outlined in the
       appropriate methods manual and as detailed in Section 4.4.3 and 4.4.4 of this study plan.

(4)     All calculations and data produced in this study must be capable of being verified through an
       independent review of the final data package by an analyst familiar with WET testing.

(5)     Interlaboratory CVs must be calculated from a minimum of six complete and useable data sets for
       each WET test method evaluated in the study.  Therefore, EPA's objective is to increase the
       number of laboratories participating in the study sufficiently beyond six to assure that at least six
       sets of complete and useable data are available after outliers and non-usable data are removed.
       To meet this DQO, EPA will directly support a minimum of nine participant laboratories. In
       addition, non-EPA-sponsored laboratories will be included in the study (up to 20 laboratories in
       the base study design and additional laboratories in the  extended design).

(6)     Participant laboratories must represent a cross-section of the laboratory community qualified to
       conduct WET tests using the proper test procedures and QA/QC provisions detailed in the
       method manuals.

To meet these DQOs, each participating laboratory will be required to have a comprehensive QA program
in place and operating throughout this study.
                                              A-7

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SECTION 3: STUDY MANAGEMENT
The WET Study will be directed by EPA with contractual support by DynCorp Information &
Engineering Technology, Inc. under the Sample Control Center (SCC) contract (EPA Contract No. 68-C-
98-139). Overall management and technical oversight of this study will be provided by EPA Office of
Water Engineering and Analysis Division's Analytical Methods Staff (AMS) and EPA's Office of
Research and Development (ORD) staff. Laboratory procurement, day-to-day management, coordination
of study activities, data review, and preparation of the final study report will be performed by SCC under
AMS and ORD guidance.  Referee laboratories will also be contracted to support the study through the
preparation and distribution of blind test samples to participant laboratories conducting WET tests in the
WET Study. The general responsibilities of each party contributing to the WET Study are detailed in
Table 4.

Table 4.  General Responsibilities of Parties Contributing to the WET Intel-laboratory Variability
Study.
Organization
EPA
SCC
Referee Laboratory
Participant Laboratory
Responsibilities
 Assemble a WET technical workgroup from OW, AMS, ORD, and Office of
General Council (OGC) staff. This workgroup will be responsible for developing
and finalizing the study plan and providing technical oversight during the study.
 Provide overall management for the study (AMS).
 Secure funding for the study.
 Manage and approve the production of study reports.
 Support WET technical workgroup in development of study plan.
 Draft statements of work (SOW) and standard operating procedures (SOP) for
referee and participant laboratories.
 Procure referee and participant laboratories (Phase 1 of the study).
 Coordinate and provide day-to-day management of referee and participant
laboratories during study Phases 2, 3, and 4.
 Track sample shipment/receipt during study Phase 4.
 Review, validate, and analyze study data.
 Provide draft interim and final study reports to EPA.
 Collect real-world samples.
 Conduct preliminary testing on real-world and synthetic test samples (Phase 2).
 Prepare, package, and distribute test samples (Phase 3) to laboratories
participating in the base and extended study design.
 Conduct WET tests concurrently with interlaboratory testing (Phase 4).
 Conduct WET tests during interlaboratory testing and report results to SCC
(Phase 4).
SECTION 4: TECHNICAL APPROACH

4.1    Phase 1 - Laboratory Procurement

The purpose of Phase 1 is to contract referee and participant laboratory support for the WET Study.  EPA
will attempt to maximize the number of qualified laboratories participating in the study and select
laboratories that are representative of laboratories throughout the United States that routinely conduct
WET tests for permittees. At the same time, EPA will only select laboratories that possess the capacity
and capabilities, experience and proficiency, and quality assurance and quality control necessary to meet
the needs of the study. To achieve these goals, EPA will identify and solicit a large number of
laboratories, but select participant laboratories only from those that meet prequalification requirements.
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A smaller more select list of laboratories that possess exceptional qualifications (based on EPA technical
staff recommendations) will be solicited for the referee laboratory positions, since the responsibilities of
the referee laboratory are demanding and critical to successful implementation of the WET Study.

4.1.1   Identification of Potential Laboratories

Laboratories participating in the WET Study may include EPA, state, academic, municipal, industrial
and/or private laboratories.  A list of potential participant laboratories will be identified from a variety of
sources, including EPA and State environmental agencies, the Society of Environmental Toxicology and
Chemistry (SETAC), reviews of the public literature, the Directory of Environmental Laboratories1, and
EPA's Discharge Monitoring Report Quality Assurance (DMRQA) list of laboratories conducting testing
for the DMRQA program. A list of laboratories interested in participating without EPA sponsorship was
also provided by the petitioners.  All laboratories included in the compiled potential laboratory list will be
solicited as participant laboratories.  A subset of potential referee laboratories will be selected from the
laboratory list based on EPA technical staff recommendations.

4.1.2  Selection of Referee Laboratories

At least one referee laboratory for Round 1 and at least one referee laboratory for both Round 2 and 3 will
be required to conduct preliminary testing, collect and prepare blind test samples, distribute test samples
to participant laboratories, and conduct WET tests concurrently with participant laboratories during Phase
4. Potential referee laboratories will be forwarded a bid solicitation package that includes the following
documents: (1) referee laboratory prequalification document, (2) SOW, including a preliminary study
schedule,  and (3) referee laboratory bid sheet. Referee laboratories must meet all of the prequalification
requirements given in Section 4.1.4 for participant laboratories.  In addition to the requirements for
participant laboratories, the referee laboratory must submit three client references and provide
background information on potential real-world effluent and receiving water sample sources.  Referee
laboratory prequalification materials will be evaluated based on the rejection criteria listed in Section
4.1.4 and the additional reference and sample source requirements. The capacity and capabilities of
potential referee laboratories will be highly scrutinized to ensure that the laboratory can meet the  sample
collection, preparation, distribution, and testing requirements of the study. Potential referee laboratories
will be initially screened based on the prequalification requirements.  For each WET test method, the
referee laboratory that meets the prequalification requirements and has the lowest bid will be selected.

4.1.3  Selection of Participant Laboratories

All laboratories identified as described in Section 4.1.1 will receive a solicitation package from SCC that
includes the following documents: (1) a detailed cover letter describing the solicitation, (2) participant
laboratory prequalification document, (3) SOW, including a preliminary study schedule, and (4)
participant laboratory bid sheet.

All laboratories seeking to participate in the WET Study  must prequalify for each WET test method they
would like to conduct according to the requirements in Section 4.1.4. From the pool of prequalified
laboratories submitting bids, the nine lowest cost laboratories will be selected for EPA-sponsorship to
support each WET test method.  An additional maximum of 11 laboratories (for each WET test method)
will be randomly selected from the pool  of prequalified laboratories to participate in the base study design
at their own cost or an external sponsor's cost (non-EPA sponsorship).  The 9 EPA-sponsored
laboratories and the 11 randomly chosen non-EPA-sponsored laboratories will constitute the 20
'Directory of Environmental Laboratories, DynCorp, 1996.
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laboratories included in the base study design for each WET test method. All remaining prequalified
laboratories not selected for the base study design yet willing to participate without EPA sponsorship may
participate in the extended study design.

Laboratories participating in the base design will each test four blind test samples received as whole
volume samples or ampules. Laboratories participating in the extended design will each test two or three
blind test samples received as test ampules.  SCC will  formally notify all laboratories of their selection
and level of participation.

4.1.4  Prequalification Requirements

The prequalification process consists of submitting information that documents WET testing experience,
proficiency, capacity, and quality control. Laboratories may choose to prequalify to perform one or more
of the twelve WET test methods listed in Table 1. The entire prequalification process must be completed
for each WET method potential participant laboratories are interested in performing. Laboratories may
not qualify to fill both the  referee and participant laboratory role for the same test species in the study.

Laboratories must be willing and able to abide by the  statement of work and preliminary study schedule
for the conduct and timing of each WET test method for which prequalification materials are submitted.
Participant laboratories must have the capacity and capability to accommodate the testing schedule. It
may be necessary for participant laboratories to limit the number of test methods for which they submit
prequalification materials if laboratory facilities cannot meet the demands of the full testing schedule.
Laboratories should recognize that selection for participation is more likely for those methods that are less
common, however, laboratories must be prepared to perform all methods for which they submit
prequalification information.

To prepare a complete prequalification package, laboratories must address all prequalification
requirements, attach all required documentation, provide an explanation for the omission of any requisite
information, and submit the material in accordance with the turnaround requirements  in Section 4.1.5  of
this document.  Laboratories also must complete the attached laboratory bid sheet based on the
performance of the tasks outlined in the participant laboratory SOW.

Prequalification materials must document that the potential participant laboratory has the capacity and
capabilities to perform the  necessary tasks in this study, experience and proficiency in conducting the
WET test methods, and established quality assurance and quality control practices.  To demonstrate these
aspects, each potential participant laboratory must provide the following:

General information

(1)     Information (on a cover page) including the laboratory name, address, telephone number, fax
       number, e-mail address, contact person, and additional contacts for day-to-day sample tracking
       and technical issues if different from primary contact.

(2)     A statement on the number of tests that the laboratory can conduct at one time with the proposed
       staff, including the number of tests using a single test method and the number of tests using
       multiple test  methods (e.g., three C.  dubia survival and reproduction tests, three fathead minnow
       survival and growth tests, and two of each simultaneously). This information will not affect
       prequalification, but may be used for evaluating alternate study schedules if the preliminary study
       schedule must be further compressed.
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Capacity and capabilities

(3)     A statement that the combination of facilities, equipment, staff and laboratory capabilities are
       sufficient to meet study needs. In determining the sufficiency of laboratory capabilities, attention
       must be payed to the preliminary testing schedule. Participant laboratories must have the
       equipment, organisms, and personnel to accommodate this testing schedule. It may be necessary
       for participant laboratories to limit the number of test methods for which they submit
       prequalification materials if laboratory facilities cannot meet the demands of the full testing
       schedule.

(4)     Detailed information on the type and  size of laboratory and test equipment used for conducting
       each test method. Include information on temperature control, sample storage, water purification
       devices (i.e., Millipore Milli-Q filtration and ion exchange), and dilution water sources.
       Laboratories must provide summaries of routine water quality monitoring data on dilution water
       and water used for culturing or maintaining each species (e.g., 3-4 months of pH, alkalinity,
       hardness, and salinity measurements on dilution and culture waters).

(5)     A statement that the laboratory can receive next day deliveries (including Saturday deliveries) via
       overnight carriers (i.e., Fed Ex, UPS,  etc.) and initiate a test on the same day as receipt.

(6)     A list of laboratory staff able to participate in the  study, including resumes and titles.

(7)     Information on the source  of organisms. This information must include whether organisms are
       cultured in-house or obtained externally.  If cultured in-house, provide standard operating
       procedures for organism culturing (as required in number 9 below), provide a summary of how
       culture performance is assessed, and provide data on culture performance. For example, provide
       Ceriodaphnia dubia brood board monitoring data for the past six month or records of Pimephales
       promelas egg production.  If obtained from an external source, include source, number of
       organisms that can be obtained from this source on a given day, age of obtained organisms, and
       organism holding and maintenance conditions.

Experience and proficiency

(8)     Copies of internal Standard Operating Procedures (SOPs) for conducting each of the test methods
       for which prequalification is sought. Internal laboratory SOPs for each test method must be in
       place with dates of SOP origination.

(9)     Copies of supporting internal laboratory SOPs for organism culturing, food preparation, and
       dilution water preparation for each species and  each method.

(10)   A statement on the number of effluent tests conducted in the last year using each of the WET test
       methods for which prequalification is sought. Include the frequency with which test acceptability
       criteria were met in these tests and the average control response measured in these tests.

(11)   A statement regarding State or regional certifications. Does the given State or region certify
       toxicity testing laboratories?  If so, is the laboratory currently certified? Provide documentation
       of current certifications.
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Quality assurance/quality control

(12)   Evidence that the laboratory maintains control (cusum) charts for reference toxicant tests for each
       method. The laboratory must submit the most current control chart for each test method,
       covering at least 12-24 data points and showing control limits. The raw data (actual data sheets
       and summarized data) for each data point also must be provided. Data charts with NOEC and/or
       IC25 for the same test values should be provided or describe why one is used rather than the other.
       Explanations must be included if methods used to develop control charts using reference
       toxicants deviate from promulgated methods or from the previous edition of a relevant test
       protocol.

(13)   Evidence that reference toxicant tests are  conducted at the appropriate frequency (e.g., monthly
       for tests that are routinely run for NPDES permits). Along with control chart information
       described above, provide a statement on the frequency of reference toxicant testing. If control
       charts (particularly for less common test methods) are composed of fewer than 12-24 data points,
       include an explanation.

(14)   Copies of internal laboratory SOPs for conducting reference toxicant tests and constructing
       control charts.  This information must include a narrative explanation of the width of the control
       limits for the laboratory and a statement of corrective action for any toxicity test endpoint value
       that falls outside the control  limits.

(15)   Results of the most recent DMRQA study, if the lab participated. The laboratory must also
       readily provide data point(s) for each method performed for the previous year's DMRQA  study.
       If the laboratory did not participate, a narrative statement to that  effect must be included.

(16)   A signed statement of accuracy and completeness. The following statement should be included
       with the prequalification  information and signed and dated by an authorized representative of the
       laboratory: "I certify that the information provided in this prequalification package is complete
       and accurate to the best of my knowledge."

Rejection of laboratories would be based on the following:

(1)     Combination of facilities, equipment, staff and lab capacity and capabilities were insufficient to
       meet study needs.

(2)     Organism source information was not provided, culture and or collection  information  was
       severely lacking, or source information was inadequate to assess the health of the organisms
       routinely used.

(3)     Internal laboratory SOPs for each method were vague and could  not be discerned and/or were
       generally insufficient to support performance of the methods in accordance with specific
       instructions provided by EPA.

(4)     Statements regarding the  number of effluent tests conducted per year, test acceptability rates,
       average control response, and/or State certifications were not provided, did not adequately
       demonstrate proficiency in the test method, or did not adequately demonstrate that the laboratory
       is representative of laboratories throughout the United States that routinely conduct WET testing
       for permittees.
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(5)     Control charts were not adequately maintained for reference toxicant tests, or data were not
        provided (cusum chart for each endpoint and raw data for each data point). Control charts should
        cover 12-24 data points for each species and test method, or an acceptable explanation given.

(6)     Reference toxicant tests were not conducted at the appropriate frequency (monthly for tests that
        are routinely run for permits) and a satisfactory explanation was not provided.

(7)     No acceptable explanation or evidence of corrective action was provided for any control chart
        value falling outside the control limits.

(8)     Laboratory did not provide the most recent DMRQA study results, or an acceptable explanation
        for non-passing results was not provided. If the laboratory did not participate in the DMRQA
        study, the laboratory did not include an acceptable explanation as to why they did  not participate.

(9)     No signed statement of accuracy and completeness was included.

4.1.5   Prequalification Information Turnaround Requirements

All required prequalification information must be received by SCC in accordance with the turnaround
requirements listed below to be considered valid.

       Prequalification information should address each item listed in Section 4.1.4 and the order and
        format of submitted information should follow the list in Section 4.1.4.

        The laboratory must submit two copies of all prequalification information and a completed
        participant laboratory bid response sheet (if seeking EPA sponsorship) to SCC at the address
        provided below within 15  business days (three calendar weeks) of receipt of the bid solicitation
        package.

Participant laboratory procurement for this study will be conducted by SCC. Laboratories should submit
prequalification information to the following  address:

        DynCorp I&ET, Sample Control Center Contract
        6101 Stevenson Avenue
        Alexandria, VA 22304
        Attn: Robert Brent

Laboratories will be required to assume responsibility for ensuring that prequalification materials are
received within the 15 business day deadline.

4.2     Phase 2 - Preliminary Testing

The referee laboratories that are contracted to support each Round of the  study will be responsible for
conducting preliminary testing for each WET test method. This preliminary testing will be completed
two weeks prior to commencement of each testing round. A four part preliminary testing scheme will be
instituted to accomplish the following goals of preliminary testing:
(1)     Determine the suitability of selected real-world sample matrices (i.e., effluent, receiving water)
        for use in the study through characterization of physical, chemical, and toxicological properties of
        the test sample
(2)     Determine the appropriate spiking concentrations for reference toxicant samples to achieve the
        desired range of toxicity

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(3)     Determine the persistence of toxicity in real-world test samples
(4)     Assess whether test samples will provide the desired range of sample toxicity following shipping
        and handling.

4.2.1    Part 1- Characterization of Physical, Chemical, and Toxicological Properties of
        Real-World Matrix Types

Part 1 of preliminary testing will verify that selected real-world sample matrices are acceptable for study
use by assessing the physical, chemical, and toxicological characteristics of the samples.  Selection of
potential real-world effluent and receiving water sample sources will begin with the list submitted by
referee laboratories as part of prequalification materials and a review of historical information from the
source (if available).  Through consultation with SCC and EPA, a preliminary selection of the real-world
sample sources will be made for each test species.  Following this determination, the referee laboratory
will initiate Part 1 of preliminary testing.

Following sample collection, physical and chemical analysis of both the effluent sample and the receiving
water sample including alkalinity, hardness, pH, temperature, total residual chlorine, total ammonia,
dissolved oxygen, total dissolved solids, total suspended solids, total organic carbon, biological oxygen
demand, and chemical oxygen demand will be conducted. For samples that are to be used in marine tests
(Round 2 and 3), salinity also will be measured.

Following chemical and physical characterization of the sample, a single background definitive test using
each of the test species will be conducted on a sample from each real-world source. For acute and chronic
methods using the same species, the conduct of the acute background definitive test may be omitted
(acute results may be obtained from measurements nested within the chronic test). If historical
information (chemistry analysis or toxicological analysis) on the real-world matrix source is available,
this information will be submitted along with results of the background testing.  Following completion of
analysis and historical data gathering, all historical and current information will be provided to SCC and
EPA to accept or reject the sample source for use as the real-world sample matrix.

4.2.2    Part 2 - Determination of the Toxicity of Spiked Reference Toxicants in the
        Sample Matrix

The goal of Part 2 of preliminary testing is to determine the spiking concentration of reference toxicants
to achieve the desired range of toxicity for reference toxicant samples. It may also be necessary to spike
real-world matrix samples to achieve the desired range of toxicity. In Part 2 preliminary testing, a range-
finding test using each WET test method will be conducted on each sample that is to be spiked.  The
range-finding test will use a range of spiking concentrations, and results will be used to isolate the
appropriate spiking level to achieve the desired range of toxicity.

4.2.3    Part 3 - Holding Time Testing

Part 3 of preliminary testing will determine the persistence of toxicity in the real-world samples.  Excess
volume of the real-world samples will be retained from Part 1 (if real-world sample is to be unspiked) or
Part 2 (if real-world sample is to be spiked) of preliminary testing and stored in the dark at 4C. Following
storage for 7 days,  a second test (using each WET test method for which the given sample is to be used)
will be conducted and results compared to that of the initial test.  The results of holding time testing will
provide valuable information on the persistence of sample toxicity that will allow determinations of
appropriate holding times for real-world samples.  This information will be useful in the timing and
scheduling of sample preparation for interlaboratory testing.  This information may also be useful in the
event that participant laboratories do not receive samples or are not able to conduct testing on the day

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specified in the final study schedule. If Part 3 testing reveals that significant changes to toxicity occur
during sample holding, the real-world sample sources may be reconsidered at this time.

4.2.4  Part 4 - Definitive Testing

Part  4 of preliminary testing will validate that the samples and spiking concentrations (if applicable) are
appropriate for use in the study. Each sample type that will be used in interlaboratory testing will be
prepared or collected, packaged, and shipped exactly as described for interlaboratory testing (Phase 4).
The  samples will be shipped by the referee laboratory round-trip back to the referee laboratory.  Upon
receipt, the referee laboratory will then conduct the definitive WET tests as described for interlaboratory
testing (Phase 4).  If samples produce the desired and expected range of toxicity in Part 4 preliminary
testing, then the sample selection and preparation will be validated and preliminary testing is complete.  If
WET test values are not within the target range, SCC and EPA will be consulted and additional testing
may be conducted to determine more appropriate spiking concentrations or sample sources.

4.3    Phase 3 - Sample Preparation, Packaging, and Distribution

4.3.1  Description of Test Samples

As mentioned in Section 1, a "test sample" is a single bulk sample preparation (i.e, matrix, recipe) that is
provided to a participant laboratory. Aliquots of the single  bulk sample will be used for test initiation  and
renewal(s) for the WET test method under study.

Four types of test  samples will be tested using each WET test method. The four test sample types
include: reference toxicants, industrial and/or municipal wastewater effluents, ambient receiving water,
and method "blanks"( i.e., moderately hard reagent water prepared as explained in the test method
manuals).  Within each test sample type, EPA will select specific test samples that reflect the precision of
the tests and not the variability  of the toxicant or sample. Test samples also will be selected to exhibit a
range of toxicity across test sample types. Preliminary  testing (Phase 2) will validate the selection of real-
world samples and spiking concentrations for reference toxicants.

EPA will randomly distribute "blind" test samples to each laboratory for evaluation. Each participant
laboratory will receive some combination of the four test sample types.  The combination of blind test
samples received at any given laboratory may include duplicates of one or more test sample types and
may exclude one or more test sample types.  Neither EPA,  EPA's authorized representatives, nor selected
referee laboratories shall disclose the nature, number, or composition of any of the  various samples
distributed to laboratories participating in the studies.

4.3.2    Collection of Real- World Samples

The  referee laboratories will collect real-world samples for the industrial and/or municipal wastewater
effluent and ambient receiving water test sample types.  Sample collection will be conducted to supply
sufficient test sample volume for preliminary testing (Phase 2) and interlaboratory testing (Phase 4).
Samples will be collected in accordance with the procedures detailed in specific instructions provided to
the referee laboratories, the referee laboratory SOW, and Section 8 of the methods manuals. All real-
world samples will be collected as grab samples. Grab samples of effluent will be collected from the
designated NPDES sampling locations using a peristaltic pump.  Between sampling events the sampling
hose will be cleaned and rinsed thoroughly.  Prior to the collection of a sample during each sampling
period, three hose volumes of the sample will be pumped, purged, and disposed.
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Real-world samples will be collected in pre-rinsed polyethylene containers of the appropriate size to
accommodate the necessary volume of sample.  Alternatively, multiple smaller polyethylene containers
may be used to ease in the collection and transport of samples, provided that the individual containers are
combined and homogenized in a bulk container prior to sample preparation. Immediately following
sample collection, samples will be refrigerated and placed in the dark or in darkened containers.

The referee laboratory will use an SCC-assigned episode number to track each sampling event. All
samples will be identified with a five-digit EPA sample number and documented on EPA traffic reports.
Sample numbers, sample labels, and EPA traffic reports will be provided to the referee laboratory by SCC
along with detailed instructions for sample documentation.

The referee laboratory SOW and specific instructions provided to the referee laboratories will give
detailed instructions about the volume of each real-world test samples that should be collected for the
WET methods included in this study.

4.3.3     Preparation of Test Samples

The referee laboratories will prepare test samples for use in Phase 2 preliminary testing and Phase 4
interlaboratory testing.  For Phase 4, the referee laboratory will prepare four bulk test samples that will be
divided and distributed to the participant laboratories for test initiation and test renewals (if necessary).  A
portion of each bulk test sample will also remain in the referee laboratory for WET testing to be
conducted by the referee laboratory.  An additional portion (20%) will remain in the referee laboratory
until all shipped samples (including renewals) have been documented  as arriving in good condition at the
participant laboratories.  This is to ensure that extra sample is available in the case of loss  or damage
during shipment.

Test samples will be prepared in large, thoroughly cleaned, and rinsed, polyethylene containers or tanks.
Containers may be reused for preparation of separate bulk samples provided that they are properly
cleaned before reuse. Containers will be cleaned according to recommendations for cleaning of
laboratory apparatus stated in the WET methods manuals. Containers may be reused for preparation  of an
identical sample following only a rinse with deionized water.  Similar type containers will be used
to prepare samples for preliminary testing and for interlaboratory testing.

Test samples will be prepared in bulk in large containers or tanks that  satisfy the volume requirements of
the test sample needed for interlaboratory testing (Phase 4).  Ideally, each of the bulk samples (for all
laboratories and all renewals) should be prepared in a single batch container. For several tests, however,
the minimum prepared volumes may be too large to be prepared in a single batch container. Under these
circumstances the samples for test initiation and each renewal may be  prepared individually.

Bulk samples will be mixed thoroughly using a paddle or impeller to ensure homogenization prior to
division of test sample aliquots for shipment.  For spiked test samples, the bulk volume will be
homogenized prior to spiking, following spiking, and prior to division of test sample aliquots for
shipment. The bulk samples will be prepared and mixed at least 12 hours, but not more than 36 hours
prior to division of sample aliquots and shipment to participating laboratories. The holding time
requirements may be relaxed if Part 3 preliminary testing indicates that the toxicity of samples is
persistent during sample holding. During bulk sample mixing and holding, samples will remain
refrigerated at 4C in the dark.
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4.3.4     Packaging and Distribution of Test Samples

After bulk test samples have been prepared according to Section 4.3.3, each bulk test sample will be
divided into individual test sample aliquots for shipment to participant laboratories. Test sample aliquots
will be divided into containers appropriate for the individual test sample volumes.  Sample containers will
be pre-rinsed with the  sample, filled, and then sealed with zero head-space.  All samples for a given test
method will be shipped in the same container style and size. Samples will be cooled to 4C  2C prior to
shipment and then packed in coolers (e.g., 28, 48, 54-qt) containing ice packs (i.e., blue ice).  Depending
on the test method being performed by an individual participant laboratory, multiple test samples may be
shipped in one cooler.  The maximum volume of sample that can be shipped in one cooler (about 54 qt) is
approximately 21-L. Test sample volumes above 21-L will exceed the maximum weight limit for
overnight shipping. Test sample volumes above 21-L will be sent  in separate coolers. Ideally, duplicate
test sample aliquots will  be shipped in the same cooler; if test sample volume prohibits shipping
duplicates in the same  cooler, they will be  shipped under the same airbill to ensure they are shipped
together.  An EPA traffic report form and any additional information for participant laboratories regarding
test sample preparation or testing (such as reconstitution instructions for ampule samples) will be included
with each sample shipment. Referee laboratories will follow guidelines and recommendations for sample
shipment given in Section 8 of the method manuals and the referee laboratory SOW that will be provided
by SCC.

SCC will provide the referee laboratory with a list of participant laboratories for each method. The list of
participant laboratories will include addresses and contacts, as well as specifications for the test samples
each participant laboratory is to receive.  The referee laboratory will ship aliquots of test samples to each
participating laboratory that is conducting the given test.  Samples for testing at the referee laboratory will
be prepared and shipped round-trip back to the referee laboratory. Testing will be  scheduled to occur
simultaneously at each participant laboratory, so samples will be shipped overnight to arrive at each
participant laboratory on the day of scheduled testing.

4.3.5     Sample Tracking

Sample Labeling:  Each WET test method will receive an EPA episode number to designate samples
prepared  for that test method.  Each sample aliquot that is prepared and shipped will be assigned a unique
sample number. Duplicate samples will  receive different sample numbers to retain the blind sample
aspect of the study design.  For tests that require additional shipments for sample renewal, the sample
number will be the same for each initiation and renewal shipment  with the addition of a letter (A, B, and
C) after the sample number to designate the sample for use as initiation (A), renewal 1 (B), or renewal 2
(C).  The sample number will appear clearly  and permanently on each container and on each EPA traffic
report form included with the shipment.

Referee Laboratory Tracking:  SCC will provide referee laboratories with EPA traffic report forms that
must accompany each  sample shipped. The referee laboratory will clearly indicate on the traffic report
form the episode number, sample number, name and address of the referee laboratory, name and address
of the participant laboratory, date shipped, airbill number, tests requested, and pre-shipment sample
information (sample preparation date and initial water chemistry). A traffic report form specific to each
sample will be placed in  a waterproof enclosure (i.e., Ziploc bag) and packed in the cooler with the
respective sample.

For each  shipment event, the referee laboratory will also complete a sample shipment documentation
form. The form will be faxed along with a copy of the airbill to SCC immediately after sample pickup by

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the overnight carrier. This form will document the identity of each sample that is shipped. Information
reported on this form will include:
   sample number - the unique identifying number for each sample aliquot
    sample description - identifies the sample as either blank, spiked effluent, spiked effluent duplicate,
    spiked receiving water, reference toxicant, or reference toxicant duplicate
    participant laboratory name - the name of the laboratory that the sample is shipped to
   airbill number - the overnight shipping service's number that identifies each individual shipment
    size of test containers - the size of the test container in which the sample is shipped
   cooler number - a unique identifying number for the cooler in which the sample is shipped. Each
    cooler used in the study should be permanently numbered and labeled (with the referee laboratory
    name and address) to assist in locating lost coolers and to assist in retrieving coolers from participant
    laboratories.
   comments - any miscellaneous comment related to sample shipment.

Participant Laboratory Tracking: Upon receipt of each sample, participant laboratories will be
responsible for determining that the sample arrived in  satisfactory condition and for documenting receipt
of the sample, post-shipment sample water quality (temperature, pH, dissolved oxygen, and conductivity
or salinity), and any problems on the EPA traffic report form. Laboratories will be required to fax the
completed traffic report form to SCC immediately upon sample receipt and retain a copy for inclusion in
the data report. If individual test samples are unusable or not received, the participant laboratories must
contact SCC on the day of expected shipment arrival for problem resolution.

4.4     Phase 4 - Interlaboratory Testing

The general conduct of interlaboratory testing will proceed as described in Section 1 of this  study plan.
Round 1 will include the acute and short-term chronic Ceriodaphnia dubia and Pimephales promelas
tests and the short-term chronic Selenastrum capricornutum test. Round 2 includes the acute and  short-
term chronic Menidia beryllina tests, the acute Holmesimysis costata test, and the short-term chronic
Champia parvula test. Round 3 includes the acute and short-term chronic Cyprinodon variegatus tests
and the short-term chronic Mysidopsis bahia test.  Participant and referee laboratories will conduct
interlaboratory testing simultaneously according to the final study schedule.

4.4.1    Study Initiation

Following prequalification, EPA will notify participant laboratories that have been selected to take part in
the WET Study. This notification will be accompanied by a final study schedule. EPA will provide
adequate time for laboratories to prepare for study initiation.

4.4.2  Preliminary Study Schedule

The interlaboratory testing phase of the WET Study will be conducted from approximately August 1999
to February 2000, with final data reports from each participant laboratory due 30 days following
termination of the round.  A preliminary schedule for the timing of each round is provided in Table 5.
Note: This is a preliminary schedules for planning purposes only; a final study schedule will be provided
to participant laboratories with bid acceptance notification.  The structure of the schedule will remain  the
same, but dates may be slightly altered.  Testing will be scheduled to occur simultaneously at each
participant laboratory, so adherence to the finalized schedule is mandatory for all participant laboratories.
Samples will arrive at each participant laboratory on the day scheduled for test commencement.
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In order to meet project deadlines, it is necessary to overlap Rounds 1 and 2 of the study causing some
marine methods to be conducted concurrently with freshwater methods. Within each round, the study
schedule was designed to allow the conduct of only one WET test method at a time, however, one test
method may begin on the day that another test method ends. During the study, samples will be
distributed in pairs and numbered  1-4 for each test method. Testing of samples #1 and 2 will be
conducted concurrently, and testing of samples #3 and 4 will be conducted concurrently.
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Table 5. Preliminary Schedule for WET Intel-laboratory Study
Approximate Date
6/11/99 - 7/5/99
6/11/99
7/5/99
8/9/99
8/24/99 - 10/25/99
8/24/99 - 8/26/99
8/26/99 - 8/28/99
8/31/99-9/4/99
9/9/99 - 9/13/99
9/14/99 - 9/21/99
9/21/99-9/28/99
9/28/99 - 10/6/99
10/7/99- 10/11/99
10/12/99 - 10/20/99
10/21/99- 10/25/99
11/24/99
8/24/99 - 10/30/99
8/24/99 - 9/2/99
9/9/99-9/18/99
9/21/99-9/25/99
9/28/99 - 10/2/99
10/5/99 - 10/12/99
10/12/99 - 10/19/99
10/19/99 - 10/23/99
10/26/99 - 10/30/99
11/29/99
1/11/00 - 2/19/00
1/11/00- 1/18/00
1/18/00- 1/25/00
1/25/00 - 2/1/00
2/1/00 - 2/8/00
2/8/00 - 2/12/00
2/15/00-2/19/00
3/20/00
Activity
Participant laboratory prequalification
DynCorp SCC solicits participant labs
Prequalification materials due
DynCorp SCC to award participant labs
Round 1 Testing
Conduct Cladoceran, Ceriodaphnia dubia, Acute Test with samples #1&2
Conduct Cladoceran, Ceriodaphnia dubia, Acute Test with samples #3&4
Conduct Green Alga, Selenastrum capricornutum, Growth Test with samples #1&2
Conduct Green Alga, Selenastrum capricornutum, Growth Test with samples #3&4
Conduct Fathead Minnow, Pimephales promelas, Larval Survival and Growth Test with samples #1&2
Conduct Fathead Minnow, Pimephales promelas, Larval Survival and Growth Test with samples #3&4
Conduct Cladoceran, Ceriodaphnia dubia, Survival and Reproduction Test with samples #1&2
Conduct Fathead Minnow, Pimephales promelas, Acute Test with samples #1&2
Conduct Cladoceran, Ceriodaphnia dubia, Survival and Reproduction Test with samples #3&4
Conduct Fathead Minnow, Pimephales promelas, Acute Test with samples #3&4
Round 1 data due
Round 2 Testing
Conduct Red Macroalga, Champiaparvula, Reproduction Test with samples #1&2
Conduct Red Macroalga, Champiaparvula, Reproduction Test with samples #3&4
Conduct Mysid, Holmesimysis costata, Acute Test with samples #1&2
Conduct Mysid, Holmesimysis costata, Acute Test with samples #3&4
Conduct Inland Silverside, Menidia beryllina, Larval Survival and Growth Test with samples #1&2
Conduct Inland Silverside, Menidia beryllina, Larval Survival and Growth Test with samples #3&4
Conduct Inland Silverside, Menidia beryllina, Acute Test with samples #1&2
Conduct Inland Silverside, Menidia beryllina, Acute Test with samples #3&4
Round 2 data due
Round 3 Testing
Conduct Sheepshead Minnow, Cyprinodon variegatus, Larval Survival and Growth Test with samples #1&2
Conduct Mysid, Mysidopsis bahia, Survival, Growth, and Fecundity Test with samples #1&2
Conduct Sheepshead Minnow, Cyprinodon variegatus, Larval Survival and Growth Test with samples #3&4
Conduct Mysid, Mysidopsis bahia, Survival, Growth, and Fecundity Test with samples #3&4
Conduct Sheepshead Minnow, Cyprinodon variegatus, Acute Test with samples #1&2
Conduct Sheepshead Minnow, Cyprinodon variegatus, Acute Test with samples #3&4
Round 3 data due
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4.4.3        General Testing Requirements

Each laboratory selected to participate in the base study design will receive four blind test samples (as
whole volume samples or ampules) for each method they are prequalified to perform. Additionally,
sample aliquots of each test sample type will be analyzed in the referee laboratories. Each laboratory
participating in the extended study design will receive two or three blind test samples (as ampules) for
each method they are prequalified to perform. Instructions will be included for reconstituting the ampule
samples. Whole volume samples and reconstituted ampule samples should be treated as if they are
effluent samples being tested for compliance monitoring purposes. Except where indicated in Sections
4.4.3 and 4.4.4 of this study plan and specific instructions provided to participant laboratories, each test
will be conducted in accordance with the general guidance and method specific requirements for effluent
testing included in the methods manuals.

EPA acknowledges that the promulgated WET methods distinguish between requirements (indicated by
the compulsory terms "must" and "shall") and recommendations and guidance (indicated by discretionary
terms "should" and "may"). The latter terms indicate that the analyst has flexibility to optimize successful
test completion and when standardization is necessary to assure the predictability of the methods to
provide reliable results. Additionally, the method manuals allow variations of the methods which are
typically fixed in the permit; therefore, for the purposes of this study, a set of variables will be defined by
EPA (for example, dilution water, salinity, and acute test duration). Any deviation from defined test
procedures and/or conditions, such as the necessity to change reagents, equipment, test conditions, or
other specified test parameters must be reported to SCC, recorded at the time of modification, noted in
telephone  logs of communications, documented in a memorandum, and approved by EPA.

Additional WET test requirements and general requirements listed in the methods manuals of special note
are provided below:

(1)     Personnel that conduct tests must be the same personnel that routinely conduct the WET tests at
        that laboratory facility and who were identified in the prequalification materials.  If these
        individuals cannot be available during any part of the study, the laboratory must contact SCC.
        Personnel conducting the tests must be identified clearly and consistently in records.

(2)     To coordinate testing at participant laboratories, testing of each sample with each method must be
        initiated on the precise day specified in the finalized study schedule. The  finalized study
        schedule will be distributed to participating laboratories prior to commencement of each study
        round and in ample time to prepare for testing.  Samples should be tested within 36 hours from
        the time  of sample preparation (determined in this study as the time at which individual sample
        aliquots were divided from the bulk test sample for distribution to participant laboratories).
        Deviation from this schedule must be reported to SCC immediately for approval.

(3)     Physical and chemical properties of the test samples must be in the ranges specified in this study
        plan, the SOW, specific instructions, and the methods manuals. Method specific instructions for
        any adjustments to the test samples prior to sample use (such as reconstitution of ampule samples
        or salinity adjustments) will be provided to the testing laboratories with the sample.  Test samples
        received at participant laboratories must be refrigerated (at 4C  2C) immediately upon receipt
        and throughout the period of testing.  The temperature of the refrigeration unit should be
        routinely or continuously monitored to ensure that these sample holding requirements are met.
                                              A-21

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(4)     Measurement of test conditions (pH, conductivity or salinity, total alkalinity, total hardness, and
       dissolved oxygen) shall be performed for each method by the participant laboratories following
       guidance in method manuals.

(5)     The specified dilution and control waters (listed in Tables 6-17 for each test method) must be
       used and prepared according to instructions in Section 7 of the methods manuals.  Marine waters
       must also be prepared to meet the salinity ranges for each test (given in Tables 11  - 17).

(6)     All WET tests are to be definitive tests with a control and a minimum of five test concentrations
       prepared using a dilution factor of 0.5.

(7)     All tests must be conducted using the number of replicates and number of test containers per
       concentration as specified in Section 4.4.4.

(8)     Test chambers used within a test must be of the same type, size,  shape, and material.  The
       material must be allowed by the method manuals for the method used.

(9)     Test vessels shall be randomized  in accordance with the method manuals. In addition, block
       randomization and use of known  parentage will be required for the Ceriodaphnia dubia survival
       and reproduction test as described in Method 1002.0. The Agency plans to amend Method
       1002.0 (Ceriodaphnia dubia Survival and Reproduction test) to require that test organisms be
       allocated among test replicates so that offspring of each female are evenly distributed among test
       replicates ("blocking-by-parentage").

(10)   The Ceriodaphnia dubia Survival and Reproduction Test (Method 1002.0), which would
       otherwise be terminated after 3 broods according to  Section 13.12.1 of that method, must be
       conducted for  8 days,  with endpoints including survival, number of young per day, and number of
       broods recorded each day. These  readings are to be made at the end of the 6th, 7th and 8th day
       (specifically, at 144 hours, at 168 hours, and at 192 hours, respectively, from test initiation). This
       will be done to assess  the effect of that test acceptance criterion on test results. No test shall be
       terminated prior to the eighth day for any reason, including a failure to meet test acceptance
       criteria. The additional measurements on days 6, 7,  and 8 should be included as raw data in the
       final data report, but should not affect the data analysis of test results. The analysis of data from
       the C. dubia chronic test shall be  conducted as specified in the method manual using the three
       brood approach.

(11)   The Green Algae, Selenastrum capricornutum, Growth Test shall be  conducted simultaneously
       with and without EDTA for each sample.  For laboratories participating in the base study design,
       four samples will be tested with and without EDTA  for a total of eight analyses. For laboratories
       participating in the extended study design, two or three samples will be tested with and without
       EDTA for a total of four or six analyses.

(12)   Daily observation of mortality and removal of dead  organisms for each test is required, except for
       the Selenastrum capricornutum and Champiaparvula tests. Daily young counts are required for
       the Ceriodaphnia dubia survival and reproduction test, along with determining the number of
       broods at each count.

(13)   If test results indicate too great of toxicity (i.e., control mortalities, or complete mortality in all
       concentrations), the laboratories must contact SCC immediately  and then investigate possible

                                             A-22

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       causes, first by checking for transcription and calculation mistakes, and then by investigating
       possible contamination in dilution waters, organism cultures, equipment, or other procedural
       steps.

(14)   If any test that has been initiated fails to be completed for any reason, the laboratory must contact
       SCC immediately for problem resolution and scheduling of additional testing. The incomplete
       test data and the reason for not completing the test must be fully documented in the final report.

(15)   Each laboratory shall be required to report all data obtained during the course of testing,
       including the response of control samples.

(16)   Laboratories must perform all QA/QC tests listed in Section 4 of the method manuals.
       Laboratories that purchase organisms must supply QA/QC from the test organism supplier and
       follow method manuals for the appropriate QA/QC for purchasing organisms.

(17)   A reference toxicant QC test must be performed for each test method in the month that testing for
       this study occurs. Results of this test must be submitted with the final data package.

(18)   Data and statistical analyses must be submitted in hard copy in the standardized format specified
       in Section 5 of this study plan. All bench sheets and raw data, including sample tracking and
       chemistry analysis data must be submitted. Data must also be submitted electronically according
       to an electronic template (Microsoft Excel spreadsheet, or equivalent) that will be provided by
       SCC prior to test initiation.

(19)   Data analysis must be performed in accordance with the statistical programs specified in the
       methods manuals. Statistical methods and programs used must be reported along with sample
       calculations.

(20)   An LC50 must be reported for each  acute test. An NOEC and LC50 for survival, and an NOEC and
       IC25 for growth/reproduction must be reported as appropriate for each short-term chronic test as
       described in the method manuals and Table 2 of this study plan. The laboratories must report
       individual toxicity endpoints; laboratories are not allowed to average or perform other data
       manipulations unless required by a methods's instructions.

4.4.4   Method-Specific Requirements

The summary of test conditions for the twelve WET methods to be evaluated in the WET Study are
provided in Tables 6 - 17. These tables are extracted from the summary test condition tables in the
methods manuals and modified to fit the scope of this study.  Items that are bold italic in these tables
represent conditions standardized for the purposes of this study where method manuals provide a range.
Final SOPs for sample preparation (i.e., reconstitution of ampules) and test conduct will be provided to
each participant laboratory prior to study initiation.
                                              A-23

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Table 6.  Fathead Minnow, Pimephales promelas, Acute Test. Summary of test conditions and test acceptability criteria for fathead minnow,
Pimephales promelas, acute toxicity tests with effluents and receiving waters	
1. Test type:                                            Static-renewal
1. Test duration:                                         96 h
3. Temperature:                                         251C
4. Light quality:                                         Ambient laboratory illumination
5. Light intensity:                                       10-20 uE/m2/s (50-100 ft-c) (ambient laboratory levels)
6. Photoperiod:                                         16 h light, 8 h darkness
7. Test chamber size:                                    250 mL
8. Test solution volume:                                  200 mL
9. Renewal of test solutions:                              At 48 h
10. Age of test organisms:                                1-14 days; 24-h range in age
11. No. organisms per test chamber:                       10
12. No. replicate chambers per concentration:               2
13. No. organisms per concentration:                       20
14. Feeding regime:                                     Artemia nauplii are made available while holding prior to the test; add 0.2 mL Artemia nauplii
                                                       concentrate 2 h prior to test solution renewal at 48 h
15. Test chamber cleaning:                               Cleaning not required
16. Test solution aeration:                                None, unless DO concentration falls below 4.0 mg/L; rate should not exceed 100 bubbles/min
17. Dilution water:                                      Moderately hard synthetic water prepared using MILLIPORE MILLI-Q or equivalent
                                                       deionized water and reagent grade chemicals (see Methods Manual Section 7, Dilution Water)
18. Test concentrations:                                  Five concentrations and a control
19. Dilution factor                                      > 0.5
20. Endpoint:                                           Mortality (LC50)
21. Sample handling and holding requirements:              Samples treated as effluent samples for NPDES monitoring. Samples are to be used on the day
                                                       specified by the finalized interlab oratory study testing schedule
22. Sample volume required:                              2 Lfor effluents
23. Test acceptability criterion:	90% or greater survival  in controls	
NOTE: Test vessels shall be randomized in accordance with the method manuals.
                                                                    A-24

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Table 7.  Fathead Minnow, Pimephales promelas, Larval Survival and Growth Test. Summary of test conditions and test acceptability criteria
for fathead minnow, Pimephales promelas, larval survival and growth toxicity tests with effluents and receiving waters	
1. Test type:
2. Temperature:
3. Light quality:
4. Light intensity:
5. Photoperiod:
6. Test chamber size:
7. Test solution volume:
8. Renewal of test solutions:
9. Age of test organisms:
10. No. larvae per test chamber:
11. No. replicate chambers per concentration:
12. No. larvae per concentration:
13. Source of food:
14. Feeding regime:
15. Cleaning:
16. Aeration:
17. Dilution water:

18. Test concentrations:
19. Dilution factor
20. Test duration:
21. Endpoints:
22. Test acceptability criteria:

23. Sample handling and holding requirements:

24. Sample volume required:	
Static renewal
251C
Ambient laboratory illumination
10-20 nE/m2/s (50-100 ft-c) (ambient laboratory levels)
16 h light, 8 h darkness
500 ml
250 ml
Daily
Newly hatched larvae less than 24h old. If shipped, not more than 48h old, 24h range in age
10
4
40
Newly \\atc\\edArtemia nauplii (less than 24 h old)
Feed 0.1 g newly hatched (less than 24-h old) brine shrimp nauplii three times daily at 4-h
intervals or, as a minimum, 0.15 g twice daily, 6 hbetween feedings (at the beginning of the work
day prior to renewal, and at the end of the work day following renewal). Sufficient nauplii are
added to provide an excess. Larvae fish are not fed during the final 12 h of the test
Siphon daily,  immediately before test solution renewal
None, unless DO concentration falls below 4.0 mg/L. Rate should not exceed 100 bubbles/min
Moderately hard synthetic water prepared using MILLIPORE MILLI-Q or equivalent
deionized water and reagent grade chemicals (see Methods Manual Section 7, Dilution Water)
Five concentrations and a control
>0.5
7 days
Survival and growth (weight as mean per original)
80% or greater survival in controls; average dry weight per surviving organism in control
chambers equals or exceeds 0.25 mg/surviving
Samples treated as effluent samples for NPDES monitoring. Samples are to be used on the day
specified by the finalized interlab oratory study testing schedule
2.5 L/day
NOTE: Test vessels shall be randomized in accordance with the method manuals.
                                                                    A-25

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Table 8.  Cladoceran, Ceriodaphnia dubia, Acute Test. Summary of test conditions and test acceptability criteria for daphnid, Ceriodaphnia
dubia acute toxicity tests with effluents and receiving waters	
1. Test type:                                             Static non-renewal
1. Test duration:                                         48 h
3. Temperature:                                         251C
4. Light quality:                                         Ambient laboratory illumination
5. Light intensity:                                        10-20 uE/m2/s (50-100 ft-c) (ambient laboratory levels)
6. Photoperiod:                                          16 h light, 8 h darkness
7. Test chamber size:                                     30 mL
8. Test solution volume:                                  75 mL
9. Renewal of test solutions:                              None
10. Age of test organisms:                                Less than 24-h old
11. No. organisms per test chamber:                        5
12. No. replicate chambers per concentration:               4
13. No. organisms per concentration:                       20
14. Feeding regime:                                      Feed YCT and Selenastmm while holding prior to the test; newly-released young should have
                                                       food available a minimum of 2 h prior to use in a test.
15. Test chamber cleaning:                               Cleaning not required
16. Test chamber aeration:                                None
17. Dilution water:                                       Moderately hard synthetic water prepared using MILLIPORE MILLI-Q or equivalent
                                                       deionized water and reagent grade chemicals (see Methods Manual Section 7, Dilution Water)
18. Test concentrations:                                  Five concentrations and a control
19. Dilution factor                                       > 0.5
20. Endpoint:                                           Mortality (LC50)
21.  Sample handling and holding requirements:             Samples treated as effluent samples for NPDES monitoring. Samples are to be used on the day
                                                       specified by the finalized interlab oratory study testing schedule
22. Sample volume required:                              1 L
23. Test acceptability  criterion:	90% or greater survival in controls	
NOTE: Test vessels shall be randomized in accordance with the method manuals.
                                                                    A-26

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Table 9.  Cladoceran, Ceriodaphnia dubia, Survival and Reproduction Test. Summary of test conditions and test acceptability criteria for
daphnid, Ceriodaphnia dubia, survival and reproduction toxicity tests with effluents and receiving waters
1. Test type:
2. Temperature (C):
3. Light quality:
4. Light intensity:
5. Photoperiod:
6. Test chamber size:
7. Test solution volume:
8. Renewal of test solutions:
9. Age of test organisms:
10. No. neonates per test chamber:1
11. No. replicate test chambers per concentration:
12. No. neonates per test concentration:
13. Feeding regime:
14. Cleaning:
15. Aeration:
16. Dilution water:

17. Test concentrations:
18. Dilution factor:
19. Test duration:2
20. Endpoints:
21. Test acceptability criteria:

22.  Sample handling and holding requirements:

23. Sample volume required:	
Static renewal
25 1C
Ambient laboratory illumination
10-20 uE/m2/s, or 50-100 ft-c (ambient laboratory levels)
16 h light, 8 h dark
30 mL
15 ml
Daily
Less than 24 h; and all released within a 8-h period
1
10
10
Feed 0.1 mL each of YCT and algal suspension per test chamber daily.
Use freshly cleaned glass beakers or new plastic cups daily
None
Moderately hard synthetic water prepared using MILLIPORE MILLI-Q or equivalent
deionized water and reagent grade chemicals (see Methods Manual Section 7, Dilution Water)
Five concentrations and a control
>0.5
8 days
Survival and reproduction
80% or greater survival and an average of 15 or more young per surviving female in the control
solutions. 60% of surviving control organisms must produce three broods.
Samples treated as effluent samples for NPDES monitoring.  Samples are to be used on the day
specified by the finalized interlab oratory study testing schedule
1 L/day	
1 Test vessels shall be randomized in accordance with the method manuals. In addition, block randomization and use of known parentage will be required for the Ceriodaphnia
survival and reproduction test as described in the manual and guidance will be reiterated in the specific instructions provided to the laboratories.
2 The Ceriodaphnia dubia test which would otherwise be terminated after 3 broods according to methods manual Section 13.12.1 of that Method must be conducted for 8 days,
with endpoints (survival and number of young per day and number of broods at each recording interval) recorded at the end of the 6th, 7th and 8th day (specifically, at 144, 168,
and 192 hours, respectively, from test initiation). No test shall be terminated prior to the eighth day for any reason, including a failure to meet test acceptance criteria.
                                                                       A-27

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Table 10.  Green Alga, Selenastrum capricornutum, Growth Test. Summary of test conditions and test acceptability criteria for green alga,
Selenastrum capricornutum, growth toxicity tests with effluents and receiving waters. Test will be conducted with EDTA and without EDTA.
1. Test type:                                           Static non-renewal
2. Temperature:                                        25  1C
3. Light quality:                                        "Cool white" fluorescent lighting
4. Light intensity:                                       86  8.6 uE/m2/s (400  40 ft-c or 4306 lux)
5. Photoperiod:                                         Continuous illumination
6. Test chamber size:                                    250 mL
7. Test solution volume:                                 100 mL
8. Renewal of test solutions:                              None
9. Age of test organisms:                                 4 to 7 days
10. Initial cell density in test chambers:                    10,000 cells/mL
11. No. replicate chambers per concentration:               4
12. Shaking rate:                                        100 cpm continuous
13. Aeration:                                           None
14. Dilution water:                                      Algal stock culture medium, moderately hard synthetic water prepared using MILLIPORE
                                                      MILLI-Q or equivalent deionized water and reagent grade chemicals(see Methods Manual
                                                      Section 7, Dilution Water)
15. Test concentrations:                                  Five concentrations and a control
16. Test dilution factor:                                  >0.5
17. Test duration:                                       96 h
18. Endpoint:                                          Growth (cell counts)
19. Test acceptability criteria:                             1 X 106 cells/mL with EDTA or 2 X 105 cells/mL without EDTA in the controls; Variability of
                                                      controls should not exceed 20%
20.  Sample handling and holding requirements:             Samples treated as effluent samples for NPDES monitoring. Samples are to be used on the day
                                                      specified by the finalized interlaboratory study testing schedule
21. Sample volume required:	2 L	
NOTE: Test vessels shall be randomized in accordance with the method manuals.
                                                                   A-28

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Table 11. Inland Silverside, Menidia beryllina , Acute Test. Summary of test conditions and test acceptability criteria for inland silverside,
Menidia beryllina, acute toxicity test with effluents and receiving waters	
1. Test type:                                             Static-renewal
2. Test duration:                                         96 h
3. Temperature:                                          25 C  1 C
4. Light quality:                                          Ambient laboratory illumination
5. Light intensity:                                        10-20 A(E/m2/s (50-100 ft-c) (ambient laboratory levels)
6. Photoperiod:                                          16 h light, 8 h darkness
7. Test chamber size:                                     250 mL
8. Test solution volume:                                  200 mL
9. Renewal of test solutions:                              At 48 h
10. Age of test organisms:                                 9-14 days; 24-h range in age
11. No. organisms per test chamber:                        10
12. No. replicate chambers per concentration:                2
13. No. organisms per concentration:                       20
14. Feeding regime:                                     Artemia nauplii are made available while holding prior to the test; add 0.2 mL Artemia nauplii
                                                        concentrate  2 h prior to test solution renewal at 48 h
15. Test chamber cleaning:                                Cleaning not required
16. Test solution aeration:                                 None, unless DO concentration falls below 4.0 mg/L; rate should not exceed 100 bubbles/min
17. Dilution water:                                       25%o (2%o) Bioassay Grade Forty Fathoms artificial seawater prepared with MILLIPORE
                                                       MILLI-Q or equivalent deionized water (see Methods Manual Section 7, Dilution Water)
18. Test concentrations:                                   Five concentrations and a control
19. Dilution factor:                                       > 0.5
20. Endpoint:                                           Mortality (LC50)
21. Sample handling and holding requirements:              Samples treated as effluent samples for NPDES monitoring.  Samples are to be used on the day
                                                        specified by the finalized interlab oratory study testing schedule
22. Sample volume required:                              1 Lfor effluents
23. Test acceptability criterion:                             90% or greater survival in controls
24. Salinity:	25%0(2%0)	
NOTE: Test vessels shall be randomized in accordance with the method manuals.
                                                                     A-29

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Table 12.  Inland Silverside, Menidia beryllina, Larval Survival and Growth Test. Summary of test conditions and test acceptability criteria
for the inland silverside, Menidia beryllina, larval survival and growth test with effluents and receiving waters	
1.  Test type:
2.  Salinity:
3.  Temperature:
4.  Light quality:
5.  Light intensity:
6.  Photoperiod:
7.  Test chamber size:
8.  Test solution volume:
9.  Renewal of test solutions:
10. Age of test organisms:
11. No. larvae per test chamber:
12. No. replicate chambers per concentration:
13. No. larvae per concentration:
14. Source of food:

15. Feeding regime:
16.
17.
Cleaning:
Aeration:
18. Dilution water:

19. Test concentrations:
20. Dilution factor:
21. Test duration:
22. Endpoints:
23. Test acceptability criteria:
24. Sample handling and holding requirements:

25. Sample volume required:	
Static renewal
25%0(2%<)
251C
Ambient laboratory illumination
10-20 uE/m2/s (50-100 ft-c) (Ambient laboratory levels)
16 h light, 8 h darkness
1 L containers
750 mL/replicate (loading and DO restrictions must be met)
Daily
7-11 days post hatch; 24-h range in age
10
4
40
Newly \\atc\\edArtemia nauplii (survival of 7-9 days old Menidia beryllina larvae improved by
feeding 24 h oldArtemia)
Feed 0.10 g wet weight^4rte/w/'a nauplii per replicate on days 0-2; Feed 0.15 g wet weight Artemia
nauplii per replicate on days 3-6
Siphon daily, immediately before test solution renewal and feeding
None, unless DO concentration falls below 4.0 mg/L, then aerate all chambers. Rate should be
less than 100 bubbles/min.
25%o (2%o) Bioassay  Grade Forty Fathoms artificial seawater prepared with MILLIPORE
MILLI-Q or equivalent deionized water (see Methods Manual Section 7, Dilution Water)
Five concentrations and a control
>0.5
7 days
Survival and growth (weight)
80% or greater survival in controls, 0.50 mg average dry weight of control larvae when larvae
dried immediately after test termination, or 0.43 mg or greater average dry weight of control
larvae, preserved not more than 7 days in 4% formalin or 70% ethanol
Samples treated as effluent samples for NPDES monitoring. Samples are to be used on the day
specified by the finalized interlab oratory study testing schedule
6 L/day
NOTE: Test vessels shall be randomized in accordance with the method manuals.
                                                                     A-30

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Table 13.  Mysid, Holmesimysis costata, Acute Test. Summary of test conditions and test acceptability criteria for mysid, Holmesi'mysis costata,
acute toxicity tests with effluents and receiving waters. The acute test procedure described in the Acute Methods Manual for Mysidopsis bahia
will be used for this test with a salinity of 32%o  ( 2%o) and a temperature of 12 C  1 C.	
1. Test type:
2. Test duration:
3. Temperature:
4. Light quality:
5. Light intensity:
6. Photoperiod:
7. Test chamber size:
8. Test solution volume:
9. Renewal of test solutions:
10. Age of test organisms:
11. No. organisms per test chamber:
12. No. replicate chambers per concentration:
13. No. organisms per concentration:
14. Feeding regime:

15. Test chamber cleaning:
16. Test solution aeration:
17. Dilution water:
18. Test concentrations:
19. Dilution factor:
20. Endpoint:
21. Sample handling and holding requirements:

22. Sample volume required:
23. Test acceptability  criterion:
24. Salinity:	
Static-renewal
96 h
12C1C
Ambient laboratory illumination
10-20 A(E/m2/s (50-100 ft-c) (ambient laboratory levels)
16 h light, 8 h darkness
250 ml
200 ml
At48h
1-5 days; 24-h range in age
10
4
40
Artemia nauplii are made available while holding prior to the test; feed 0.2 mL of concentrated
suspension of Artemia nauplii < 24-h old, daily (approximately 100 nauplii per mysid)
Cleaning not required
None, unless DO concentration falls below 4.0 mg/L; rate should not exceed 100 bubbles/min
32%o salinity  natural seawater
Five concentrations and a control
>0.5
Mortality (LC50)
Samples treated as effluent samples for NPDES monitoring.  Samples are to be used on the day
specified by the finalized interlaboratory study testing schedule
1 Lfor effluents
90% or greater survival in controls
32%0(2%<)	
NOTE: Test vessels shall be randomized in accordance with the method manuals.
                                                                    A-31

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Table 14.  Red Macroalga, Champia parvula, Reproduction Test. Summary of test conditions and test acceptability criteria for the red
macroalga, Champia parvula, sexual reproduction test	
1. Test type:                                            Static non-renewal
2. Salinity:                                              30%o(2%0)
3. Temperature:                                         231C
4. Photoperiod:                                         16 h light, 8 h darkness
5. Light intensity:                                       75 uE/m2/s (500 ft-c)
6. Light source:                                         Cool-white fluorescent lights
7. Test chamber size:                                     200 mL polystyrene cups, or 250 mL Erlenmeyer flasks
8. Test solution volume:                                  100 mL
9. No. organisms per test chamber:                        5 female branch tips and 1 male plant
10. No. replicate chambers per concentration:               4
11. No. organisms per concentrations:                      24
12. Dilution water:                                      30%o salinity natural seawater
13. Test concentrations:                                  Five concentrations and a control
14. Test dilution factor:                                  >0.5
15. Test duration:                                       2 day exposure to effluent, followed by 5 to 7-day recovery period in control medium for
                                                       cyctocarp development
16. Endpoints:                                          Reduction in cystocarp production compared to controls
17. Test acceptability criteria:                             80% or greater survival, and an average of 10 cystocarps per plant in controls
18. Sample handling and holding requirements:             Samples treated as effluent samples for NPDES monitoring.  Samples are to be used on the day
                                                       specified by the finalized interlab oratory study testing schedule
19. Sample volume required:	2 L per test	
NOTE: Test vessels shall be randomized in accordance with the method manuals.
                                                                    A-32

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Table 15.  Sheepshead Minnow, Cyprinodon variegatus, Acute Test. Summary of test conditions and test acceptability criteria for sheepshead
minnow, Cyprinodon variegatus, acute toxicity tests with effluents and receiving waters	
1. Test type:                                            Static renewal
1. Test duration:                                         96 h
3. Temperature:                                         25 C  1 C
4. Light quality:                                         Ambient laboratory illumination
5. Light intensity:                                       10-20 uE/m2/s or (50-100 ft-c) (ambient laboratory levels)
6. Photoperiod:                                          16 h light, 8 h darkness
7. Test chamber size:                                     250 mL
8. Test solution volume:                                  200 mL
9. Renewal of test solutions:                              At 48 h
10. Age of test organisms:                                1-14 days; 24-h range in age
11. No. organisms per test chamber:                       10
12. No. replicate chambers per concentration:               2
13. No. organisms per concentration:                       20
14. Feeding regime:                                      Artemia nauplii are made available while holding prior to the test; add 0.2 mL Artemia nauplii
                                                       concentrate 2 h prior to test solution renewal at 48 h
15. Test chamber cleaning:                                Cleaning not required
16. Test solution aeration:                                None, unless DO concentration falls below 4.0 mg/L; rate should not exceed 100 bubbles/min
17. Dilution water:                                      25 %o  2%oBioassay Grade Forty Path oms artificial seawater prepared with MILLIPORE
                                                       MILLI-Q or equivalent deionized water (see Methods Manual Section 7, Dilution Water)
18. Test concentrations:                                  Five concentrations and a control
19. Dilution factor                                      > 0.5
20. Endpoint:                                           Mortality (LC50)
21. Sample handling and holding requirements:              Samples treated as effluent samples for NPDES monitoring. Samples are to be used on the day
                                                       specified by the finalized interlab oratory study testing schedule
22. Sample volume required:                              1 Lfor effluents
23. Test acceptability criterion:                            90% or greater survival  in controls
24. Salinity:	25%0(2%0)	
NOTE: Test vessels shall be randomized in accordance with the method manuals.
                                                                    A-33

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Table 16.  Sheepshead Minnow, Cyprinodon variegatus, Larval Survival And Growth Test. Summary of test conditions and test acceptability
criteria for the sheepshead minnow, Cyprinodon variegatus, larval survival and growth test with effluents and receiving waters	
1. Test type:
2. Salinity:
3. Temperature:
4. Light quality:
5. Light intensity:
6. Photoperiod:
7. Test chamber size:
8. Test solution volume:
9. Renewal of test solutions:
10. Age of test organisms:
11. No. larvae per test chamber:
12. No. replicate chambers per concentration:
13. No. larvae per concentration:
14. Source of food:
15. Feeding regime:

16. Cleaning:
17. Aeration:

18. Dilution water:

19. Test concentrations:
20. Dilution factor:
21. Test duration:
22. Endpoints:
23. Test acceptability criteria:
24. Sample handling and holding requirements:

25. Sample volume required:	
Static renewal
25%0(2%0)
251C
Ambient laboratory illumination
10-20 nE/m2/s (50-100 ft-c) (ambient laboratory levels)
16 h light, 8 h darkness
600 mL beaker
500 mL/replicate (loading and DO restrictions must be met)
Daily
Newly hatched larvae (less than 24 h old; 24-h range in age)
10
4
40
Newly \\atc\\edArtemia nauplii, (less than 24-h old)
Feed once a day 0.10 g wet weight Artemia nauplii per replicate on Days 0-2; Feed 0.15 g wet
weight Artemia nauplii per replicate on Days 3-6
Siphon daily, immediately before test solution renewal and feeding
None, unless DO falls below 4.0 mg/L, then aerate all chambers. Rate should be less than 100
bubbles/min
25%o (2%o) Bioassay Grade Forty Fathoms artificial seawater prepared with MILLIPORE
MILLI-Q or equivalent deionized water (see Methods Manual Section 7, Dilution Water)
Five concentrations and a control
>0.5
7 days
Survival and growth (weight)
80% or greater survival in controls; average dry weight per surviving organism in control
chambers should be 0.60 mg or greater, if unpreserved, or 0.50 mg or greater after no more than 7
days in 4% formalin or 70% ethanol
Samples treated as effluent samples for NPDES monitoring. Samples are to be used on the day
specified by the finalized interlab oratory study testing schedule
6 L per day	
NOTE: Test vessels shall be randomized in accordance with the method manuals.
                                                                    A-34

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Table 17.  Mysid Shrimp, Mysidopsis bahia, Survival, Growth, and Fecundity Test. Summary of test conditions and test acceptability criteria
for the mysid, Mysidopsis bahia, seven day survival, growth, and fecundity test with effluents and receiving waters	
1. Test type:
2. Salinity:
3. Temperature:
4. Light quality:
5. Light intensity:
6. Photoperiod:
7. Test chamber:
8. Test solution volume:
9. Renewal of test solutions:
10. Age of test organisms:
11. No. organisms per test chamber:
12. No. replicate chambers per concentration:
13. No. larvae per concentration:
14.  Source of food:
15.  Feeding regime:
16.  Cleaning:
17.  Aeration:
18.  Dilution water:

19.  Test concentrations:
20.  Dilution factor:
21.  Test duration:
22.  Endpoints:
23.  Test acceptability criteria:

24.   Sample handling and holding requirements:

25.  Sample volume required:	
Static renewal
25%0 (2%<)
26 1C
Ambient laboratory illumination
10-20 nE/m2/s (50-100 ft-c.) (ambient laboratory levels)
16 h light, 8 h darkness, with phase in/out period
8 oz plastic disposable cups, or 400 mL glass beakers
150 mL per replicate
Daily
7 days
5
8
40
Newly \\atc\\edArtemia nauplii (less than 24 h old)
Feed 150 24 h old nauplii per mysid daily, half after test solution renewal and half after 8-12 h.
Pipette excess food from cups daily immediately before test solution renewal and feeding.
None unless DO falls below 4.0 mg/L, then gently aerate in all cups
25%o (2%o) Bioassay  Grade Forty Fathoms artificial seawater prepared with MILLIPORE
MILLI-Q or equivalent deionized water (see Methods Manual Section 7, Dilution Water)
Five concentrations and a control
>0.5
7 days
Survival, growth, and egg development
80% or greater survival, average dry weight 0.20 mg or greater in controls; fecundity may be
used if 50% or more of females in controls produce eggs
Samples treated as effluent samples for NPDES monitoring. Samples are to be used on the
day specified by the finalized Inter lab oratory study testing schedule
3 L per day	
NOTE: Test vessels shall be randomized in accordance with the method manuals.
                                                                     A-35

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SECTION 5:    DATA REPORTING AND EVALUATION

Each referee and participant laboratory will be required to submit data reports in a hard copy format that
is consistent with the applicable methods manual. Submission of data reports will be required within 30
calendar days of the completion of each testing round. At a minimum, this report should follow the data
reporting format outlined in Table 18 and include all laboratory bench sheets. Laboratories also will be
required to submit selected data in an electronic format (Microsoft Excel spreadsheet, or equivalent) that
will allow SCC to create a database of study results. This database will facilitate automated review and
statistical analysis of study results. Specific instructions regarding the electronic format will be provided
to referee and participant laboratories prior to study initiation. Raw data will be made available in the
public record.

Upon receipt of each laboratory data package, SCC will review the results to ensure that they were
generated in accordance with the required procedures. Data generated by all qualified participating
laboratories will be considered in the evaluation of the test methods and will be compiled in a study
database and statistically analyzed to determine the interlaboratory variability of the acute and short-term
chronic methods under study. Statistical methods appropriate to the data received will be used in the
analysis process. This may include outlier analysis if warranted by the data.  Data also will be assessed to
determine the success rate for test initiation and test completion for each method and the rate at which the
tests indicate "toxicity" is present when measuring non-toxic samples. Overall, EPA will evaluate the
study results to draw conclusions about the performance of standardized WET tests. Participant
laboratories that fail to initiate tests in Phase 4 or fail to complete tests due to reasons unrelated to the test
methods themselves (i.e., laboratory error, sample receipt problems) will not be included in the success
rate calculations nor statistical analyses. SCC will assemble background information and study data into a
final study report for review by EPA staff.

EPA will evaluate results from the WET Study in accordance with the criteria for evaluating the adequacy
of biological methods described in "Availability, Adequacy, and Comparability for the Analysis of
Pollutants Established Under Section 304(h) of the Federal Water Pollution Control Act," EPA/600/9-
87/030 (September 1988), and, to the extent applicable, the "Data Quality Objectives" guidance (from
EPA's Permit Writers' Guide dated November 1990 and Guidance for Planning for Data Collection,
EPA/QA/G-4).

Note: Laboratories may not independently publish the results of analyses they are paid by EPA to
perform under this study plan.

Table 18. Data Reporting Format.	

Section 1 - Summary Page
     1.1  Laboratory name
     1.2  Laboratory address and phone number
     1.3  Name  and signature of laboratory QA Officer, certifying that data have been internally reviewed and that
         personnel meticulously followed the methods, and the procedures are deemed to be compliant with the
         methods and acceptable for reporting purposes.
     1.4  Laboratory contact responsible for study
     1.5  Analyst(s) who performed WET tests (full name)
     1.6  Toxicity tests performed
     1.7  Detailed explanations of any difficulties encountered and any approved modifications to the techniques
         specified in the SOW, specific instructions, or the methods manuals.
     1.8  Number of successful tests completed
                                               A-36

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Section 2 - Sample Information
    2.1   Number of samples received and EPA sample number assigned to each sample
    2.2   Dates of sample receipt
    2.3   Sample temperature when received at laboratory
    2.4   Physical and chemical data of sample contents (as required in appropriate method)
    2.5   Dilution water
          2.5.1    Source and time frame water is used or how maintained
          2.5.2    Collection or preparation date(s), where  applicable
          2.5.3    Pretreatment information
          2.5.4    Physical and chemical characteristics (pH, hardness, conductivity, salinity, etc.)
    2.6   Sample storage information
    2.7   Sample preparation for testing information

Section 3 - Test Conditions
    3.1   Toxicity test method used (title, number, source)
    3.2   Endpoint(s) of test(s)
    3.3   Deviations from reference method(s), if any, and  reason(s)
    3.4   Date and time test(s) started, date and time samples were prepared and solutions transferred for renewals
    3.5   Date and time test(s) terminated
    3.6   Type and volume of test chambers
    3.7   Volume of solution used per chamber
    3.8   Number of organisms per test chamber
    3.9   Number of replicate test chambers per treatment
    3.10   Feeding frequency and amount and type of food (be specific with sources, concentrations of foods (i.e,
          algae concentration, YCT solids level, preparation dates)
    3.11   Acclimation of test organisms (temperature mean and range and, where applicable, salinity mean and
          range)
    3.12   Test temperature (mean and range)
    3.13   Test salinity, where applicable (mean and range)
    3.14   Specify if aeration was needed
    3.15   Specify if organisms were dried immediately for weighing or preserved prior to drying
    3.16   Specify how food was prepared and sources of food. Include test results that validate the quality of
          batch food preparations (i.e., Ceriodaphnia dubia tests on YCT preparation)
    3.17   Describe how routine chemistries on new solutions were made (in actual test chamber or in beakers after
          dispensing)
    3.18   Describe how randomization was conducted, especially blocking and known parentage. Report how
          brood distinctions were made and male (if any) identification was made

Section 4 - Test Organisms
    4.1   Scientific name of test species, verification of species documented
    4.2   Age (life stage) of test species (be specific for all species). Age at time of test initiation (for example, for
          C. dubia be sure to clarify the window of age of the neonates as well as the overall age of the animals)
    4.3   Mean length and weight (where applicable)
    4.4   Source and QA/QC test conditions
    4.5   Holding Conditions
    4.6   Diseases and treatment (where applicable)
    4.7   Taxonomic key used for species identification

Section 5 - Quality Assurance
    5.1   Reference toxicant used routinely; source; date received; lot number
    5.2   Date and time of most recent reference toxicant test; test results and current control (cusum) chart
          including 20 most recent data points
    5.3   Dilution water used in reference toxicant tests (with characteristics provided)
    5.4   Physical and chemical methods used
    5.5   Reference toxicant results (NOEC, IC25, or LC50 where applicable, LOEC or EC50)


                                                 A-37

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Section 6 - Results
    6.1   Copies of all bench sheets. Be sure to count and notate broods for reproduction test with Ceriodaphnia
    6.2   Raw toxicity data in tabular form, including daily records of affected organisms in each replicate at each
          concentration (including controls) and plots of toxicity data
    6.3   Table of endpoints (LC50, IC25, NOEC for each endpoint) and confidence limits (where applicable)
    6.4   Statistical methods and software used to calculate endpoints
    6.5   Summary table of physical and chemical data
                                                  A-38

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        Appendix B:

   Participant Laboratory
Standard Operating Procedures

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DynCorp
litanisrtiii G Bircrjjnse TasMiijiy
TO:         Participant Laboratories for the Ceriodaphnia dubia Acute Test Method

FROM:      Robert Brent, WET Study Coordinator

DATE:      October 25, 1999

SUBJECT:   Final Guidance and SOP for the Ceriodaphnia dubia Acute Test
Enclosed is the final standard operating procedure (SOP) for laboratories participating in the
Ceriodaphnia dubia acute test method in EPA's WET Interlaboratory Variability Study. This
SOP is a supplement to the statement of work (SOW) that was distributed on July 9, 1999 with
the solicitation for this study. The SOP details the sample distribution and testing schedule and
provides important information for completing participant laboratory tasks, such as instructions
for the reconstitution of ampule samples. Participant laboratories should follow the guidance in
the enclosed SOP, the SOW, and the method manuals.

Also enclosed is the electronic data reporting format disk for the Ceriodaphnia dubia acute
method. A description and instructions for use of the electronic data report form are provided in
the SOP.

Please ensure that the enclosed SOP and disk pertaining to the Ceriodaphnia dubia acute test
method are distributed to laboratory  staff that will be performing the test method in the study.
CDA Participant Lab SOP
B-l

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                      STANDARD OPERATING PROCEDURE
           Participant Laboratory Support for EPA's WET Interlaboratory Study

                              Ceriodaphnia dubia Acute Method
Preamble:
This standard operating procedure document (SOP) is a supplement to the participant laboratory
Statement of Work (SOW).  This SOP details specific information in the SOW regarding the conduct of
the Ceriodaphnia dubia acute test method in the WET Interlaboratory Variability Study. All
modifications to the SOP must be approved by DynCorp prior to implementation.

All data deliverables submitted for this Interlaboratory study become the property of EPA and may be
incorporated into the public record. Laboratories may not independently publish the results of analyses
for which they are paid to perform under this SOP.

1.0 Sample Distribution and Testing Schedule

Participant laboratory testing for the Ceriodaphnia dubia acute method will occur between November 9
and 13, 1999 with final reports due 30 days following termination of all tests (December 13, 1999). The
schedule for the testing is provided below. The date ranges on the schedule indicate the test start dates
and test completion dates.  Samples will  be shipped FedEx Priority Overnight to arrive at the participant
laboratory on the test start date by 10:OOAM. All tests must be initiated on the day of sample arrival.
Testing is scheduled to occur simultaneously at each participant laboratory,  so adherence to the testing
schedule is mandatory for all participant laboratories.

Laboratories participating in the base study design will receive 4 blind test samples  (reflected in the
schedule) that may be whole volume or ampule samples. Two samples will arrive on  11/9/99 for test
initiation on that day, and two samples will arrive on 11/11/99 for test initiation on that day.  Laboratories
participating in  the extended study design will each receive three ampule samples.  Some laboratories will
receive two samples on 11/9/99 and one  on  11/11/99, while other laboratories in the extended design will
receive one on 11/9/99 and two on 11/11/99. Laboratories participating in the extended design will be
notified by fax prior to study initiation to confirm the number of samples (one or two) that should be
expected for each testing period.

Each sample aliquot that is prepared and shipped will be assigned a unique sample number. The sample
number will appear clearly and permanently on each container and on an EPA traffic report form that will
accompany each sample. For whole volume samples, one aliquot will be received on test Day 0. This
aliquot shall be  used for test initiation on Day 0. For ampule samples, one aliquot will be received on test
Day 0. This aliquot shall be reconstituted on Day 0, and the reconstituted sample shall be used for test
initiation.
CDA Participant Lab SOP                      B-2

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Table 1. Schedule for Ceriodaphnia dubia Acute Testing.
Date
(start date - finish date)
11/9/99- 11/11/99
11/11/99- 11/13/99
12/13/99
Activity
Conduct Cladoceran, Ceriodaphnia dubia, Acute Test with samples #1&2
Conduct Cladoceran, Ceriodaphnia dubia, Acute Test with samples #3&4
Cladoceran, Ceriodavhnia dubia. Acute Test data due
2.0 Sample Traffic Reporting Tasks

2.1    Sample Receipt Confirmation

An EPA traffic report form (Appendix B) will be received with each sample. The traffic report form will
document important sample collection, shipment, and receipt information.  Portions of the form will be
completed by the referee laboratory prior to shipment and will indicate the episode number, sample
number, referee laboratory information, participant laboratory shipping information, sample pre-shipment
information, and the requested analysis.  Immediately upon receipt of the sample, participant laboratories
will be responsible for determining that the sample arrived in satisfactory condition and for documenting
receipt of the sample, post-shipment sample water quality (temperature, pH, dissolved oxygen, and
conductivity or salinity), and any problems on the EPA traffic report form. The participant laboratory
shall complete the traffic report form by recording the required information in the "For Participant Lab
Use Only" box and in the "Post-Shipment" column of the "Sample Collection/Receipt Information" box.

For ampule samples, the pre-shipment and post-shipment measurement of pH, dissolved oxygen, and
conductivity or salinity shall be omitted. This will avoid possible contamination of the ampule sample
prior to reconstitution. Pre-shipment and post-shipment temperature shall be measured in a temperature
check sample. An additional sample container labeled "temperature check" will be included with each
cooler shipment of ampules. The temperature shall be measured in this container and recorded on the
EPA traffic report form as a surrogate measure of temperature for all ampule samples within that cooler.
The temperature check shall be discarded after temperature measurement, and must not be used in any
way for WET testing.

Immediately upon completion of the EPA traffic report form, the participant laboratory shall fax the
completed form to DynCorp and retain a copy for inclusion in the final data report. Receipt of the faxed
traffic report form by DynCorp will be confirmation of successful sample arrival, so it is important that
the form be completed and faxed immediately.  Forms should be faxed by 11:OOAM (local laboratory
time) to facilitate sample tracking and potential problem resolution. The EPA traffic report form shall be
faxed to:

                                       Brian Rusignuolo
                                      Sample  Coordinator
                                         DynCorp SCC
                                      fax:  (703)461-8056
                                     phone: (703)461-2401
CDA Participant Lab SOP                       B-J

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2.2    Problem Resolution

Prior to test initiation, notify Brian Rusignuolo of any special shipment receiving instructions (i.e., hold
shipment at airport or FedEx shipment center for pickup). Notification must also be made if any specific
instructions are required for Saturday deliveries.

Participant laboratories should be expecting the arrival of samples based on the provided schedule. If
samples do not arrive as expected, if samples are damaged in shipment, if samples arrive without
accompanying traffic report forms, if sample numbers are not clearly identified on samples, or if any
other sample shipment or receipt problem is encountered immediately notify Brian Rusignuolo by phone
at (703)461-2401.

Brian must be notified by  11:OOAM (local laboratory time) if samples fail to arrive by the morning FedEx
shipment or if other shipment or sample receipt problems are encountered. DynCorp will track lost
shipments and instruct referee laboratories to resend test samples for arrival the following day if
necessary.

3.0 WET Test Analysis

3.1    Sample Preparation

3.1.1  Whole Volume Samples

Whole volume samples will be received in cubitainers with sufficient volume for test conduct and
required water chemistry analysis. No sample preparation or adjustment steps (e.g. pH adjustment)
should be conducted prior to test initiation.  The whole volume sample shall be treated as a typical 100%
effluent sample received for NPDES compliance monitoring. Test concentrations of 100%, 50%, 25%,
12.5%, and 6.25% sample shall be prepared from the whole volume sample for use in the test. These test
concentrations and a dilution water control shall be prepared using moderately hard synthetic freshwater
as the dilution water (prepared according to Section 7 of the method manuals).

3.1.2  Ampule Samples

Ampule samples will be received as small volume liquid samples in 125ml plastic containers.  Prior to
test initiation the ampule samples must be reconstituted according to the instructions in Appendix A to
provide the necessary volume for testing.  The reconstituted sample shall then be treated as atypical
100% effluent sample received for NPDES compliance monitoring. Test concentrations of 100%, 50%,
25%, 12.5%, and 6.25% sample shall be prepared from this reconstituted sample for use in the test. These
test concentrations and a dilution water control shall be prepared using moderately hard synthetic
freshwater as the dilution water (prepared according to Section 7 of the method manuals).

3.2    Test Conduct

This section of the SOP reiterates testing requirements from Section 3 of the Participant Laboratory
Statement of Work that must be followed for the conduct of the Ceriodaphnia dubia acute test method.
Except where indicated in Sections 3.2.1 and 3.2.2 of this SOP, each test shall be conducted in
accordance with the general guidance and method specific requirements for effluent testing included in
the methods manuals.
CDA Participant Lab SOP                        B-4

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3.2.1   General Testing Requirements

EPA acknowledges that the promulgated WET methods distinguish between requirements (indicated by
the compulsory terms "must" and "shall") and recommendations and guidance (indicated by discretionary
terms "should" and "may"). The latter terms indicate that the analyst has flexibility to optimize successful
test completion and when standardization is necessary to assure the predictability of the methods to
provide reliable results. Additionally, the method manuals allow variations of the methods which are
typically fixed in the permit; therefore, for the purposes of this study, a set of variables will be defined by
EPA (for example, dilution water, salinity, and acute test duration). Any deviation from defined test
procedures and/or conditions, such as the necessity to change reagents, equipment, test conditions, or
other specified test parameters must be reported to SCC, recorded at the time of modification, noted in
telephone logs of communications, documented in a memorandum, and approved by EPA.

Additional WET test requirements and general requirements listed in the methods manuals of special note
are provided below:

(1)    Personnel that conduct tests must be the same personnel that routinely conduct the WET tests at
       that laboratory facility and who were identified in the prequalification materials. If these
       individuals cannot be available during any part of the study, the laboratory must contact SCC.
       Personnel conducting the tests must be identified clearly and consistently in records.

(2)    To coordinate testing at participant laboratories, testing of each sample with each method must be
       initiated on the precise day specified in the finalized study schedule.  Samples should be tested
       within 36 hours from the time of sample preparation (determined in this study as the time at
       which individual sample aliquots were divided from the bulk test sample for distribution to
       participant laboratories). Deviation from this schedule must be reported to SCC immediately for
       approval.

(3)    Physical and chemical properties of the test samples must be in the ranges specified in this SOP,
       the SOW, specific instructions, and the methods manuals. Method specific instructions for any
       adjustments to the test samples prior to sample use (such as reconstitution of ampule samples or
       salinity adjustments) are provided herein.  Test samples received at participant laboratories must
       be refrigerated (at 4C  2C) immediately upon receipt and throughout the period of testing.
       The temperature of the refrigeration unit should be routinely or continuously monitored to ensure
       that these sample holding requirements are met.

(4)    Measurement of test conditions (pH, conductivity or salinity, total alkalinity, total hardness, and
       dissolved oxygen) shall be performed for each method by the participant laboratories following
       guidance in method manuals.

(5)    The specified dilution and control waters must be used and prepared according to instructions in
       Section 7  of the methods manuals.

(6)    All WET tests are to be definitive tests with a control and a minimum of five test concentrations
       prepared using a dilution factor of 0.5.

(7)    All tests must be conducted using the number of replicates and number of test containers per
       concentration as specified in Section 3.2.2.


CDA Participant Lab SOP                       B-5

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(8)    Test chambers used within a test must be of the same type, size, shape, and material. The
       material must be allowed by the method manuals for the method used.

(9)    Test vessels shall be randomized in accordance with the method manuals.

(10)   Daily observation of mortality and removal of dead organisms for each test is required.

(11)   If test results indicate too great of toxicity (i.e., control mortalities, or complete mortality in all
       concentrations), the laboratories must contact SCC immediately and then investigate possible
       causes, first by checking for transcription and calculation mistakes, and then by investigating
       possible contamination in dilution waters, organism cultures, equipment, or other procedural
       steps.

(12)   If any test that has been initiated fails to be completed for any reason, the laboratory must contact
       SCC immediately for problem resolution and scheduling of additional testing.  The incomplete
       test data and the reason for not completing the test must be fully documented in the  final report.

(13)   Each laboratory shall be required to report all data obtained during the course of testing,
       including the response of control samples.

(14)   Laboratories must perform all QA/QC tests listed in Section 4 of the method manuals.
       Laboratories that  purchase organisms must supply QA/QC from the test organism supplier and
       follow method manuals for the appropriate QA/QC for purchasing organisms.

(15)   A reference toxicant QC test must be performed for each test method in the month that testing for
       this study occurs. Results of this test must be submitted with the final data package.

(16)   Data and statistical analyses must be submitted in hard copy in the standardized format specified
       in Section 5 of the SOW. All bench sheets and raw data, including sample tracking and chemistry
       analysis data must be submitted. Data must also be submitted electronically according to an
       electronic template (Microsoft Excel spreadsheet) that is provided with this SOP.

(17)   Data analysis must be performed in accordance with the statistical programs specified in the
       methods manuals. Statistical methods and programs used must be reported along with sample
       calculations.

(18)   An LC50 must be  reported for each acute test. The laboratories must report individual toxicity
       endpoints; laboratories are not allowed to average or perform other data manipulations unless
       required by a methods's instructions.

3.2.2   Method-Specific  Requirements

Table 2 provides a summary of test conditions that shall be followed for the conduct of all Ceriodaphnia
dubia acute tests performed in the WET Interlaboratory Study. This table is extracted from the summary
test condition table in the  method manual and modified to fit the scope of this study. Items that are bold
italic in this table represent conditions standardized for the purposes of this study where method manuals
provide a range.
CDA Participant Lab SOP                       B-6

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Table 2.  Cladoceran, Ceriodaphnia dubia, Acute Test. Summary of test conditions and test acceptability criteria for daphnid, Ceriodaphnia
dubia acute toxicity tests with effluents and receiving waters	
1. Test type:                                            Static non-renewal
1. Test duration:                                        48 h
3. Temperature:                                        251C
4. Light quality:                                        Ambient laboratory illumination
5. Light intensity:                                       10-20 uE/m2/s (50-100 ft-c) (ambient laboratory levels)
6. Photoperiod:                                         16 h light, 8 h darkness
7. Test chamber size:                                    30 mL
8. Test solution volume:                                 75 mL
9. Renewal of test solutions:                              None
10. Age of test organisms:                                Less than 24-h old
11. No. organisms per test  chamber:                       5
12. No. replicate chambers per concentration:               4
13. No. organisms per concentration:                      20
14. Feeding regime:                                     Feed YCT and Selenastmm while holding prior to the test; newly-released young should have
                                                      food available a minimum of 2 h prior to use in a test.
15. Test chamber cleaning:                              Cleaning not required
16. Test chamber aeration:                               None
17. Dilution water:                                      Moderately hard synthetic water prepared using MILLIPORE MILLI-Q or equivalent
                                                      deionized water and reagent grade chemicals (see Methods Manual Section 7, Dilution Water)
18. Test concentrations:                                 Five concentrations and a control
19. Dilution factor                                      0.5
20. Endpoint:                                          Mortality (LC50)
21.  Sample handling and holding requirements:             Samples treated as effluent samples for NPDES monitoring. Samples are to be used on the day
                                                      specified by the finalized interlaboratory study testing schedule
22. Sample volume required:                             1 L
23. Test acceptability criterion:	90% or greater survival in controls	
NOTE: Test vessels shall be randomized in accordance with the method manuals.
CDA Participant Lab SOP                                            B-7

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3.2.3    Data Analysis

For the Ceriodaphnia dubia acute test method, the 48 hour LC50 shall be calculated and reported. Data
analysis and statistical procedures should be conducted according to the method manuals.

3.2.4    Problem Resolution

In the event that problems are encountered during the WET test analysis contact Brian Rusignuolo at
(703)461-2401 for guidance.  Brian will direct your call as appropriate for the resolution of technical
issues.

3.2.5    Sample Disposal

Following the termination of the test, any excess sample shall be  disposed of according to standard
laboratory procedures for disposal of effluent samples.

4.0 Additional Information  on Data Reporting Deliverables

According to the Participant Laboratory Statement of Work, the submission of three deliverables are
required: 1). Narrative summary of testing, 2). Hardcopy results synopsis and full report, and 3).
Electronic results synopsis.  Deliverables #1 and 2 shall be submitted according to the requirements
specified in the SOW. This section provides additional instructions for the submission of the electronic
results synopsis.

Enclosed with this package is  a computer diskette that contains the template for the electronic submission
of results from the Ceriodaphnia dubia acute test method.  The disk contains a Microsoft Excel 97
spreadsheet file named CDA	.xls.  The CDA indicates that this template is for the Ceriodaphnia dubia
acute test method, and the number following is a unique identifying number for your laboratory.  If your
laboratory does not have the hardware or software capabilities to  view and enter data into this file, please
contact Brian Rusignuolo at (703)461-2401 to arrange distribution of an alternative version of the
electronic template.

Notice the following characteristics of the electronic template file:

    1.   The file contains four worksheet pages labeled "Sample #1", "Sample #2", "Sample #3", and
        "Sample #4". The results from each of the samples that your laboratory tested in this study
        should be entered on a separate worksheet page within the same file.
    2.   Each worksheet page  contains six information boxes (general information, sample
        collection/receipt information, test information, biological data, water quality data, and
        summarized test results) in which data should be entered. The seventh information box (data
        quality flags) is for SCC use only and will aid in the automated review and quality control check
        of data.
    3.   Cells that are highlighted in pale yellow indicate required information.  Cells highlighted in blue
        indicate optional information that may be entered if available. Cells highlighted in red are for
        SCC use only.
    4.   The file has been protected such that data can only be entered in yellow or blue cells.  No other
        cells can be changed.
CDA Participant Lab SOP

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To complete the electronic results synopsis data deliverable:

    1.  Record information into a separate worksheet page for each sample tested.  If participating in the
       extended study design, the last worksheet page (Sample #4) may be left empty.
    2.  Record requested information or data in all required cells (pale yellow).
    3.  Record requested information or data in optional cells (blue) if data is available.
    4.  Check entered data against hardcopy bench sheets to ensure accuracy.
    5.  Save the file onto the diskette provided.  Do not change the file name.
    6.  Submit the diskette with the other data reporting deliverables by the due date to:

                              DynCorp I&ET, Sample Control Center
                              6101 Stevenson Avenue
                              Alexandria, VA 22304
                              Phone:(703)461-2064
                              Fax:(703)461-8056
                              Attention: Robert Brent

5.0 Coolers

All coolers will include a prepaid return FedEx label for returning the cooler to the referee laboratory.
Fill out the sender portion of the FedEx label and place it on the cooler.  Coolers will be returned by 2
Day shipment, as already marked on the prepared label. Coolers must be returned to the referee
laboratory within 7 days of sample receipt. Please empty and wipe out the cooler prior to returning it.
CDA Participant Lab SOP                       B-9

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           Appendix A:  Instructions for Reconstitution of Liquid Ampule Sample for
                          Cladoceran, Ceriodaphnia, dubia, Acute Test

For each sample, a single liquid ampule will be received.  The container shall be reconstituted and used to
initiate the test.  Follow the directions below for the reconstitution of the sample ampule.

1.   Volumetrically add 100 mL of the liquid ampule sample to approximately 600mL of moderately-hard
    synthetic freshwater (MHSF) prepared using MILLIPORE MILLI-Q or equivalent deionized water
    and reagent grade chemicals according to Section 7 of the methods manuals.

2.   Mix by swirling or gently shaking.

3.   Bring final volume to 1L (measured using volumetric glassware) with MHSF dilution water.

4.   Mix again by swirling and gently shaking.

5.   Place reconstituted sample in a plastic container of appropriate volume. A container that minimizes
    head space (i.e. cubitainer) is recommended.

6.   This 1L sample is the whole volume reconstituted sample and should be treated as a typical effluent
    sample. It should be used directly for the 100% effluent test concentration and diluted appropriately
    to prepare other test concentrations (50%, 25%, 12.5%, and 6.25%).

7.   Use the reconstituted sample for test initiation. Store sample at 4C.

8.   Perform the Cladoceran, Ceriodaphnia dubia, Acute Test as described in the method manuals, the
    SOW for this study, and this SOP.
CDA Participant Lab SOP                       B-10

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Appendix B: EPA Traffic Report
^EPA
United States
Environmental Protection Agency
Washington, DC 20460
WET Interlaboratory Variability Study Traffic Report
USEPA ENGINEERING AND ANALYSIS DIVISION
SAMPLE CONTROL CENTER
Referee Laboratory Information
Name:
Address:
City:
State:

Sampler name:
EPISODE NO:
SAMPLE NO:
DynCorp - Sample Control Center
Fax completed form immediately 6101 Stevenson Avenue
upon completion, and include Alexandria, VA 22304
hardcopy in final data report to: phone: (703) 461 -21 00
fax: (703)461-8056
Participant Lab Shipping Information
Lab Name:
Address:
City:
State:
Airbill no:
Date shipped:
SAMPLE COLLECTION /
Requested Information
1.
2.
3.
4.
5.
6.
7.
8.
Sample use description
Sample collection/receipt date
Sample collection/receipt time
Sampler / recipient signature
PH
Temperature
Conductivity (freshwater methods) / Salinity
(marine methods)
Dissolved Oxygen concentration
FOR PARTICIPANT LAB USE ONLY
Received by:
Sample condition on receipt:
RECEIPT INFORMATION
Pre-Shipment
I 	 (initiation I 	
renewal 1 I 	 Irenewal 2







Post-Shipment








REQUESTED ANALYSES
9.
I 	 I Pimephales promelas Acute
I 	 I Pimephales promelas Chronic
I 	 I Ceriodaphnia dubia Acute
I 	 I Ceriodaphnia dubia Chronic
I 	 I Selenastrum capricornutum Chronic
I 	 I Menidia beryllina Acute
I 	 I Menidia beryllina Chronic
I 	 I Holmesmysis costata Acute
I 	 I Champia parvula Chronic

I 	 I Cyprinodon variegatus Acute
I 	 I Cyprinodon variegatus Chronic
I 	 I Mysidopsis bahia Chronic


CDA Participant Lab SOP
B-ll

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DynCorp
litanisrtiii G Bircrjjnse TasMiijiy
TO:          Participant Laboratories for the Ceriodaphnia dubia Chronic Test Method

FROM:       Robert Brent, WET Study Coordinator

DATE:       October 8, 1999

SUBJECT:    Additional Question from the WET Participant Laboratory Meeting
On October 6, 1999, the meeting notes from the WET Participant Laboratory Meeting were distributed
along with the final guidance SOP and electronic data reporting disk for the Ceriodaphnia dubia chronic
test method.  Unfortunately, one question and answer from the meeting pertaining to the Ceriodaphnia
dubia chronic test method was inadvertently omitted from the meeting notes. The question and answer
are provided below:

   Q: Is the addition of selenium to synthetic freshwater required in the preparation of dilution water for
Ceriodaphnia dubia tests?

   A:  The addition of selenium is not specifically required in this study, but is recommended.
Laboratories should follow their standard procedures and the method manuals for preparation of synthetic
freshwater.

Thank you for your attention to this addition, and I apologize for the omission of this question in the
meeting notes.
CDC Participant Lab SOP
B-12

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                      STANDARD OPERATING PROCEDURE
          Participant Laboratory Support for EPA's WET Interlaboratory Study

                   Ceriodaphnia dubia Survival and Reproduction Method
Preamble:
This standard operating procedure document (SOP) is a supplement to the participant laboratory
Statement of Work (SOW).  This SOP details specific information in the SOW regarding the conduct of
the Ceriodaphnia dubia survival and reproduction test method in the WET Interlaboratory Variability
Study.  All modifications to the SOP must be approved by DynCorp prior to implementation.

All data deliverables submitted for this interlaboratory study become the property of EPA and may be
incorporated into the public record. Laboratories may not independently publish the results of analyses
for which they are paid to perform under this SOP.

1.0 Sample Distribution and Testing Schedule

Participant laboratory testing for the Ceriodaphnia dubia survival and reproduction method will occur
between October 12 and November 3, 1999 with final reports due 30 days following termination of all
tests (December 3, 1999). The schedule for the testing is provided below. The date ranges on the
schedule indicate the test start dates and test completion dates. Samples will be shipped FedEx Priority
Overnight to arrive at the participant laboratory on the test start date by 10:OOAM.  All tests must be
initiated on the day of sample arrival. Testing is  scheduled to occur simultaneously at each participant
laboratory, so adherence to the testing schedule is mandatory for all participant laboratories.

Laboratories participating in the base study design will receive 4 blind test samples (reflected in the
schedule) that may be whole volume or ampule samples. Two samples will arrive on 10/12/99 for test
initiation on that day, and two samples will arrive on 10/26/99 for test initiation on that day. Laboratories
participating in the extended study design will each receive three ampule samples.  Some laboratories will
receive two samples on 10/12/99 and one on 10/26/99, while other laboratories in the extended design
will receive one on 10/12/99 and two on 10/26/99. Laboratories participating in the extended design will
be notified by fax prior to study initiation to confirm the number of samples (one or two) that should be
expected for each testing period.

Each sample aliquot that is prepared and shipped will be assigned a unique sample number. The sample
number will appear clearly and permanently on each container and on an EPA traffic report form that will
accompany each sample. For tests that require additional shipments for sample renewal, the sample
number shall be the same for each initiation and renewal shipment with the addition of a letter (A, B, or
C) after the sample number to designate the sample for use  as initiation (A), renewal 1 (B), or renewal 2
(C).

For whole volume samples, separate aliquots will be received on test Day 0, Day 2, and Day 4. The first
aliquot (identified with the sample number and the letter "A") shall be used for test initiation on Day 0
and renewal on Day 1. The second aliquot (identified with the sample number and the letter "B") shall be
used for test renewals on Day 2 and Day 3. The final aliquot (identified with the sample number and the
letter "C") shall be used for test renewal on Day 4, Day 5, Day 6, and Day 7.  This sample shipment
schedule mimics the typical schedule for chronic monitoring of effluent for compliance.

CDC Participant Lab  SOP                      B-13

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For ampule samples, three separate ampules containers (marked with the sample number followed by A,
B, or C) will be received in a single shipment on test Day 0. The container marked "A" shall be
reconstituted on test Day 0 and used for test initiation and renewal on Day 1. The other aliquots of the
sample shall be refrigerated and stored until use on Day 2 and Day 4, respectively. The container marked
"B" shall be reconstituted on test Day 2 and used for renewal on Day 2 and Day 3. The container marked
"C" shall be reconstituted on Day 4 and used for renewal on Day 4, Day 5, Day 6, and Day 7. The
sample reconstitution schedule for ampules attempts to mimic the typical sample shipment schedule for
chronic monitoring of effluents for compliance.
Table 1. Schedule for Ceriodaphnia dubia Survival and Reproduction Testing.
Date
(start date - finish date)
10/12/99 - 10/20/99
10/26/99- 11/3/99
12/3/99
Activity
Conduct Cladoceran, Ceriodaphnia dubia, Survival and Reproduction Test with samples #1&2
Conduct Cladoceran, Ceriodaphnia dubia, Survival and Reproduction Test with samples #3&4
Cladoceran, Ceriodaphnia dubia, Survival and Reproduction Test data due
2.0 Sample Traffic Reporting Tasks

2.1    Sample Receipt Confirmation

An EPA traffic report form (Appendix B) will be received with each sample. The traffic report form will
document important sample collection, shipment, and receipt information.  Portions of the form will be
completed by the referee laboratory prior to shipment and will indicate the episode number, sample
number, referee laboratory information, participant laboratory shipping information, sample pre-shipment
information, and the requested analysis.  Immediately upon receipt of the sample, participant laboratories
will be responsible for determining that the sample arrived in satisfactory condition and for documenting
receipt of the sample, post-shipment sample water quality (temperature, pH, dissolved oxygen, and
conductivity or salinity), and any problems on the EPA traffic report form. The participant laboratory
shall complete the traffic report form by recording the required information in the "For Participant Lab
Use Only" box and in the "Post-Shipment" column of the "Sample Collection/Receipt Information" box.

For ampule samples, the pre-shipment and post-shipment measurement of pH, dissolved oxygen, and
conductivity or salinity shall be omitted. This will avoid possible contamination of the ampule sample
prior to reconstitution. Pre-shipment and post-shipment temperature shall be measured in a temperature
check sample. An additional sample container labeled "temperature check" will be included with each
cooler shipment of ampules. The temperature shall be measured in this container and recorded on the
EPA traffic report form  as a surrogate measure of temperature for all ampule samples within that cooler.
The temperature check shall be discarded after temperature measurement, and must not be used in any
way for WET testing.

Immediately upon completion of the EPA traffic report form, the participant laboratory shall fax the
completed form to DynCorp and retain a copy for inclusion in the final data report. Receipt of the faxed
traffic report form by DynCorp will be confirmation of successful sample arrival, so it is important that
the form be completed and faxed immediately. Forms should be faxed by 11:00 AM (local laboratory
CDC Participant Lab SOP                       B-14

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time) to facilitate sample tracking and potential problem resolution. The EPA traffic report form shall be
faxed to:

                                       Brian Rusignuolo
                                      Sample Coordinator
                                         DynCorp SCC
                                      fax: (703)461-8056
                                     phone: (703)461-2401

2.2    Problem Resolution

Prior to test initiation, notify Brian Rusignuolo of any special shipment receiving instructions (i.e., hold
shipment at airport or FedEx shipment center for pickup). Notification must also be made if any specific
instructions are required for Saturday deliveries.

Participant laboratories should be expecting the arrival of samples based on the provided schedule.  If
samples do not arrive as expected, if samples are damaged in shipment, if samples arrive without
accompanying traffic report forms, if sample numbers are not clearly identified on samples, or if any
other sample shipment or receipt problem is encountered immediately notify Brian Rusignuolo by phone
at (703)461-2401.

Brian must be notified by 11:00 AM (local laboratory time) if samples fail to arrive by the morning
FedEx shipment or if other shipment or sample receipt problems are encountered. DynCorp will track
lost shipments and instruct referee laboratories to resend test samples for arrival the following day if
necessary. If renewal shipments do not arrive on  the expected day, DynCorp will provide guidance for
test renewal on a case-by-case basis. Depending on the volume of sample remaining from previous
shipments, laboratories may be instructed to conduct full renewals with the remaining sample, conduct
partial  renewals with the remaining sample, or omit the sample renewal for that day but carefully record
dissolved oxygen throughout the day and remove excess food and dead organisms from the test
containers.

3.0 WET Test Analysis

3.1    Sample Preparation

3.1.1   Whole Volume Samples

Whole volume samples will be received in cubitainers with sufficient volume for test conduct and
required water chemistry analysis. No sample preparation or adjustment steps (e.g. pH adjustment)
should be conducted prior to test initiation. The whole volume sample shall be treated as a typical 100%
effluent sample received for NPDES compliance monitoring. Test concentrations of 100%, 50%, 25%,
12.5%, and 6.25% sample shall be prepared from the whole volume sample for use in the test.  These test
concentrations and a dilution water control shall be prepared using moderately hard synthetic freshwater
as the dilution water (prepared according to Section  7 of the method manuals).
CDC Participant Lab SOP                       B-15

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3.1.2   Ampule Samples

Ampule samples will be received as small volume liquid samples in 125ml plastic containers. Prior to
test initiation the ampule samples must be reconstituted according to the instructions in Appendix A to
provide the necessary volume for testing. The reconstituted sample shall then be treated as atypical
100% effluent sample received for NPDES compliance monitoring. Test concentrations of 100%, 50%,
25%, 12.5%, and 6.25% sample shall be prepared from this reconstituted sample for use in the test. These
test concentrations and a dilution water control shall be prepared using moderately hard synthetic
freshwater as the dilution water (prepared according to Section 7 of the method manuals).

3.2    Test Conduct

This section of the SOP reiterates testing requirements from Section 3 of the Participant Laboratory
Statement of Work that must be followed for the conduct of the Ceriodaphnia dubia survival and
reproduction test method. Except where indicated in Sections 3.2.1 and 3.2.2 of this SOP, each test shall
be conducted in accordance with the general guidance and method specific requirements for effluent
testing included in the methods manuals.

3.2.1   General Testing Requirements

EPA acknowledges that the promulgated WET methods distinguish between requirements (indicated by
the compulsory terms "must" and "shall") and recommendations and guidance (indicated by  discretionary
terms "should" and "may"). The latter terms indicate that the analyst has flexibility to optimize successful
test completion and when standardization is necessary to assure the predictability of the methods to
provide reliable results. Additionally, the method manuals allow variations of the methods which are
typically fixed in the permit; therefore, for the purposes of this study, a set of variables will be defined by
EPA (for example, dilution water, salinity, and acute test duration). Any deviation from defined test
procedures and/or conditions, such as the necessity to change reagents,  equipment, test conditions, or
other specified test parameters must be reported to SCC, recorded at the time of modification, noted in
telephone logs of communications, documented in a memorandum, and approved by EPA.

Additional WET test requirements and general requirements listed in the methods manuals of special note
are provided below:

(1)    Personnel that conduct tests must be the same personnel that routinely conduct the WET tests at
       that laboratory facility and who were  identified in the prequalification materials. If these
       individuals cannot be available during any part of the study, the laboratory must contact SCC.
       Personnel conducting the tests must be identified clearly and consistently in records.

(2)    To coordinate testing at participant  laboratories, testing of each sample with each method must be
       initiated on the  precise day specified in the finalized study schedule.  Samples should be tested
       within 36  hours from the time of sample preparation (determined in this study as the  time at
       which individual sample aliquots were divided from the bulk test sample for distribution to
       participant laboratories).  Deviation from this schedule must be reported to SCC immediately for
       approval.

(3)    Physical and chemical properties of the test samples must be in the ranges specified in this SOP,
       the SOW, specific instructions, and the methods manuals. Method specific instructions for any


CDC Participant Lab  SOP                      B-16

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       adjustments to the test samples prior to sample use (such as reconstitution of ampule samples or
       salinity adjustments) are provided herein. Test samples received at participant laboratories must
       be refrigerated (at 4C  2C) immediately upon receipt and throughout the period of testing.
       The temperature of the refrigeration unit should be routinely or continuously monitored to ensure
       that these sample holding requirements are met.

(4)     Measurement of test conditions (pH, conductivity or salinity, total alkalinity, total hardness, and
       dissolved oxygen) shall be performed for each method by the participant laboratories following
       guidance in method manuals.

(5)     The specified dilution and control waters must be used and prepared according to instructions in
       Section 7 of the methods manuals.

(6)     All WET tests are to be definitive tests with a control and a minimum of five test concentrations
       prepared using a dilution factor of 0.5.

(7)     All tests must be conducted using the number of replicates and number of test containers per
       concentration as specified in Section 3.2.2.

(8)     Test chambers used within a test must be of the same type, size, shape, and material. The
       material must be allowed by the method  manuals for the method used.

(9)     Test vessels shall be randomized in accordance with the method manuals.  In addition, block
       randomization and use of known parentage will be required for the Ceriodaphnia dubia survival
       and reproduction test as described in Method 1002.0.

(10)   The Ceriodaphnia dubia Survival and Reproduction Test (Method 1002.0), which would
       otherwise be terminated after 3 broods according to Section 13.12.1 of that method, must be
       conducted for 8 days, with endpoints including survival, number of young per day, and number of
       broods recorded each day. These readings are to be made at the end of the 6th, 7th and 8th day
       (specifically, at 144 hours, at 168 hours,  and at 192 hours, respectively, from test initiation). This
       will be done to assess the effect of that test acceptance criterion on test results. No test shall be
       terminated prior to the eighth day for any reason, including a failure to meet test acceptance
       criteria. The additional measurements on days 6, 7, and 8 should be included as raw data in the
       final data report, but should not affect the data analysis of test results.  The analysis of data from
       the C. dubia chronic test shall be conducted as specified in the method manual using the three
       brood approach.

(11)   Daily observation of mortality and removal of dead organisms for each test is required. Daily
       young counts are required for the Ceriodaphnia dubia survival and reproduction test, along with
       determining the number of broods at each count.

(12)   If test results indicate too great of toxicity (i.e., control mortalities, or complete mortality in all
       concentrations), the laboratories must contact SCC immediately and then investigate possible
       causes,  first by checking for transcription and calculation mistakes, and then by investigating
       possible contamination in dilution waters, organism cultures, equipment, or other procedural
       steps.
CDC Participant Lab SOP                       B-17

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(13)   If any test that has been initiated fails to be completed for any reason, the laboratory must contact
       SCC immediately for problem resolution and scheduling of additional testing.  The incomplete
       test data and the reason for not completing the test must be fully documented in the final report.

(14)   Each laboratory shall be required to report all data obtained during the course of testing,
       including the response of control samples.

(15)   Laboratories must perform all QA/QC tests listed in Section 4 of the method manuals.
       Laboratories that purchase organisms must supply QA/QC from the test organism supplier and
       follow method manuals for the appropriate QA/QC for purchasing organisms.

(16)   A reference toxicant QC test must be performed for each test method in the month that testing for
       this study occurs. Results of this test must be submitted with the final data package.

(17)   Data and statistical analyses must be submitted in hard copy in the standardized format specified
       in Section 5 of the SOW. All bench sheets and raw data, including sample tracking and chemistry
       analysis data must be submitted. Data must also be submitted electronically according to an
       electronic template (Microsoft Excel spreadsheet) that is provided with this SOP.

(18)   Data analysis must be performed in accordance with the statistical programs specified in the
       methods manuals.  Statistical methods and programs used must be reported along  with sample
       calculations.

(19)   An NOEC and LC50 for survival, and an NOEC and IC25 for growth/reproduction must be
       reported as appropriate for each short-term chronic test as described in the method manuals. The
       laboratories must report individual toxicity endpoints; laboratories are not allowed to average or
       perform other data manipulations unless required by a methods's instructions.

3.2.2   Method-Specific Requirements

Table 2 provides a summary  of test conditions that shall be followed for the conduct of all Ceriodaphnia
dubia survival and reproduction tests performed in the WET Interlaboratory Study. This table is extracted
from the summary test condition table in the method manual and modified to fit the scope  of this study.
Items that are bold italic in this table  represent conditions standardized for the purposes of this study
where method manuals provide a range.
CDC Participant Lab SOP                      B-18

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Table 2.  Cladoceran, Ceriodaphnia dubia, Survival and Reproduction Test. Summary of test conditions and test acceptability criteria for
daphnid, Ceriodaphnia dubia, survival and reproduction toxicity tests with effluents and receiving waters
1. Test type:
2. Temperature (C):
3. Light quality:
4. Light intensity:
5. Photoperiod:
6. Test chamber size:
7. Test solution volume:
8. Renewal of test solutions:
9. Age of test organisms:
10. No. neonates per test chamber:1
11. No. replicate test chambers per concentration:
12. No. neonates per test concentration:
13. Feeding regime:
14. Cleaning:
15. Aeration:
16. Dilution water:

17. Test concentrations:
18. Dilution factor:
19. Test duration:2
20. Endpoints:
21. Test acceptability criteria:

22.  Sample handling and holding requirements:

23. Sample volume required:	
Static renewal
25 1C
Ambient laboratory illumination
10-20 uE/m2/s, or 50-100 ft-c (ambient laboratory levels)
16 h light, 8 h dark
30 mL
15 ml
Daily
Less than 24 h; and all released within a 8-h period
1
10
10
Feed 0.1 mL each of YCT and algal suspension per test chamber daily.
Use freshly cleaned glass beakers or new plastic cups daily
None
Moderately hard synthetic water prepared using MILLIPORE MILLI-Q or equivalent
deionized water and reagent grade chemicals (see Methods Manual Section 7, Dilution Water)
Five concentrations and a control
0.5
8 days
Survival and reproduction
80% or greater survival and an average of 15 or more young per surviving female in the control
solutions. 60% of surviving control organisms must produce three broods.
Samples treated as effluent samples for NPDES monitoring.  Samples are to be used on the day
specified by the finalized interlaboratory study testing schedule
1 L/day	
1 Test vessels shall be randomized in accordance with the method manuals. In addition, block randomization and use of known parentage will be required for the Ceriodaphnia
survival and reproduction test as described in the manual and guidance will be reiterated in the specific instructions provided to the laboratories.
2 The Ceriodaphnia dubia test which would otherwise be terminated after 3 broods according to methods manual Section 13.12.1 of that Method must be conducted for 8 days,
with endpoints (survival and number of young per day and number of broods at each recording interval) recorded at the end of the 6th, 7th and 8th day (specifically, at 144, 168,
and 192 hours, respectively, from test initiation). No test shall be terminated prior to the eighth day for any reason, including a failure to meet test acceptance criteria.
CDC Participant Lab SOP
              B-19

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3.2.3   Data Analysis

The Ceriodaphnia dubia survival and reproduction test shall not be terminated until day 8, however,
survival and reproduction endpoints should be calculated according to the method manuals using the three
brood approach. Raw data beyond the three brood time period should be recorded and reported, yet this
data should not be included in endpoint determination. EPA will perform additional analysis using this
data.

For survival endpoints, a NOEC and an LC50 should be calculated at the time of normal test termination
(after 60% of controls have had three broods).  For reproduction endpoints, a NOEC and an IC25 should
be calculated at the time of normal test termination (after 60% of controls have had three broods). Data
analysis and statistical procedures should be conducted according to the methods manuals.
3.2.4   Problem Resolution

In the event that problems are encountered during the WET test analysis contact Brian Rusignuolo at
(703)461-2401 for guidance. Brian will direct your call as appropriate for the resolution of technical
issues.

3.2.5   Sample Disposal

Following the termination of the test, any excess sample should be disposed of according to standard
laboratory procedures for disposal of effluent samples.

4.0 Additional Information on Data Reporting Deliverables

According to the Participant Laboratory Statement of Work, the submission of three deliverables are
required: 1). Narrative summary of testing, 2). Hardcopy results synopsis and full report, and 3).
Electronic results synopsis.  Deliverables #1 and 2 shall be submitted according to the requirements
specified in the SOW. This section provides additional instructions for the submission of the electronic
results synopsis.

Enclosed with this package is a computer diskette that contains the template for the electronic submission
of results from the Ceriodaphnia dubia survival and reproduction test method.  The disk contains a
Microsoft Excel 97 spreadsheet file named CDC	.xls. The CDC indicates that this template is for the
Ceriodaphnia dubia chronic test method, and the number following is a unique identifying number for
your laboratory. If your laboratory does not have the hardware or software capabilities to view and enter
data into this file, please contact Brian Rusignuolo at (703)461-2401 to arrange distribution of an
alternative version of the electronic template.

Notice the following  characteristics of the electronic template file:

1. The file contains four worksheet pages labeled "Sample #1", "Sample #2", "Sample #3", and "Sample
#4". The results from each of the samples that your laboratory tested in this study should be entered on a
separate worksheet page within the  same file.
    2.  Each worksheet page contains  seven information boxes (general information, sample
    collection/receipt information, test information, biological data, water quality data, summarized
    biological data, and summarized test results) in which data should be entered.  The eighth information
CDC Participant Lab SOP                      B-20

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    box (data quality flags) is for SCC use only and will aid in the automated review and quality control
    check of data.
    3.  Cells that are highlighted in pale yellow indicate required information.  Cells highlighted in blue
    indicate optional information that may be entered if available.  Cells highlighted in red are for SCC
    use only.
    4.  The file has been protected such that data can only be entered in yellow or blue cells. No other
    cells can be changed.

To complete the electronic results synopsis data deliverable:

    1.  Record information into a separate worksheet page for each sample tested. If participating in the
       extended study design, the last worksheet page (Sample #4) may be left empty.
    2.  Record requested information or data in all required cells (pale yellow).
    3.  Record requested information or data in optional cells (blue) if data is available.
    4.  Check entered data against hardcopy bench sheets to ensure accuracy.
    5.  Save the file onto the diskette provided.  Do not change the file name.
    6.  Submit the diskette with the other data reporting deliverables by the due date to:

                              DynCorp I&ET, Sample Control Center
                              6101 Stevenson Avenue
                              Alexandria, VA 22304
                              Phone:(703)461-2064
                              Fax:(703)461-8056
                              Attention: Robert Brent

5.0 Coolers

All coolers will include a prepaid return FedEx label for returning the cooler to the referee laboratory.
Fill out the sender portion of the FedEx label and place it on the cooler. Coolers will be returned by 2
Day shipment, as already marked on the prepared label. Coolers must be returned to the referee
laboratory within 7 days of sample receipt. Please empty and wipe out the cooler prior to returning it.
CDC Participant Lab SOP                       B-21

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        Appendix A: Instructions for Reconstitution of Liquid Ampule Sample for
            Cladoceran, Ceriodaphnia dubia, Survival and Reproduction Test

For each sample, three liquid ampules will be received (marked with the sample number
followed by an "A", "B", or "C"). The three containers shall be reconstituted as described below
to mimic the sample shipment schedule for effluent samples.  The container marked "A" shall be
reconstituted on the day of test initiation (Day 0) and used for renewal on Day 1. The container
marked "B" shall be reconstituted on Day 2 and used for renewals on Day 2 and Day 3. The
container marked "C" shall be reconstituted on Day 4 and used for renewals on Day 4, Day 5,
Day 6, and Day 7. Follow the directions below for the reconstitution of each  sample ampule.

1.  Volumetrically add 100 mL of the liquid ampule sample to approximately 1L of moderately-
    hard synthetic freshwater (MHSF) prepared using MILLIPORE MILLI-Q or equivalent
    deionized water and reagent grade chemicals according to Section 7 of the methods manuals.

2.  Mix by swirling or gently shaking.

3.  Bring final volume to 3L (measured using volumetric glassware) with MHSF dilution water.

4.  Mix again by swirling and gently shaking.

5.  Place reconstituted sample in a plastic container of appropriate volume. A container that
    minimizes head space (i.e. cubitainer) is recommended.

6.  This 3L sample is the whole volume reconstituted sample and should be treated as a typical
    effluent sample. It should be used directly for the 100% effluent test concentration and
    diluted appropriately to prepare other test concentrations (50%, 25%, 12.5%, and 6.25%).

7.  Use the reconstituted sample for test initiation and test renewal on Day 1.  Store sample at
    4C.

8.  Perform the Cladoceran,  Ceriodaphnia dubia, Survival and Reproduction Test as described
    in the SOW for this study and the methods manuals.

9.  Follow Steps 1 through 6 with each sample container to prepare the reconstituted sample  on
    Day 2  and Day 4 for subsequent daily renewals.
CDC Participant Lab SOP                     B-22

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Appendix B: EPA Traffic Report
United States
PDA Environmental Protection Agency
'c tn^ Washington, DC 20460
EPISODE NO:
SAMPLE NO:
DynCorp - Sample Control Center
WET Interlaboratory Variability Study Traffic Report Fax completed form immediately 6101 Stevenson Avenue
USEPA ENGINEERING AND ANALYSIS DIVISION upon completion, and include Alexandria, VA 22304
SAMPLE CONTROL CENTER hardcopy in final data report to: phone: (703) 461 -21 00
fax: (703)461-8056
Referee Laboratory Information Participant Lab Shipping Information
Name: Lab Name:
Address: Address:
City: City:
State: State:
Airbill no:
Sampler name: Date shipped:
FOR PARTICIPANT LAB USE ONLY
Received by:
Sample condition on receipt:
SAMPLE COLLECTION / RECEIPT INFORMATION
Requested Information
1.
2.
3.
4.
5.
6.
7.
8.
Sample use description
Sample collection/receipt date
Sample collection/receipt time
Sampler / recipient signature
PH
Temperature
Conductivity (freshwater methods) / Salinity
(marine methods)
Dissolved Oxygen concentration
Pre -Shipment
I 	 (initiation I 	 Irenewal 1 I 	 Irenewal 2







Post-Shipment








REQUESTED ANALYSES
9.
I 	 I Pimephales promelas Acute
I 	 I Pimephales promelas Chronic
I 	 I Ceriodaphnia dubia Acute
I 	 I Ceriodaphnia dubia Chronic
I 	 I Selenastrum capricornutum Chronic
I 	 I Menidia beryllina Acute
I 	 I Menidia beryllina Chronic
I 	 I Holmesmysis costata Acute
I 	 I Champia parvula Chronic

I 	 I Cyprinodon variegatus Acute
I 	 I Cyprinodon variegatus Chronic
I 	 I Mysidopsis bahia Chronic


CDC Participant Lab SOP
B-23

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DynCorp
litanisrtiii G Bircrjjnse TasMiijiy
TO:          Participant Laboratories for the Fathead Minnow Acute Test Method

FROM:       Robert Brent, WET Study Coordinator

DATE:       October 13, 1999

SUBJECT:    Final Guidance and SOP for the Fathead Minnow Acute Test
Enclosed is the final standard operating procedure (SOP) for laboratories participating in the fathead
minnow acute test method in EPA's WET Interlaboratory Variability Study. This SOP is a supplement to
the statement of work (SOW) that was distributed on July 9, 1999 with the solicitation for this study.  The
SOP details the sample distribution and testing schedule and provides important information for
completing participant laboratory tasks, such as instructions for the reconstitution of ampule samples.
Participant laboratories should follow the guidance in the enclosed SOP, the SOW, and the method
manuals.

Also enclosed is the electronic data reporting format disk for the fathead minnow acute method. A
description and instructions for use of the electronic data report form are provided in the  SOP.

Please ensure that the enclosed SOP and disk pertaining to the fathead minnow acute test method are
distributed to laboratory staff that will be performing the test method in the study.
FHMA Participant Lab SOP                     B-24

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                      STANDARD OPERATING PROCEDURE
          Participant Laboratory Support for EPA's WET Interlaboratory Study

                             Pimephales promelas Acute Method
Preamble:
This standard operating procedure document (SOP) is a supplement to the participant laboratory
Statement of Work (SOW).  This SOP details specific information in the SOW regarding the conduct of
the Pimephales promelas acute test method in the WET Interlaboratory Variability Study. All
modifications to the SOP must be approved by DynCorp prior to implementation.

All data deliverables submitted for this Interlaboratory study become the property of EPA and may be
Incorporated Into the public record. Laboratories may not Independently publish the results of analyses
for which they are paid to perform under this SOP.

1.0 Sample Distribution and Testing Schedule

Participant laboratory testing for the Pimephales promelas acute method will occur between October 21
and November 8,  1999 with final reports due 30 days following termination of all tests (December 8,
1999). The schedule for the testing is provided below. The date ranges on the schedule indicate the test
start dates and test completion dates.  Samples will be shipped FedEx Priority Overnight to arrive at the
participant laboratory on the test start date by 10:OOAM.  All tests must be initiated on the day of sample
arrival. Testing is scheduled to occur simultaneously at each participant  laboratory, so adherence to the
testing schedule is mandatory for all participant laboratories.

Laboratories participating in the base study design will receive 4 blind test samples (reflected in the
schedule) that may be whole volume or ampule samples. Two samples will  arrive on 10/21/99 for test
initiation on that day, and two  samples will arrive on 11/4/99 for test initiation on that day. Laboratories
participating in the extended study design will each receive three ampule samples.  Some laboratories will
receive two samples on 10/21/99 and one on 11/4/99, while other laboratories in the extended design will
receive one on 10/21/99 and two on 11/4/99. Laboratories participating in the extended design will be
notified by fax prior to study initiation to confirm the number of samples (one or two) that should be
expected for each testing period.

Each sample aliquot that is prepared and shipped will be assigned a unique sample number.  The sample
number will appear clearly and permanently on each container and on an EPA traffic report form that will
accompany each sample. For whole volume samples, one aliquot will be received on test Day 0. This
aliquot shall be used for test initiation on Day 0 and test renewal at 48 hours. For ampule samples, one
aliquot will be received on test Day 0. This aliquot shall be reconstituted on Day 0, and the reconstituted
sample shall be used for test initiation on Day 0 and test renewal at 48 hours.

Table 1. Schedule for Pimephales promelas Acute Testing.
Date
(start date - finish date)
10/21/99 - 10/25/99
11/4/99- 11/8/99
12/8/99
Activity
Conduct Fathead minnow, Pimephales promelas, Acute Test with samples #1&2
Conduct Fathead minnow, Pimephales promelas, Acute Test with samples #3&4
Fathead minnow, Pimephales promelas, Acute Test data due
FHMA Participant Lab SOP                     B-25

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2.0 Sample Traffic Reporting Tasks

2.1    Sample Receipt Confirmation

An EPA traffic report form (Appendix B) will be received with each sample. The traffic report form will
document important sample collection, shipment, and receipt information. Portions of the form will be
completed by the referee laboratory prior to shipment and will indicate the episode number, sample
number, referee laboratory information, participant laboratory shipping information, sample pre-shipment
information, and the requested analysis.  Immediately upon receipt of the sample, participant laboratories
will be responsible for determining that the sample arrived in satisfactory condition and for documenting
receipt of the sample, post-shipment sample water quality (temperature, pH, dissolved oxygen, and
conductivity or salinity), and any problems on the EPA traffic report form. The participant laboratory
shall complete the traffic report form by recording the required information in the "For Participant Lab
Use Only" box and in the "Post-Shipment" column of the "Sample Collection/Receipt Information" box.

For ampule samples, the pre-shipment and post-shipment measurement of pH, dissolved oxygen, and
conductivity or salinity shall be omitted. This will avoid possible contamination of the ampule sample
prior to reconstitution. Pre-shipment and post-shipment temperature shall be measured in a temperature
check sample. An additional sample container labeled "temperature check" will be included with each
cooler shipment of ampules. The temperature shall be measured in this container and recorded on the
EPA traffic report form as a surrogate measure of temperature for all ampule samples within that cooler.
The temperature check shall be discarded after temperature measurement, and must not be used in any
way for WET testing.

Immediately upon completion of the EPA traffic report form, the participant laboratory shall fax the
completed form to DynCorp and retain a copy for inclusion in the final data report. Receipt of the faxed
traffic report form by DynCorp will be confirmation of successful sample arrival, so it is important that
the form be completed and faxed immediately. Forms should be faxed by 11:00 AM (local laboratory
time) to facilitate sample tracking and potential problem resolution. The EPA traffic report form shall be
faxed to:

                                       Brian Rusignuolo
                                      Sample  Coordinator
                                         DynCorp  SCC
                                      fax: (703)461-8056
                                     phone: (703)461-2401

2.2    Problem Resolution

Prior to test initiation, notify Brian Rusignuolo of any special shipment receiving instructions (i.e., hold
shipment at airport or FedEx shipment center for pickup). Notification must also be made if any specific
instructions are required for Saturday deliveries.

Participant laboratories should be expecting the arrival of samples based on the provided schedule.  If
samples do not arrive as expected, if samples are damaged in shipment, if samples arrive without
accompanying traffic report forms, if sample numbers are not clearly identified on samples, or if any
other sample shipment or receipt problem is encountered immediately notify Brian Rusignuolo by phone
at (703)461-2401.

Brian must be notified by 11:00 AM (local laboratory time) if samples fail to arrive by the morning
FedEx shipment or if other shipment or sample receipt problems are encountered. DynCorp will track

FHMA Participant Lab SOP                    B-26

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lost shipments and instruct referee laboratories to resend test samples for arrival the following day if
necessary.

3.0 WET Test Analysis

3.1    Sample Preparation

3.1.1   Whole Volume Samples

Whole volume samples will be received in cubitainers with sufficient volume for test conduct and
required water chemistry analysis.  No sample preparation or adjustment steps (e.g. pH adjustment)
should be conducted prior to test initiation. The whole volume sample shall be treated as a typical 100%
effluent sample received for NPDES compliance monitoring. Test concentrations of 100%, 50%, 25%,
12.5%, and 6.25% sample shall be prepared from the whole volume sample for use in the test. These test
concentrations and a dilution water control shall be prepared using moderately hard synthetic freshwater
as the dilution water (prepared according to Section 7 of the method manuals).

3.1.2   Ampule Samples

Ampule samples will be received as small volume liquid samples in 125ml plastic containers. Prior to
test initiation the ampule samples must be reconstituted according to the instructions in Appendix A to
provide the necessary volume for testing.  The reconstituted sample shall then be treated as atypical
100% effluent sample received for NPDES compliance monitoring. Test concentrations of 100%, 50%,
25%,  12.5%, and 6.25% sample shall be prepared from this reconstituted sample for use in the test.  These
test concentrations and a dilution water control shall be prepared using moderately hard synthetic
freshwater as the dilution water (prepared according to Section 7 of the method manuals).


3.2    Test Conduct

This section of the SOP reiterates testing requirements from Section 3 of the Participant Laboratory
Statement of Work that must be followed for the conduct of the Fathead minnow, Pimephales promelas,
Acute test method. Except where indicated in Sections 3.2.1 and 3.2.2 of this SOP, each test shall be
conducted in accordance with the general guidance and method specific requirements for effluent testing
included  in the methods manuals.

3.2.1   General Testing Requirements

EPA acknowledges that the promulgated WET methods distinguish between requirements (indicated by
the compulsory terms "must" and "shall") and recommendations and guidance (indicated by discretionary
terms "should" and "may"). The  latter terms indicate that the analyst has flexibility to optimize successful
test completion and when standardization is necessary to assure the predictability of the methods to
provide reliable results. Additionally, the method manuals allow variations of the methods which are
typically fixed in the permit; therefore, for the purposes of this study, a set of variables will be defined by
EPA (for example, dilution water, salinity, and acute test duration). Any deviation from defined test
procedures and/or conditions, such as the necessity to change reagents, equipment, test conditions, or
other specified test parameters must be reported to SCC, recorded at the time of modification, noted in
telephone logs of communications, documented in a memorandum, and approved by EPA.

Additional WET test requirements and general requirements listed in the methods manuals of special note
are provided below:

FHMA Participant Lab SOP                     B-27

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(1)    Personnel that conduct tests must be the same personnel that routinely conduct the WET tests at
       that laboratory facility and who were identified in the prequalification materials. If these
       individuals cannot be available during any part of the study, the laboratory must contact SCC.
       Personnel conducting the tests must be identified clearly and consistently in records.

(2)    To coordinate testing at participant laboratories, testing of each sample with each method must be
       initiated on the precise day specified in the finalized study schedule.   Samples should be tested
       within 36 hours from the time of sample preparation (determined in this study as the time at
       which individual sample aliquots were divided from the bulk test sample for distribution to
       participant laboratories). Deviation from this schedule must be reported to SCC immediately for
       approval.

(3)    Physical and chemical properties of the test samples must be in the ranges specified in this SOP,
       the SOW, specific instructions, and the methods manuals. Method specific instructions for any
       adjustments to the test samples prior to sample use (such as reconstitution of ampule samples or
       salinity adjustments) are provided herein.  Test samples received at participant laboratories must
       be refrigerated (at 4C  2C) immediately upon receipt and throughout the period of testing.
       The temperature of the refrigeration unit should be routinely or continuously monitored to ensure
       that these sample holding requirements are met.

(4)    Measurement of test conditions (pH, conductivity or salinity, total alkalinity, total  hardness, and
       dissolved oxygen) shall be performed for each method by the participant laboratories following
       guidance in method manuals.

(5)    The specified dilution and control waters must be used and prepared according to instructions in
       Section 7 of the methods manuals.

(6)    All WET tests are to be definitive tests with a control and a minimum of five test concentrations
       prepared using a dilution factor of 0.5.

(7)    All tests must be conducted using the number of replicates and number of test containers per
       concentration as specified in Section 3.2.2.

(8)    Test chambers used within a test must be of the same type, size, shape, and material. The
       material must be allowed by the method manuals for the method used.

(9)    Test vessels shall be randomized in accordance with the method manuals.

(10)   Daily observation of mortality and removal of dead organisms for each test is required.

(11)   If test results indicate too great of toxicity (i.e., control mortalities, or complete mortality in all
       concentrations), the laboratories must contact SCC immediately and then  investigate possible
       causes, first by checking for transcription and calculation mistakes, and then by investigating
       possible contamination in  dilution waters, organism cultures, equipment, or other procedural
       steps.

(12)   If any test that has been initiated fails to be completed for any reason, the  laboratory must contact
       SCC immediately for problem resolution and scheduling of additional testing. The incomplete
       test data and the reason for not completing the test must be fully documented in the final report.
FHMA Participant Lab SOP                     B-28

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(13)   Each laboratory shall be required to report all data obtained during the course of testing,
       including the response of control samples.

(14)   Laboratories must perform all QA/QC tests listed in Section 4 of the method manuals.
       Laboratories that purchase organisms must supply QA/QC from the test organism supplier and
       follow method manuals for the appropriate QA/QC for purchasing organisms.

(15)   A reference toxicant QC test must be performed for each test method in the month that testing for
       this study occurs. Results of this test must be submitted with the final data package.

(16)   Data and statistical analyses must be submitted in hard copy in the standardized format specified
       in Section 5 of the SOW. All bench sheets and raw data, including sample tracking and chemistry
       analysis data must be submitted. Data must also be submitted electronically according to an
       electronic template (Microsoft Excel spreadsheet) that is provided with this SOP.

(17)   Data analysis must be performed in accordance with the statistical programs specified in the
       methods manuals.  Statistical methods and programs used must be reported along with sample
       calculations.

(18)   An IC25 must be reported for each chronic test.  The laboratories must report individual toxicity
       endpoints; laboratories are not allowed to average or perform other data manipulations unless
       required by a methods's instructions.

3.2.2   Method-Specific Requirements

Table 2 provides a summary of test conditions that shall be followed for the conduct of all fathead
minnow acute tests performed in the WET Interlaboratory Study. This table is extracted from the
summary test condition table in the method manual and modified to fit the scope of this study. Items that
are bold italic in this table represent conditions standardized for the purposes of this study where method
manuals provide a range.
FHMA Participant Lab SOP                      B-29

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Table 2.  Fathead Minnow, Pimephales promelas, Acute Test. Summary of test conditions and test acceptability criteria for fathead minnow,
Pimephales promelas, acute toxicity tests with effluents and receiving waters	
1. Test type:                                            Static-renewal
2. Test duration:                                         96 h
3. Temperature:                                          25  1 C
4. Light quality:                                         Ambient laboratory illumination
5. Light intensity:                                        10-20 uE/m2/s (50-100 ft-c) (ambient laboratory levels)
6. Photoperiod:                                          16 h light, 8 h darkness
7. Test chamber size:                                     250 mL
8. Test solution volume:                                  200 mL
9. Renewal of test solutions:                              At 48 h
10. Age of test organisms:                                1-14 days; 24-h range in age
11. No. organisms per test chamber:                        10
12. No. replicate chambers per concentration:                2
13. No. organisms per concentration:                       20
14. Feeding regime:                                      Artemia nauplii are made available while holding prior to the test; add 0.2 mL Artemia nauplii
                                                       concentrate 2 h prior to test solution renewal at 48 h
15. Test chamber cleaning:                                Cleaning not required
16. Test solution aeration:                                None, unless DO concentration falls below 4.0 mg/L; rate should not exceed 100 bubbles/min
17. Dilution water:                                       Moderately hard synthetic water prepared using MILLIPORE MILLI-Q or equivalent
                                                       deionized water and reagent grade chemicals (see Methods Manual Section 7, Dilution Water)
18. Test concentrations:                                  Five concentrations and a control
19. Dilution factor                                       0.5
20. Endpoint:                                           Mortality (LC50)
21. Sample handling and holding requirements:              Samples treated as effluent samples for NPDES monitoring. Samples are to be used on the day
                                                       specified by the finalized interlaboratory study testing schedule
22. Sample volume required:                              2 Lfor effluents
23. Test acceptability criterion:	90% or greater survival in controls	
NOTE: Test vessels shall be randomized in accordance with the method manuals.
FHMA Participant Lab SOP                                          B-30

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3.2.3   Data Analysis

For the Fathead minnow acute test method, the 96 hour LC50 and NOEC shall be calculated and reported.
Data analysis and statistical procedures should be conducted according to the method manuals.

3.2.4   Problem Resolution

In the event that problems are encountered during the WET test analysis contact Brian Rusignuolo at
(703)461-2401 for guidance. Brian will direct your call as appropriate for the resolution of technical
issues.

3.2.5   Sample Disposal

Following the termination of the test, any excess sample shall be disposed of according to standard
laboratory procedures for disposal of effluent samples.

4.0 Additional Information on Data Reporting Deliverables

According to the Participant Laboratory Statement of Work, the submission of three deliverables are
required: 1). Narrative summary of testing, 2). Hardcopy results synopsis and full report, and 3).
Electronic results synopsis.  Deliverables #1 and 2  shall be submitted according to the requirements
specified in the SOW. This section provides additional instructions for the submission of the electronic
results synopsis.

Enclosed with this package  is a computer diskette that contains the template for the electronic submission
of results from the Fathead minnow acute test method.  The disk contains a Microsoft Excel 97
spreadsheet file named FFA	.xls. The FFA indicates that this template is for the Fathead minnow
acute test method, and the number following is a unique identifying number for your laboratory. If your
laboratory does not have the hardware  or software capabilities to view and enter data into this file, please
contact Brian Rusignuolo at (703)461-2401 to arrange distribution of an alternative version of the
electronic template.

Notice the following characteristics of the electronic template file:

    1.  The file contains four worksheet pages labeled "Sample #1", "Sample #2", "Sample #3", and
       "Sample #4". The results from each of the samples that your laboratory tested in this study
       should be entered on a separate worksheet page within the same file.
    2.  Each worksheet page contains  six information boxes (general information, sample
       collection/receipt information, test information, biological data, water quality data, and
       summarized test results) in which data should be entered. The  seventh information box (data
       quality flags) is for SCC use only and will  aid in the automated review and quality control check
       of data.
    3.  Cells that are highlighted in pale yellow indicate required information.  Cells highlighted in blue
       indicate optional information that may be entered if available.  Cells highlighted in red are for
       SCC use only.
    4.  The file has been protected such that data can only be entered in yellow or blue cells.  No other
       cells can be changed.

To complete the electronic results synopsis data deliverable:
FHMA Participant Lab SOP                     B-31

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    1.  Record information into a separate worksheet page for each sample tested.  If participating in the
       extended study design, the last worksheet page (Sample #4) may be left empty.
    2.  Record requested information or data in all required cells (pale yellow).
    3.  Record requested information or data in optional cells (blue) if data is available.
    4.  Check entered data against hardcopy bench sheets to ensure accuracy.
    5.  Save the file onto the diskette provided.  Do not change the file name.
    6.  Submit the diskette with the other data reporting deliverables by the due date to:

                              DynCorp I&ET, Sample Control Center
                              6101 Stevenson Avenue
                              Alexandria, VA 22304
                              Phone:(703)461-2064
                              Fax:(703)461-8056
                              Attention: Robert Brent

5.0 Coolers

All coolers will include a prepaid return FedEx label for returning the cooler to the referee laboratory.
Fill out the sender portion of the FedEx label and place it on the cooler.  Coolers will be returned by 2
Day shipment, as already marked on the prepared label. Coolers must be returned to the referee
laboratory within 7 days of sample receipt. Please empty and wipe out the cooler prior to returning it.
FHMA Participant Lab SOP                     B-32

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        Appendix A:  Instructions for Reconstitution of Liquid Ampule Sample for
                   Fathead Minnow, Pimephales promelas, Acute Test

For each sample, a single liquid ampule will be received. The container shall be reconstituted
and used to initiate the test. The same reconstituted sample shall be used for test renewal at 48
hours.  Follow the directions below for the reconstitution of the sample ampule.

1.   Volumetrically add 100 mL of the liquid ampule sample to approximately 1L of
    moderately-hard synthetic freshwater (MHSF) prepared using MILLIPORE MILLI-Q or
    equivalent deionized water and reagent grade chemicals according to Section 7 of the
    methods manuals.

2.   Mix by swirling or gently shaking.

3.   Bring final volume to 4L (measured using volumetric glassware) with MHSF dilution water.

4.   Mix again by swirling and gently shaking.

5.   Place reconstituted sample in a plastic container of appropriate volume. A container that
    minimizes head space (i.e. cubitainer) is recommended.

6.   This 4L sample is the whole volume reconstituted sample and should be treated as a typical
    effluent sample. It should be used directly for the 100% effluent test concentration and
    diluted appropriately to prepare other test concentrations (50%, 25%, 12.5%, and 6.25%).

7.   Use the reconstituted sample for test initiation and test renewal at 48 hr. Store  sample at
    4C.

8.   Perform the Fathead Minnow, Pimephales promelas., Acute Test as described in the SOW
    for this study and the methods manuals.
FHMA Participant Lab SOP                   B-33

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Appendix B: EPA Traffic Report
United States
PDA Environmental Protection Agency
'c tn^ Washington, DC 20460
EPISODE NO:
SAMPLE NO:
DynCorp - Sample Control Center
WET Interlaboratory Variability Study Traffic Report Fax completed form immediately 6101 Stevenson Avenue
USEPA ENGINEERING AND ANALYSIS DIVISION upon completion, and include Alexandria, VA 22304
SAMPLE CONTROL CENTER hardcopy in final data report to: phone: (703) 461 -21 00
fax: (703)461-8056
Referee Laboratory Information Participant Lab Shipping Information
Name: Lab Name:
Address: Address:
City: City:
State: State:
Airbill no:
Sampler name: Date shipped:
FOR PARTICIPANT LAB USE ONLY
Received by:
Sample condition on receipt:
SAMPLE COLLECTION / RECEIPT INFORMATION
Requested Information
1.
2.
3.
4.
5.
6.
7.
8.
Sample use description
Sample collection/receipt date
Sample collection/receipt time
Sampler / recipient signature
PH
Temperature
Conductivity (freshwater methods) / Salinity
(marine methods)
Dissolved Oxygen concentration
Pre -Shipment
I 	 (initiation I 	 Irenewal 1 I 	 Irenewal 2







Post-Shipment








REQUESTED ANALYSES
9.
I 	 I Pimephales promelas Acute
I 	 I Pimephales promelas Chronic
I 	 I Ceriodaphnia dubia Acute
I 	 I Ceriodaphnia dubia Chronic
I 	 I Selenastrum capricornutum Chronic
I 	 I Menidia beryllina Acute
I 	 I Menidia beryllina Chronic
I 	 I Holmesmysis costata Acute
I 	 I Champia parvula Chronic

I 	 I Cyprinodon variegatus Acute
I 	 I Cyprinodon variegatus Chronic
I 	 I Mysidopsis bahia Chronic


FHMA Participant Lab SOP
B-34

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DynCorp
litanisrtiii G Bircrjjnse TasMiijiy
TO:           Participant Laboratories for the Fathead Minnow Chronic Test Method

FROM:        Robert Brent, WET Study Coordinator

DATE:        September 23, 1999

SUBJECT:    Final Guidance and SOP for the Fathead Minnow Chronic Test
Enclosed is the final standard operating procedure (SOP) for laboratories participating in the fathead
minnow chronic test method of the WET Interlaboratory Variability Study.  This SOP is a supplement to
the statement of work (SOW) that was distributed on July 9, 1999 with the solicitation for this study.
Participant laboratories should follow the guidance in the enclosed SOP, the SOW, and the method
manuals.

At the Participant Laboratory Meeting held on September 16, 1999, EPA and DynCorp staff presented the
general study design, discussed participant laboratory tasks, and answered questions from participant
laboratories. Notes from the meeting, including handouts, slide copies, and a list of questions and
answers, will be provided to participant laboratories by next week. Unfortunately, the fathead chronic
test method is scheduled to begin before these meeting notes are finalized and distributed, so items from
the meeting that specifically apply to the fathead chronic test method are addressed in this memo.
Participant laboratory tasks that were discussed in the meeting presentations are covered in the SOW and
SOP provided. Specific questions and answers from the meeting that pertain to the fathead chronic test
are listed below:

(1)  Q: Is residual chlorine measurement required?
     A: The requirements of the method manuals should be followed for each test method. Residual
     chlorine measurement is not specifically required for the fathead chronic test, however, if your
     laboratory routinely tests residual chlorine on each sample, this information should be included in
     the data report deliverables.

(2)  Q: Our laboratory's moderately hard synthetic water typically has a pH of 8.0-8.2, above the range
     given in Section 7 of the method manual. Should we adjust the pH of the dilution water to within
     the given range in Table 3 (Section 7 of the method manuals)?
     A: No.  The table in Section 7 gives expected approximate ranges, not required ranges.  No
     adjustment should be made, however, the laboratory should confirm that the water is prepared
     properly and prepared using the proper chemicals (correct hydrate forms  of the chemicals).

(3)  Q: Is distilled water acceptable for use  as the base for moderately hard synthetic dilution water?
     A: No.  The method manuals state that the synthetic dilution water must be prepared from deionized
     water obtained from a Millipore Milli-Q or equivalent system. An equivalent system should be
FHMC Participant Lab SOP                     B-35

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     interpreted to imply a deionizing system that produces water of equivalent quality (i.e.,
     conductivity).

(4)  Q: Can plastic beakers be used as test containers for the fathead chronic test?
     A: Yes.  Glass or plastic may be used.

(5)  Q: Can a 500mL plastic disposable beaker be used for the fathead chronic test?
     A: In the original solicitation, the test chamber size for the fathead chronic test was stated as 500mL.
     This was an error that was corrected in the table of test conditions provided in the final SOP. The
     intended test chamber was a 600mL glass beaker that is graduated up to SOOmL (and is often referred
     to as a SOOmL beaker).  Because of the error in the original solicitation document, a test chamber
     size of 500 - 600mL will be acceptable for the study, provided that the chamber has similar
     dimensions to the glass 600mL beaker.  Glass or plastic is acceptable within these volume
     constraints.

(6)  Q: How is mean dry weight measured for the growth endpoint in the fathead chronic test?
     A: According to the method manual, the mean dry weight is measured for each replicate as the total
     dry weight of larvae (total weight minus tare weight) divided by the number of original larvae in that
     replicate. This endpoint is in essence a combined survival and growth endpoint. For the
     determination of test acceptability, the mean dry weight per surviving larvae should be calculated for
     control replicates.
FHMC Participant Lab SOP                     B-36

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                     STANDARD OPERATING PROCEDURE
          Participant Laboratory Support for EPA's WET Interlaboratory Study

                 Pimephales promelas Larval Survival and Growth Method
Preamble:
This standard operating procedure document (SOP) is a supplement to the participant laboratory
Statement of Work (SOW).  This SOP details specific information in the SOW regarding the conduct of
the Pimephales promelas larval survival and growth test method in the WET Interlaboratory Variability
Study.  All modifications to the SOP must be approved by DynCorp prior to implementation.

All data deliverables submitted for this Interlaboratory study become the property of EPA and may be
Incorporated Into the public record. Laboratories may not Independently publish the results of analyses
for which they are paid to perform under this SOP.

1.0 Sample Distribution and Testing Schedule

Participant laboratory testing for the Pimephales promelas larval survival and growth method will occur
between  September 28 and October 12,  1999 with final reports due 30 days following termination of all
tests (Novemberl2, 1999).  The schedule for the testing is provided below. The date ranges on the
schedule indicate the test start dates and test completion dates. Samples will be shipped FedEx Priority
Overnight to arrive at the participant laboratory on the test start date by 10:OOAM.  All tests must be
initiated on the day of sample arrival. Testing is scheduled to occur simultaneously at each participant
laboratory, so adherence to the testing schedule is mandatory for all participant laboratories.

Laboratories participating in the base study design will receive 4 blind test samples (reflected in the
schedule) that may be whole volume or ampule samples. Two samples will arrive on 9/28/99 for test
initiation on that day, and two samples will arrive on 10/5/99 for test initiation on that day. Laboratories
participating in the extended study design will each receive three ampule samples.  Some laboratories will
receive two samples on 9/28/99 and one on 10/5/99, while other laboratories in the extended design will
receive one on 9/28/99 and two on 10/5/99.  Laboratories participating in the extended design will be
notified by fax prior to study initiation to confirm the number of samples (one or two) that should be
expected for each testing period.

Each sample aliquot that is prepared and shipped will be assigned a unique sample number.  The sample
number will appear clearly and permanently on each container and on an EPA traffic report form that will
accompany each sample. For tests that require additional shipments for sample renewal, the sample
number shall be the same for each initiation and renewal shipment with the addition of a letter (A, B, or
C) after the sample number to designate the sample for use  as initiation (A), renewal 1 (B), or renewal 2
(C).

For whole volume samples, separate aliquots will be received on test Day 0, Day 2, and Day 4. The first
aliquot (identified with the  sample number and the letter "A") shall be used for test initiation on Day 0
and renewal on Day  1.  The second aliquot (identified with the sample number and the letter "B") shall be
used for test renewals on Day 2 and Day 3. The final aliquot (identified with the sample number and the
letter "C") shall be used for test renewal on Day 4, Day 5, and Day 6.  This sample shipment schedule
mimics the typical schedule for chronic monitoring of effluent for compliance.
FHMC Participant Lab SOP                     B-37

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For ampule samples , three separate ampules containers (marked with the sample number followed by A,
B, or C) will be received in a single shipment on test Day 0. The container marked "A" shall be
reconstituted on test Day 0 and used for test initiation and renewal on Day 1. The other aliquots of the
sample shall be refrigerated and stored until use on Day 2 and Day 4, respectively. The container marked
"B" shall be reconstituted  on test Day 2 and used for renewal on Day 2 and 3. The container marked "C"
shall be reconstituted shall be reconstituted on Day 4 and used for renewal on Day 4, Day 5 and Day 6.
The sample reconstitution schedule for ampules attempts to mimic the typical sample shipment schedule
for chronic monitoring of effluents for compliance.

Table 1.  Schedule for Pimephales promelas Larval Survival and Growth Testing.
Date
(start date - finish date)
9/28/99 - 10/5/99
10/5/99 - 10/12/99
11/12/99
Activity
Conduct Fathead minnow, Pimephales promelas, Larval Survival and Growth Test with samples #1&2
Conduct Fathead minnow, Pimephales promelas, Larval Survival and Growth Test with samples #3&4
Fathead minnow, Pimephales promelas, Larval Survival and Growth Test data due
2.0 Sample Traffic Reporting Tasks

2.1    Sample Receipt Confirmation

An EPA traffic report form (Appendix B) will be received with each sample. The traffic report form will
document important sample collection, shipment, and receipt information.  Portions of the form will be
completed by the referee laboratory prior to shipment and will indicate the episode number, sample
number, referee laboratory information, participant laboratory shipping information, sample pre-shipment
information, and the requested analysis.  Immediately upon receipt of the sample, participant laboratories
will be responsible for determining that the sample arrived in satisfactory condition and for documenting
receipt of the sample, post-shipment sample water quality (temperature, pH, dissolved oxygen, and
conductivity or salinity), and any problems on the EPA traffic report form. The participant laboratory
shall complete the traffic report form by recording the required information in the "For Participant Lab
Use Only" box and in the "Post-Shipment" column of the "Sample Collection/Receipt Information" box.

For ampule samples, the pre-shipment and post-shipment measurement of pH, dissolved oxygen, and
conductivity or salinity shall be omitted. This will avoid possible contamination of the ampule sample
prior to reconstitution. Pre-shipment and post-shipment temperature shall be measured in a temperature
check sample. An additional sample container labeled "temperature check" will be included with each
cooler shipment of ampules. The temperature shall be measured in this container and recorded on the
EPA traffic report form  as a surrogate measure of temperature for all ampule samples within that cooler.
The temperature check shall stay with the ampules and rechecked when renewals are prepared. The
temperature check shall be discarded after the final temperature measurement, and  must not be used in
any way for WET testing.

Immediately upon completion of the EPA traffic report form, the participant laboratory shall fax the
completed form to DynCorp and retain a copy for inclusion in the final data report. Receipt of the faxed
traffic report form by DynCorp will be confirmation of successful sample arrival, so it is important that
the form be completed and faxed immediately. Forms should be faxed by 11:00 AM (local laboratory
time) to facilitate sample tracking and potential problem resolution. The EPA traffic report form shall be
faxed to:
FHMC Participant Lab SOP                     B-38

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                                       Brian Rusignuolo
                                      Sample Coordinator
                                         DynCorp SCC
                                      fax: (703)461-8056
                                     phone: (703)461-2401
2.2    Problem Resolution
Prior to test initiation, notify Brian Rusignuolo of any special shipment receiving instructions (i.e., hold
shipment at airport or at FedEx shipment center for pickup). Notification must also be made if any
specific instructions are required for Saturday deliveries.

Participant laboratories should be expecting the arrival of samples based on the provided schedule.  If
samples do not arrive as expected, if samples are damaged in shipment, if samples arrive without
accompanying traffic report forms, if sample  numbers are not clearly identified on samples, or if any
other sample shipment or receipt problem is encountered immediately notify Brian Rusignuolo by phone
at (703)461-2401.

Brian must be notified by 11:00 AM (local laboratory time) if samples fail to arrive by the morning
FedEx shipment or if other shipment or sample receipt problems are encountered. DynCorp will track
lost shipments and instruct referee laboratories to resend test samples for arrival the following day if
necessary. If renewal shipments do not arrive on the expected day, DynCorp will provide guidance for
test renewal on a case-by-case basis.  Depending on the volume of sample remaining from previous
shipments, laboratories may be instructed to conduct full renewals with the remaining sample, conduct
partial  renewals with the remaining sample, or omit the sample renewal for that day but carefully record
dissolved oxygen throughout the day and  remove excess food and dead organisms from the test
containers.

3.0 WET Test Analysis

3.1    Sample Preparation

3.1.1   Whole Volume Samples

Whole volume samples will be received in cubitainers with sufficient volume for test conduct and
required water chemistry analysis. No sample preparation or adjustment steps (e.g.  pH adjustment)
should be conducted prior to test initiation. The whole volume sample shall be treated as a typical 100%
effluent sample received for NPDES compliance monitoring. Test concentrations of 100%, 50%, 25%,
12.5%, and 6.25% sample shall be prepared from the whole volume sample for use in the test. These test
concentrations and a dilution water control shall be prepared using moderately hard synthetic freshwater
as the dilution water (prepared according to Section 7 of the methods manuals).

3.1.2   Ampule Samples

Ampule samples  will be received as  small volume liquid samples in 125ml plastic containers. Prior to
test initiation the  ampule samples must be reconstituted according to the instructions in Appendix A to
provide the necessary volume for testing.  The reconstituted sample  shall then be treated as atypical
100% effluent sample received for NPDES compliance monitoring.  Test concentrations of 100%, 50%,
25%, 12.5%, and 6.25% sample shall be prepared from this reconstituted sample for use in the test.  These
test concentrations and a dilution water control shall be prepared using moderately hard synthetic
freshwater as the dilution water (prepared according to Section 7 of the methods manuals).

FHMC Participant Lab SOP                     B-39

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3.2    Test Conduct

This section of the SOP reiterates testing requirements from Section 3 of the Participant Laboratory
Statement of Work that must be followed for the conduct of the Fathead minnow, Pimephales promelas,
Chronic test method. Except where indicated in Sections 3.2.1 and 3.2.2 of this SOP, each test shall be
conducted in accordance with the general guidance and method specific requirements for effluent testing
included in the methods manuals.

3.2.1   General Testing Requirements

EPA acknowledges that the promulgated WET methods distinguish between requirements (indicated by
the compulsory terms "must" and "shall") and recommendations and guidance (indicated by discretionary
terms "should" and "may"). The latter terms indicate that the analyst has flexibility to optimize successful
test completion and when standardization is necessary to assure the predictability of the methods to
provide reliable results. Additionally, the method manuals allow variations of the methods which are
typically fixed in the permit; therefore, for the purposes of this study, a set of variables will be defined by
EPA (for example, dilution water, salinity, and acute test duration).  Any deviation from defined test
procedures and/or conditions, such as the necessity to change reagents, equipment, test conditions, or
other specified test parameters must be reported to SCC, recorded at the time of modification, noted in
telephone logs of communications, documented in a memorandum, and approved by EPA.

Additional WET test requirements and general requirements listed in the methods manuals of special note
are provided below:

(1)    Personnel that conduct tests must be the same personnel that routinely conduct the WET tests at
       that laboratory facility and who were  identified in the prequalification materials. If these
       individuals cannot be available during any part of the study, the laboratory must contact SCC.
       Personnel conducting the tests must be identified clearly and consistently in records.

(2)    To coordinate testing at participant laboratories, testing of each sample with each method must be
       initiated on the precise  day specified in the finalized study schedule. Samples should be tested
       within 36 hours from the time of sample preparation (determined in this study as the time at
       which individual sample aliquots were divided from the bulk test sample  for distribution to
       participant laboratories).  Deviation from this schedule must be reported to SCC immediately for
       approval.

(3)    Physical and chemical properties of the test samples must be in the ranges specified in this SOP,
       the SOW, specific instructions, and the methods manuals. Method specific instructions for any
       adjustments to the test samples prior to sample use (such as reconstitution of ampule samples or
       salinity adjustments) are provided herein.  Test samples received at participant laboratories must
       be refrigerated (at 4C   2C) immediately upon receipt and throughout the period of testing.
       The temperature of the  refrigeration unit should be routinely or continuously monitored to ensure
       that these sample holding requirements are met.

(4)    Measurement of test conditions (pH, conductivity or salinity, total alkalinity, total hardness, and
       dissolved oxygen) shall be performed for each method by the participant laboratories following
       guidance in method manuals.

(5)    The specified dilution and control waters must be used and prepared according to instructions in
       Section 7  of the methods manuals.
FHMC Participant Lab SOP                     B-40

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(6)    All WET tests are to be definitive tests with a control and a minimum of five test concentrations
       prepared using a dilution factor of 0.5.

(7)    All tests must be conducted using the number of replicates and number of test containers per
       concentration as specified in Section 3.2.2.

(8)    Test chambers used within a test must be of the same type, size, shape, and material. The
       material must be allowed by the method manuals for the method used.

(9)    Test vessels shall be randomized in accordance with the method manuals.

(10)   Daily observation of mortality and removal of dead organisms for each test is required.

(11)   If test results indicate too great of toxicity (i.e., control mortalities, or complete mortality in all
       concentrations), the laboratories must contact SCC immediately and then investigate possible
       causes, first by checking for transcription and calculation mistakes, and then by investigating
       possible contamination in dilution waters, organism cultures, equipment, or other procedural
       steps.

(12)   If any test that has been initiated fails to be completed for any reason, the laboratory must contact
       SCC immediately for problem resolution and scheduling of additional testing. The incomplete
       test data and the reason for not completing the test must be fully documented in the  final report.

(13)   Each laboratory shall be required to report all data obtained during the course of testing,
       including the response of control samples.

(14)   Laboratories must perform all QA/QC tests listed in Section 4 of the method manuals.
       Laboratories that purchase organisms must supply QA/QC from the test organism supplier and
       follow method manuals for the appropriate QA/QC for purchasing organisms.

(15)   A reference toxicant QC test must be performed for each test method in the month that testing for
       this study occurs. Results of this test must be submitted with the final data package.

(16)   Data and statistical analyses must be submitted in hard copy in the standardized format specified
       in Section 5 of the SOW. All bench  sheets and raw data, including sample tracking and chemistry
       analysis data must be submitted.  Data must also be submitted electronically according to an
       electronic template (Microsoft Excel spreadsheet) that is provided with this SOP.

(17)   Data analysis must be performed in accordance with the statistical programs specified in the
       methods manuals. Statistical methods and programs used must be reported along with sample
       calculations.

(18)   An IC25 must be reported for each chronic test.  The laboratories must report individual toxicity
       endpoints; laboratories are not allowed to average or perform other data manipulations unless
       required by a methods's instructions.

3.2.2   Method-Specific Requirements

Table 2 provides a summary of test conditions that shall be followed for the conduct of all fathead
minnow chronic tests performed in the WET Interlaboratory Study. This table is extracted from the
summary test condition table in the method manual and modified to fit the scope of this study. Items that

FHMC Participant Lab SOP                     B-41

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are bold italic in these tables represent conditions standardized for the purposes of this study where
method manuals provide a range.
FHMC Participant Lab SOP                     B-42

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Table 2.  Fathead Minnow, Pimephales promelas, Larval Survival and Growth Test. Summary of test conditions and test acceptability criteria
for fathead minnow, Pimephales promelas, larval survival and growth toxicity tests with effluents and receiving waters	
1. Test type:
2. Temperature:
3. Light quality:
4. Light intensity:
5. Photoperiod:
6. Test chamber size:
7. Test solution volume:
8. Renewal of test solutions:
9. Age of test organisms:
10. No. larvae per test chamber:
11. No. replicate chambers per concentration:
12. No. larvae per concentration:
13. Source of food:
14. Feeding regime:
15. Cleaning:
16. Aeration:
17. Dilution water:

18. Test concentrations:
19. Dilution factor
        0.5
20. Test duration:
        7 days
21. Endpoints:
22. Test acceptability criteria:

23. Sample handling and holding requirements:

24. Sample volume required:	
Static renewal
251C
Ambient laboratory illumination
10-20 nE/m2/s (50-100 ft-c) (ambient laboratory levels)
16 h light, 8 h darkness
600 ml
250 ml
Daily
Newly hatched larvae less than 24h old. If shipped, not more than 48h old, 24h range in age
10
4
40
Newly hatched Artemia nauplii (less than 24 h old)
Feed 0.1 g newly hatched (less than 24-h old) brine shrimp nauplii three times daily at 4-h
intervals or, as a minimum, 0.15 g twice daily, 6 h between feedings (at the beginning of the work
day prior to renewal, and at the end of the work day following renewal). Sufficient nauplii are
added to provide an excess. Larvae fish are not fed during the final 12 h of the test
Siphon daily, immediately before test solution renewal
None, unless DO concentration falls below 4.0 mg/L. Rate should not exceed 100 bubbles/min
Moderately hard synthetic water prepared using MILLIPORE MILLI-Q or equivalent
deionized water and reagent grade chemicals (see Methods Manual Section 7, Dilution Water)
Five concentrations and a control
Survival and growth (weight as mean per original)
80% or greater survival in controls; average dry weight per surviving organism in control
chambers equals or exceeds 0.25 mg/surviving
Samples treated as effluent samples for NPDES monitoring. Samples are to be used on the day
specified by the finalized interlaboratory study testing schedule
2.5 L/day
NOTE: Test vessels shall be randomized in accordance with the method manuals.
FHMC Participant Lab SOP
             B-43

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3.2.3   Data Analysis

For the Fathead minnow chronic test method, the 7 day survival LC50, 7 day survival NOEC, growth IC25,
and growth NOEC shall be calculated and reported. Data analysis and statistical procedures should be
conducted according to the method manuals.

3.2.4   Problem Resolution

In the event that problems are encountered during the WET test analysis contact Brian Rusignuolo at
(703)461-2401 for guidance.  Brian will direct your call as appropriate for the resolution of technical
issues.

3.2.5   Sample Disposal

Following the termination of the test, any excess sample shall be disposed of according to standard
laboratory procedures for disposal of effluent samples.

4.0 Additional Information  on Data Reporting Deliverables

According to the Participant Laboratory Statement  of Work, the submission of three deliverables are
required: 1). Narrative summary of testing, 2). Hardcopy results synopsis and full report, and 3).
Electronic results synopsis.  Deliverables #1 and 2  shall be submitted according to the requirements
specified in the SOW. This section provides additional instructions for the submission of the electronic
results synopsis.

Enclosed with this package is  a computer diskette that contains the template for the electronic submission
of results from the Fathead minnow chronic test method. The disk contains a Microsoft Excel 97
spreadsheet file named FHC	.xls. The FHC indicates that this template is for the Fathead minnow
chronic test method, and the number following is a unique identifying number for your laboratory. If
your laboratory does not have the hardware or software capabilities to view and enter data into this file,
please contact Brian Rusignuolo at (703)461-2401  to arrange distribution of an alternative version of the
electronic template.

Notice the following characteristics of the electronic template file:

    1.  The file contains four worksheet pages labeled  "Sample #1", "Sample #2", "Sample #3", and
       "Sample #4". The results from each of the samples that your laboratory tested in this study
       should be entered on a separate worksheet page within the same file.
    2.  Each worksheet page  contains seven information boxes (general information, sample
       collection/receipt information, test information, biological data, water quality data, weight data,
       and summarized test results) in which data should be entered. The eighth information box (data
       quality flags) is for SCC use only and will  aid in the automated review and quality control check
       of data.
    3.  Cells that are highlighted in pale yellow indicate required information.  Cells highlighted in blue
       indicate optional information that may be entered if available.  Cells highlighted in red are for
       SCC use only.
    4.  The file has been protected such that data can only be entered in yellow or blue cells. No other
       cells can be changed.


FHMC Participant Lab SOP                     B-44

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To complete the electronic results synopsis data deliverable:

    1.  Record information into a separate worksheet page for each sample tested.  If participating in the
       extended study design, the last worksheet page (Sample #4) may be left empty.
    2.  Record requested information or data in all required cells (pale yellow).
    3.  Record requested information or data in optional cells (blue) if data is available.
    4.  Check entered data against hardcopy bench sheets to ensure accuracy.
    5.  Save the file onto the diskette provided.  Do not change the file name.
    6.  Submit the diskette with the other data reporting deliverables by the due date to:

                              DynCorp I&ET, Sample Control Center
                              6101 Stevenson Avenue
                              Alexandria, VA 22304
                              Phone:(703)461-2064
                              Fax:(703)461-8056
                              Attention: Robert Brent

5.0 Coolers

All coolers will include a prepaid return FedEx label for returning the cooler to the referee laboratory.
Fill out the sender portion of the FedEx label and place it on the cooler.  Coolers will be returned by 2
Day shipment, as already marked on the prepared label. Coolers must be returned to the referee
laboratory within 7 days of sample receipt. Please empty and wipe out the cooler prior to returning it.
FHMC Participant Lab SOP                      B-45

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       Appendix A:  Instructions for Reconstitution of Liquid Ampule Sample for
        Fathead Minnow, Pimephales promelas, Larval Survival and Growth Test

For each sample, three liquid ampules will be received (marked with the sample number
followed by an "A", "B", or "C"). The three containers shall be reconstituted as described below
to mimic the sample shipment schedule for effluent samples. The container marked "A" shall be
reconstituted on the day of test initiation (Day 0) and used for renewal on Day 1. The container
marked "B" shall be reconstituted on Day 2 and used for renewals on Day 2 and Day 3.  The
container marked "C" shall be reconstituted on Day 4 and used for renewals on Day 4, Day 5,
and Day 6. Follow the directions below for the reconstitution of each sample ampule.

1. Volumetrically add 100 mL of the liquid ampule sample to approximately 1L of moderately-
   hard synthetic freshwater (MHSF) prepared using MILLIPORE MILLI-Q or equivalent
   deionized water and reagent grade chemicals according to Section 7 of the methods manuals.

2. Mix by swirling or gently shaking.

3. Bring final volume to 8L (measured using volumetric glassware) with MHSF dilution water.

4. Mix again by swirling and gently shaking.

5. Place reconstituted sample in a plastic container of appropriate volume. A container that
   minimizes head space (i.e. Cubitainer) is recommended.

6. This 8L sample is the whole volume reconstituted sample and should be treated as a typical
   effluent sample. It should be used directly for the 100% effluent test concentration and
   diluted appropriately to prepare other test concentrations (50%, 25%, 12.5%, and 6.25%).

7. Use the reconstituted sample for test initiation and test renewal on Day  1.  Store sample at
   4C.

8. Perform the Fathead Minnow, Pimephales promelas, Larval Survival and  Growth Test as
   described in the SOW for this study and the methods manuals.

9. Follow Steps 1 through 6 with each sample container to prepare the reconstituted sample on
   Day 2 and Day 4 for subsequent daily renewals.
FHMC Participant Lab SOP                   B-46

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Appendix B: EPA Traffic Report
 United States
PDA Environmental Protection Agency
CrM Washington, DC 20460
EPISODE NO:
SAMPLE NO:
DynCorp - Sample Control Center
WET Interlaboratory Variability Study Traffic Report Fax completed form immediately 6101 Stevenson Avenue
USEPA ENGINEERING AND ANALYSIS DIVISION upon completion, and include Alexandria, VA 22304
SAMPLE CONTROL CENTER hardcopy in final data report to: phone: (703) 461 -21 00
fax:(703)461-8056
Referee Laboratory Information Participant Lab Shipping Information
Name: Lab Name:
Address: Address:
City: City:
State: State:
Airbill no:
Sampler name: Date shipped:
FOR PARTICIPANT LAB USE ONLY
Received by:
Sample condition on receipt:
SAMPLE COLLECTION / RECEIPT INFORMATION
Requested Information
1.
2.
3.
4.
5.
6.
7.
8.
Sample use description
Sample collection/receipt date
Sample collection/receipt time
Sampler / recipient signature
PH
Temperature
Conductivity (freshwater methods) / Salinity
(marine methods)
Dissolved Oxygen concentration
Pre-Shipment
I 	 (initiation I 	 I renewal 1 I 	 Irenewal 2







Post-Shipment








REQUESTED ANALYSES
9.
I  I Pimephales promelas Acute
I  I Pimephales promelas Chronic
I  I Ceriodaphnia dubia Acute
I  I Ceriodaphnia dubia Chronic
I  I Selenastrum capricornutum Chronic
I  I Menidia beryllina Acute
I  I Menidia beryllina Chronic
I  I Holmesmysis costata Acute
I  I Champia parvula Chronic

I  I Cyprinodon variegatus Acute
I  I Cyprinodon variegatus Chronic
I  I Mysidopsis bahia Chronic


FHMC Participant Lab SOP
B-47

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DynCorp
litanisrtiii G Bircrjjnse TasMiijiy
TO:          Participant Laboratories for the Selenastrum capricornutum Growth Test Method
FROM:       Robert Brent, WET Study Coordinator
DATE:       February 25, 2000
SUBJECT:    Final Guidance and SOP for the Selenastrum capricornutum Growth Test

Enclosed is the final standard operating procedure (SOP) for laboratories participating in the Selenastrum
capricornutum growth test method in EPA's WET Interlaboratory Variability Study. This SOP is a
supplement to the statement of work (SOW) that was distributed on July 9, 1999 with the solicitation for
this study. The SOP details the sample distribution and testing schedule and provides important
information for completing participant laboratory tasks, such as instructions for the reconstitution of
ampule samples. Participant laboratories should follow the guidance in the enclosed SOP, the SOW, and
the method manuals.

Also enclosed is the electronic data reporting format disk for the Selenastrum capricornutum growth test
method. A description and instructions for use of the electronic data report form are provided in the SOP.

Please ensure that the enclosed SOP and disk pertaining to the Selenastrum capricornutum growth test
method are distributed to laboratory staff that will be performing the test method in the study.  Also,
please see that laboratory staff read over the SOP carefully to ensure the proper data is collected and
reported. Please note that the ampule reconstitution instructions for this method require the
addition of 200mL of the ampule sample instead of lOOmL that has been used previously for other
methods.  For this reason, ampule samples will be provided in SOOmL bottles for this method.
SCO Participant Lab SOP                      B-48

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                     STANDARD OPERATING PROCEDURE
          Participant Laboratory Support for EPA's WET Interlaboratory Study

                  Green Alga, Selenastrum capricornutum Growth Method
Preamble:
This standard operating procedure document (SOP) is a supplement to the participant laboratory
Statement of Work (SOW).  This SOP details specific information in the SOW regarding the conduct of
the Selenastrum capricornutum growth test method in the WET Interlaboratory Variability Study.  All
modifications to the SOP must be approved by DynCorp prior to implementation.

All data deliverables submitted for this interlaboratory study become the property of EPA and may be
incorporated into the public record. Laboratories may not independently publish the results of analyses
for which they are paid to perform under this SOP.

1.0 Sample Distribution and Testing Schedule

Participant laboratory testing for the green alga, Selenastrum capricornutum, growth method will occur
between March 9 and April 3, 2000 with final reports due 30 days following termination of all tests (May
3, 2000).  The testing schedule is provided below in Table 1. The date ranges on the schedule indicate the
test start dates and test completion dates. Samples will be shipped FedEx Priority Overnight to arrive at
the participant laboratory on the test start date by 10:00AM. All tests must be initiated on the day of
sample arrival. Testing is scheduled to occur simultaneously at each participant laboratory, so adherence
to the testing schedule is mandatory for all participant laboratories.

Participant laboratories in the study will receive 4 blind test samples (reflected in the schedule) that may
be whole volume or ampule samples.  One sample will arrive on 3/9/00 for test initiation on that day, the
second, third, and fourth samples will arrive on 3/16/00, 3/23/00, and 3/30/00,  respectively. On each
testing date, two side-by-side tests will be conducted on the sample received. One test will analyze
the sample with the addition of EDTA, and one test will analyze the sample without the addition  of
EDTA

Each sample that is prepared and shipped will be assigned a unique sample number.  The sample number
will appear clearly and permanently on each container  and on an EPA traffic report form that will
accompany each sample. For whole volume samples that are received, the laboratory shall split the
sample as described in Section 3.2.1 for analysis with and without EDTA. For ampule samples, the
laboratory shall reconstitute the sample according to instructions in Appendix A and then split the sample
for analysis with and without EDTA.
SCO Participant Lab SOP                      B-49

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Table 1. Schedule for Selenastrum capricornutum Growth Testing.
Date
(start date - finish date)
3/9/00 - 3/13/00
3/16/00 - 3/20/00
3/23/00 - 3/27/00
3/30/00 - 4/3/00
5/3/00
Activity
Conduct Green Alga, Selenastrum capricornutum, Growth Test with sample #1 (with and without EDTA)
Conduct Green Alga, Selenastrum capricornutum, Growth Test with sample #2 (with and without EDTA)
Conduct Green Alga, Selenastrum capricornutum, Growth Test with sample #3 (with and without EDTA)
Conduct Green Alga, Selenastrum capricornutum, Growth Test with sample #4 (with and without EDTA)
Green Alga, Selenastrum capricornutum, Growth Test data due
2.0 Sample Traffic Reporting Tasks

2.1    Sample Receipt Confirmation

An EPA traffic report form (Appendix B) will be received with each sample. The traffic report form will
document important sample collection, shipment, and receipt information.  Portions of the form will be
completed by the referee laboratory prior to shipment and will indicate the episode number, sample
number, referee laboratory information, participant laboratory shipping information, sample pre-shipment
information, and the requested analysis.  Immediately upon receipt of the sample, participant laboratories
will be responsible for determining that the sample arrived in satisfactory condition and for documenting
receipt of the sample, post-shipment sample water quality (temperature, pH, dissolved oxygen, and
conductivity or salinity), and any problems on the EPA traffic report form. The participant laboratory
shall complete the traffic report form by recording the required information in the "For Participant Lab
Use Only" box and in the "Post-Shipment" column of the "Sample Collection/Receipt Information" box.

For ampule samples, the pre-shipment and post-shipment measurement of pH, dissolved oxygen, and
conductivity or salinity shall be omitted. This will avoid possible contamination of the ampule sample
prior to reconstitution. Pre-shipment and post-shipment temperature shall be measured in a temperature
check sample. An additional sample container labeled "temperature check" will be included with each
cooler shipment of ampules. The temperature shall be measured in this container and recorded on the
EPA traffic report form as a surrogate measure of temperature for all ampule samples within that cooler.
The temperature check shall be discarded after temperature measurement, and must not be used in any
way for WET testing.

Immediately upon completion of the EPA traffic report form, the participant laboratory shall fax the
completed form to DynCorp and retain a copy for inclusion in the final data report. Receipt of the faxed
traffic report form by DynCorp will be confirmation of successful sample arrival, so it is important that
the form be completed and faxed immediately.  Forms should be faxed by 11:OOAM (local laboratory
time) to facilitate sample tracking and potential problem resolution. The EPA traffic report form shall be
faxed to:

                                       Brian Rusignuolo
                                      Sample  Coordinator
                                         DynCorp SCC
                                      fax:  (703)461-8056
                                     phone: (703)461-2401


SCO Participant Lab SOP                      B-50

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2.2    Problem Resolution

Prior to test initiation, notify Brian Rusignuolo of any special shipment receiving instructions (i.e., hold
shipment at airport or FedEx shipment center for pickup). Notification must also be made if any specific
instructions are required for Saturday deliveries.

Participant laboratories should be expecting the arrival of samples based on the provided schedule. If
samples do not arrive as expected, if samples are damaged in shipment, if samples arrive without
accompanying traffic report forms, if sample numbers are not clearly identified on samples, or if any
other sample shipment or receipt problem is encountered immediately notify Brian Rusignuolo by phone
at (703)461-2401.

Brian must be notified by 11:OOAM (local laboratory time) if samples fail to arrive by the morning FedEx
shipment or if other shipment or sample receipt problems are encountered. DynCorp will track lost
shipments and instruct referee laboratories to resend test samples for arrival the following day if
necessary.

3.0 WET Test Analysis

3.1    Dilution Water Preparation

Two separate dilution waters must be prepared for the Selenastrum growth test.  One must be prepared as
described in the methods manual with the addition of EDTA, and one must be prepared without the
addition of EDTA. Acceptable dilution waters for this test include the algal culture media or moderately
hard synthetic water with the correct addition of nutrients.

3.2    Sample Preparation

3.2.1   Whole Volume Samples

Whole volume samples will be received in 4L cubitainers with sufficient volume for the conduct of two
tests and required water chemistry analysis.  Prior to preparing test solutions, the whole volume sample
shall be split into two portions of 2L each.  One portion should be labeled "with EDTA", and 2mL (ImL
per liter) of each nutrient stock including EDTA shall be added according to the method manual. The
second portion should be labeled "without EDTA", and 2mL (ImL per liter) of each nutrient stock
excluding EDTA shall be added according to the method manual. Each sample portion shall then be
treated as a typical 100% effluent sample received for NPDES compliance monitoring.  The Selenastrum
capricornutum growth test shall be conducted on each sample portion (with and without EDTA).  Test
concentrations of 100%, 50%, 25%,  12.5%, and 6.25% sample shall be prepared from each portion using
the appropriate dilution water (Section 3.1)

3.2.2   Ampule Samples

Ampule samples will be received as small volume liquid samples in 500ml plastic bottles. Prior to test
initiation the ampule samples must be reconstituted according to the instructions in Appendix A to
provide the necessary volume for testing. According to the instructions, the reconstituted sample will
then be split into two portions and appropriate nutrients added to prepare a "with EDTA" and "without
EDTA" portion. These portions shall then be treated as a typical 100% effluent sample received for


SCO Participant Lab SOP                      B-51

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NPDES compliance monitoring. The Selenastrum capricornutum growth test shall be conducted on each
sample portion (with and without EDTA). Test concentrations of 100%, 50%, 25%, 12.5%, and 6.25%
sample shall be prepared from each portion using the appropriate dilution water (Section 3.1)

3.3     Test Conduct

This section of the SOP reiterates testing requirements from Section 3 of the Participant Laboratory
Statement of Work that must be followed for the conduct of the Selenastrum capricornutum growth test
method. Except where indicated in Sections 3.3.1 and 3.3.2 of this SOP, each test shall be conducted in
accordance with the general guidance and method specific requirements for effluent testing included in
the methods manuals.

3.3.1    General Te sting Requirements

EPA acknowledges that the promulgated WET methods distinguish between requirements (indicated by
the compulsory terms "must" and "shall") and recommendations and guidance (indicated by discretionary
terms "should" and "may"). The latter terms indicate that the analyst has flexibility to optimize successful
test completion and when standardization is necessary to assure the predictability of the methods to
provide reliable results. Additionally, the method manuals allow variations of the methods which are
typically fixed in the permit; therefore,  for the purposes of this study, a set of variables will be defined by
EPA (for example, dilution water, salinity, and acute test duration).  Any deviation from defined test
procedures and/or conditions, such as the necessity to change reagents, equipment, test conditions, or
other specified test parameters must be  reported to SCC, recorded at the time of modification, noted in
telephone  logs of communications, documented in a memorandum, and approved by EPA.

Additional WET test requirements  and  general requirements listed in the methods manuals of special note
are provided below:

(1)     Personnel that conduct tests must be the same personnel that routinely conduct the WET tests at
        that laboratory facility and who were identified  in the prequalification materials. If these
        individuals cannot be available during any part of the study, the laboratory must contact SCC.
        Personnel conducting the tests must be identified clearly and consistently in records.

(2)     To coordinate testing at participant laboratories, testing of each sample with each method must be
        initiated on the precise day specified in the finalized study schedule.  Samples should be tested
        within 36 hours from the time of sample preparation (determined in this study as the time at
        which individual sample aliquots were divided from the bulk test sample for distribution to
        participant laboratories). Deviation from this schedule must be reported to SCC immediately for
        approval.

(3)     Physical and chemical properties of the test samples must be in the ranges specified in this SOP,
        the SOW, specific instructions, and the methods manuals. Method specific instructions for any
        adjustments to the test samples prior to sample use (such as reconstitution of ampule samples or
        salinity adjustments) are provided herein.  Test samples received at participant laboratories must
        be refrigerated (at 4C  2C) immediately upon receipt and throughout the period of testing.
        The temperature of the refrigeration unit should be routinely or continuously monitored to ensure
        that these sample holding requirements are met.
SCO Participant Lab SOP                       B-52

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(4)     Measurement of test conditions (pH, salinity, total alkalinity, total hardness, and dissolved
       oxygen) shall be performed for each method by the participant laboratories following guidance in
       method manuals. NOTE: Refer to the electronic benchsheet for required and recommended
       information.

(5)     The specified dilution and control waters must be used and prepared according to instructions in
       Section 7 of the methods manuals.

(6)     All WET tests are to be definitive tests with a control and a minimum of five test concentrations
       prepared using a dilution factor of 0.5.

(7)     All tests must be conducted using the number of replicates and number of test containers per
       concentration as specified in Section 3.2.2.

(8)     Test chambers used within a test must be of the same type, size, shape, and material.  The
       material must be allowed by the method manuals for the method used.

(9)     Test vessels shall be randomized  in accordance with the method manuals.

(10)   If test results  indicate too great of toxicity (i.e., control mortalities, or complete mortality in all
       concentrations), the laboratories must contact SCC immediately and then investigate possible
       causes, first by checking for transcription and calculation mistakes, and then by investigating
       possible contamination in dilution waters, organism cultures, equipment, or other procedural
       steps.

(11)   If any test that has been initiated fails to be completed for any reason, the laboratory must contact
       SCC immediately for problem resolution and scheduling of additional testing.  The incomplete
       test data and the reason for not completing the  test must be fully documented in the final report.

(12)   The Green Algae, Selenastrum capricornutum, Growth Test shall be conducted
       simultaneously with and without EDTA for  each  sample. For participant laboratories, four
       samples will  be tested with and  without EDTA for a total of eight analyses.

(13)   Each laboratory shall be required to report all data obtained during the course of testing,
       including the response of control  samples.

(14)   Laboratories must perform all QA/QC tests listed in Section 4 of the method manuals.
       Laboratories that purchase organisms must supply QA/QC from the test organism supplier and
       follow method manuals for the appropriate QA/QC for purchasing organisms.

(15)   A reference toxicant QC test must be performed for each test method in the month that testing for
       this study occurs. Results of this  test must be submitted with the final data package.

(16)   Data and statistical analyses must be submitted in hard copy in the standardized format specified
       in Section 5 of the SOW. All bench sheets and raw data, including sample tracking and chemistry
       analysis data  must be submitted. Data must also be submitted electronically according to an
       electronic template (Microsoft Excel spreadsheet) that is provided with this SOP.
SCO Participant Lab SOP                       B-53

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(17)   Data analysis must be performed in accordance with the statistical programs specified in the
       methods manuals. Statistical methods and programs used must be reported along with sample
       calculations.

(18)   An IC25 must be reported for each chronic test. The laboratories must report individual toxicity
       endpoints; laboratories are not allowed to average or perform other data manipulations unless
       required by a methods's instructions.

3.2.2   Method-Specific Requirements

Table 2 provides a summary of test conditions that shall be followed for the conduct of all Selenastrum
capricornutum growth tests performed in the WET Interlaboratory Study. This table is extracted from the
summary test condition table in the method manual and modified to fit the scope of this study.  Items that
are bold italic in this table represent conditions standardized for the purposes of this study where method
manuals provide a range.

For the WET interlaboratory study, the algal growth endpoint must be measured as cell counts
using an approved counting method (see method manual Section 14.10.6.2). Automatic particle
counters or manual microscopic counting methods are acceptable.  If laboratories use multiple
counting methods, submission of data from each counting method is encouraged (but not required)
and would improve the evaluation of the method and allow comparison of counting techniques.
SCO Participant Lab SOP                      B-54

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Table 2. Green Alga, Selenastrum capricornutum, Growth Test. Summary of test conditions and test acceptability criteria for green alga,
Selenastrum capricornutum, growth toxicity tests with effluents and receiving waters. Test will be conducted with EDTA and without EDTA.
1. Test type:                                            Static non-renewal
2. Temperature:                                        251C
3. Light quality:                                        "Cool white" fluorescent lighting
4. Light intensity:                                       86  8.6 uE/m2/s (400  40 ft-c or 4306 lux)
5. Photoperiod:                                         Continuous illumination
6. Test chamber size:                                    250 mL
7. Test solution volume:                                 100 mL
8. Renewal of test solutions:                              None
9. Age of test organisms:                                 4 to 7 days
10. Initial cell density in test chambers:                    10,000  cells/mL
11. No. replicate chambers per concentration:               4
12. Shaking rate:                                        100 cpm continuous
13. Aeration:                                           None
14. Dilution water:                                      Algal stock culture medium, moderately hard synthetic water prepared using MILLIPORE
                                                      MILLI-Q or equivalent deionized water and reagent grade chemicals(see Methods Manual
                                                      Section 7, Dilution Water)
15. Test concentrations:                                 Five concentrations and a control
16. Test dilution factor:                                  0.5
17. Test duration:                                       96 h
18. Endpoint:                                          Growth (cell counts)
19. Test acceptability criteria:                             1 X 106 cells/mL with EDTA or 2 X 105 cells/mL without EDTA in the controls; Variability of
                                                      controls should not exceed 20%
20.  Sample handling and holding requirements:             Samples treated as effluent samples for NPDES monitoring. Samples are to be used on the day
                                                      specified by the finalized interlaboratory study testing schedule
21. Sample volume required:	2 L	
NOTE: Test vessels shall be randomized in accordance with the method manuals.
SCO Participant Lab SOP                                            B-55

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3.2.3   Data Analysis

For the Selenastrum capricornutum growth test method, the 96 hour growth IC25 and NOEC shall be
calculated and reported.  Data analysis and statistical procedures should be conducted according to the
method manuals.

3.2.4   Problem Resolution

In the event that problems are encountered during the WET test analysis contact Brian Rusignuolo at
(703)461-2401 for guidance. Brian will direct your call as appropriate for the resolution of technical
issues.

3.2.5   Sample Disposal

Following the termination of the test, any excess sample shall be disposed of according to standard
laboratory procedures for disposal of effluent samples.

4.0 Additional Information on Data Reporting Deliverables

According to the Participant Laboratory Statement of Work, the submission of three deliverables are
required: 1). Narrative summary of testing, 2). Hardcopy results synopsis and full report, and 3).
Electronic results synopsis.  This section provides instructions for the submission of each of these
deliverables.

4.1  Narrative Summary of Testing

This narrative summary shall clearly identify the laboratory, test method, samples tested, summarized test
results, and any problems associated with the samples or conduct of the tests. This summary must list any
tests that were initiated but not completed  and fully explain the reason for not completing the test. This
summary must also include a detailed written description of any approved modification to the procedures
provided in this SOW, specific instructions, or the method manuals. This will include any telephone log
and written correspondence received from the referee laboratory and/or DynCorp during the course of
testing. Lastly, this summary should also provide comments on the performance of the method.

4.2 Hardcopy Results Synopsis and Full Report

At a minimum, this report must consist of the items outlined below in section 5.0, all raw data (biological
and chemical), and laboratory bench sheets. This report must include all pertinent sample information
including copies of all completed traffic report forms, all pertinent test condition and test organism
information, all pertinent quality assurance information including results of the monthly QA/QC reference
toxicant tests, and all summarized and raw results.

4.3  Electronic Results Synopsis

Enclosed with this package is a computer diskette that contains the template for the electronic submission
of results from the Selenastrum capricornutum growth test method. The disk contains a Microsoft Excel
97 spreadsheet file named SCG	.xls.  The SCG indicates that this template is for the Selenastrum
capricornutum growth test method, and the number following  is a unique identifying number for your


SCG Participant Lab SOP                      B-56

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laboratory. If your laboratory does not have the hardware or software capabilities to view and enter data
into this file, please contact Brian Rusignuolo at (703)461-2401 to arrange distribution of an alternative
version of the electronic template. It is recommended to view the electronic benchsheet prior to
initiating the test, so the analyst can verify all the information collected on the laboratory benchsheet
will be sufficient to complete the electronic results.

Notice the following characteristics of the electronic template file:

    1.  The file contains eight worksheet pages labeled "Sample #lw/ EDTA", "Sample #lw/o
       EDTA","Sample #2 w/EDTA","Sample #2 w/o EDTA", "Sample #3 w/EDTA", "Sample #3 w/o
       EDTA", "Sample #4 w/ EDTA", and "Sample #4 w/o EDTA". The results from each of the
       samples that your laboratory tested in this study should be entered on a separate worksheet page
       within the same file.  NOTE: Each sample has two sheets, one for each sample tested with EDTA
       and one for each sample tested without EDTA.
    2.  Each worksheet page contains six information boxes (general information, sample
       collection/receipt information, test information, biological data, water quality data, and
       summarized test results) in which data should be entered.  The seventh information box  (data
       quality flags) is for SCC use only and will aid in the automated review and quality control check
       of data.
    3.  Cells that are highlighted in pale yellow indicate required information. Cells highlighted in blue
       indicate optional information that may be entered if available. Cells highlighted in red are for
       SCC use only.
    4.  The file has been protected such that data can only be entered in yellow or blue cells. No other
       cells can be changed.

To complete the electronic results synopsis data deliverable:

    1.  Record information into a separate worksheet page for each sample tested.
    2.  Record requested information or data in all required cells (pale yellow).
    3.  Record requested information or data in optional cells (blue) if data is available.
    4.  Check entered data against hardcopy bench sheets to ensure accuracy.
    5.  Save the file onto the diskette provided.  Also keep a copy of the  file for laboratory records (a
       backup in case the diskette crashes when redelivered to DynCorp).  Do not change the file name.
    6.  Submit the diskette with the other data reporting deliverables by the due date to:

                             DynCorp I&ET, Sample Control Center
                             6101 Stevenson Avenue
                             Alexandria, VA 22304
                             Phone:(703)461-2064
                             Fax:(703)461-8056
                             Attention: Robert Brent

5.0    Data Report Format

Final hardcopy data reports should be submitted in the following format:
Note:  Adapted from Section 10 of the methods manuals.

Section 1 - Summary Page


SCO Participant Lab SOP                       B-57

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    1.1   Laboratory name
    1.2   Laboratory address and phone number
    1.3   Name and signature of laboratory QA Officer, certifying that data have been internally
         reviewed and that personnel meticulously followed the methods, and the procedures are deemed
         to be compliant with the methods and acceptable for reporting purposes.
    1.4   Laboratory contact responsible for study
    1.5   Analyst(s) who performed WET tests (full names)
    1.6   Toxicity tests performed
    1.7   Detailed explanations of any difficulties encountered and any approved modifications to the
         techniques specified in this SOW, specific instructions, or the methods manuals.
    1.8   Number of successful tests completed

Section 2 - Sample Information
    2.1   Number of samples received and EPA sample number assigned to each sample. Copies of all
         completed traffic report forms should be included.
    2.2   Dates of sample receipt
    2.3   Sample temperature when received at laboratory
    2.4   Physical and chemical data of sample contents (as required in appropriate method)
    2.5   Dilution water
         2.5.1    Source and time frame water is used or how maintained
         2.5.2    Collection or preparation date(s), where applicable
         2.5.3    Pretreatment information
         2.5.4    Physical and chemical characteristics (pH, hardness, conductivity, salinity, etc.)
    2.6   Sample storage information
    2.7   Sample preparation for testing information

Section 3 - Test Conditions
    3.1   Toxicity test method used (title, number, source)
    3.2   Endpoint(s) of test(s)
    3.3   Deviations from reference method(s), if any, and reason(s)
    3.4   Date and time test(s) started, date and time samples were prepared and solutions transferred for
         renewals.
    3.5   Date and time test(s) terminated
    3.6   Type and volume  of test chambers
    3.7   Volume of solution used per chamber
    3.8   Number of organisms per test  chamber
    3.9   Number of replicate test chambers per treatment
    3.10 Feeding frequency and amount and type of food (be  specific with sources, concentrations of
         foods (i.e, algae concentration, YCT solids level, preparation dates))
    3.11 Acclimation of test organisms (temperature mean and range and, where applicable, salinity
         mean and range)
    3.12 Test temperature (mean and range)
    3.13 Test salinity, where applicable (mean and range)
    3.14 Specify if aeration was needed
    3.15 Specify if organisms were dried immediately for weighing or preserved prior to drying
    3.16 Specify how food was prepared and sources of food. Include test results that validate the
         quality of batch food preparations (i.e., Ceriodaphnia dubia tests on YCT preparation).
SCO Participant Lab SOP                       B-58

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    3.17 Describe how routine chemistries on new solutions were made (in actual test chamber or in
         beakers after dispensing)
    3.18 Describe how randomization was conducted, especially blocking and known parentage. Report
         how brood distinctions were made and male (if any) identification was made.

Section 4 - Test Organisms
    4.1   Scientific name of test species, verification of species documented
    4.2   Age (life stage) of test species (be specific for all species). Age at time of test initiation (for
         example, for C. dubia be sure to clarify the window of age of the neonates as well as the overall
         age of the animals.)
    4.3   Mean length and weight (where applicable)
    4.4   Source and QA/QC test conditions
    4.5   Holding conditions
    4.6   Diseases and treatment (where  applicable)
    4.7   Taxonomic key used for species identification

Section 5 - Quality Assurance
    5.1   Reference toxicant used routinely; source; date received; lot number
    5.2   Date and time of most recent reference toxicant test; test results and current control (cusum)
         chart including 20 most recent  data points
    5.3   Dilution water used in reference toxicant tests (with characteristics provided)
    5.4   Physical and chemical methods used
    5.5   Reference toxicant results (NOEC, IC25, or LC50 where applicable, LOEC or EC50)

Section 6 - Results
    6.1   Copies of all bench sheets. Be sure to count and notate broods for reproduction test with
         Ceriodaphnia
    6.2   Raw toxicity data in tabular form, including daily records of affected organisms in each
         replicate at each concentration  (including controls) and plots of toxicity data
    6.3   Table of endpoints (LC50, IC25, NOEC for each endpoint) and confidence limits (where
         applicable)
    6.4   Statistical methods and software used to calculate endpoints
    6.5   Summary table of physical and chemical data

6.0 Coolers

All coolers will include a prepaid return FedEx label for returning the cooler to the referee laboratory.
Fill out the sender portion of the FedEx label and place it on the cooler.  Coolers will be returned by 2
Day shipment, as already marked on the prepared label. Coolers must be returned to the referee
laboratory within 7 days of sample receipt.  Please empty and wipe out the cooler prior to returning it.
SCO Participant Lab SOP                      B-59

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       Appendix A:  Instructions for Reconstitution of Liquid Ampule Sample for
                  Green Alga, Selenastrum capricornutum Growth Test

For each sample, a single liquid ampule will be received. The container shall be reconstituted,
split into a "with EDTA" and "without EDTA" portion, and used to initiate two tests. Follow the
directions below for the reconstitution of the sample ampule.

1.  Volumetrically add 200 mL of the liquid ampule sample to approximately 1L of
   moderately-hard synthetic freshwater (MHSF) prepared using MILLIPORE MILLI-Q or
   equivalent deionized water and reagent grade chemicals according to Section 7 of the
   methods manuals.

2.  Mix by swirling or gently shaking.

3.  Bring final volume to 4L (measured using volumetric glassware) with MHSF dilution water.

4.  Mix again by swirling and gently shaking.

5.  Split the 4L reconstituted sample into two portions  of 2L each, labeling one portion "with
   EDTA" and  one portion "without EDTA."

6.  Place the reconstituted sample portions in separate plastic containers of appropriate volume.
   A container that minimizes head space (i.e. cubitainer) is recommended.

7.  To the portion labeled "with EDTA," add 2mL (ImL per liter of sample) of nutrient stock
   including EDTA.  Nutrient stock should be prepared according to the methods manual.  This
   sample portion shall be used to conduct the Selenastrum capricornutum Growth Test with
   EDTA.

8.  To the portion labeled "without EDTA," add 2mL (ImL per liter of sample) of nutrient stock
   excluding EDTA.  Nutrient stock should be prepared according to the methods manual. This
   sample portion shall be used to conduct the Selenastrum capricornutum Growth Test without
   EDTA.

9.  The two sample portions should be used directly for the 100% effluent test concentrations
   and diluted using the respective dilution water (with or without EDTA) to prepare other test
   concentrations (50%, 25%, 12.5%,  and 6.25%).

10. Perform the Green Alga, Selenastrum capricornutum Growth Test with and without EDTA
   using the respective sample portions. Perform the tests as described in the SOW for this
   study and the methods manuals.
SCO Participant Lab SOP                     B-60

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Appendix B: EPA Traffic Report
United States
PDA Environmental Protection Agency
v ti-AA Washington, DC 20460
EPISODE NO:
SAMPLE NO:
DynCorp - Sample Control Center
WET Interlaboratory Variability Study Traffic Report Fax completed form immediately 6101 Stevenson Avenue
USEPA ENGINEERING AND ANALYSIS DIVISION upon completion, and include Alexandria, VA 22304
SAMPLE CONTROL CENTER hardcopy in final data report to: phone: (703) 461 -21 00
fax: (703)461-8056
Referee Laboratory Information Participant Lab Shipping Information
Name: Lab Name:
Address: Address:
City: City:
State: State:
Airbill no:
Sampler name: Date shipped:
FOR PARTICIPANT LAB USE ONLY
Received by:
Sample condition on receipt:
SAMPLE COLLECTION / RECEIPT INFORMATION
Requested Information
1.
2.
3.
4.
5.
6.
7.
8.
Sample use description
Sample collection/receipt date
Sample collection/receipt time
Sampler / recipient signature
PH
Temperature
Conductivity (freshwater methods) / Salinity
(marine methods)
Dissolved Oxygen concentration
Pre-Shipment
I 	 (initiation I 	 I renewal 1 I 	 Irenewal 2







Post-Shipment








REQUESTED ANALYSES
9.
I  I Pimephales promelas Acute
I  I Pimephales promelas Chronic
I  I Ceriodaphnia dubia Acute
I  I Ceriodaphnia dubia Chronic
I  I Selenastrum capricornutum Chronic
I  I Menidia beryllina Acute
I  I Menidia beryllina Chronic
I  I Holmesmysis costata Acute
I  I Champia parvula Chronic

I  I Cyprinodon variegatus Acute
I  I Cyprinodon variegatus Chronic
I  I Mysidopsis bahia Chronic


SCO Participant Lab SOP
B-61

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DynCorp
litanisrtiii G Bircrjjnse TasMiijiy
TO:            Participant Laboratories for the Mysldopsls bahla Chronic Test Method

FROM:         Robert Brent, WET Study Coordinator

DATE:         February 10, 2000

SUBJECT:     Final Guidance and SOP for the Mysidopsis bahia Chronic Test
Enclosed is the final standard operating procedure (SOP) for laboratories participating in the Mysidopsis
bahia chronic test method in EPA's WET Interlaboratory Variability Study. This SOP is a supplement to
the statement of work (SOW) that was distributed on July 9, 1999 with the solicitation for this study. The
SOP details the sample distribution and testing schedule and provides important information for
completing participant laboratory tasks, such as instructions for the reconstitution of ampule samples.
Participant laboratories should follow the guidance in the enclosed SOP, the SOW, and the method
manuals.

Also enclosed is the electronic data reporting format disk for the Mysidopsis bahia chronic method.  A
description and instructions for use of the electronic data report form are provided in the SOP.

Please ensure that the enclosed SOP and disk pertaining to the Mysidopsis bahia chronic test method are
distributed to laboratory staff that will be performing the test method in the study. Also, please see that
laboratory staff read over the  SOP carefully to ensure the proper data is collected and reported.
MBC Participant Lab SOP                      B-62

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                      STANDARD OPERATING PROCEDURE
          Participant Laboratory Support for EPA's WET Interlaboratory Study

                 Mysidopsis bahia Survival, Growth, and Fecundity Method
Preamble:
This standard operating procedure document (SOP) is a supplement to the participant laboratory
Statement of Work (SOW).  This SOP details specific information in the SOW regarding the conduct of
the Mysidopsis bahia survival, growth, and fecundity test method in the WET Interlaboratory Variability
Study.  All modifications to the SOP must be approved by DynCorp prior to implementation.

All data deliverables submitted for this interlaboratory study become the property of EPA and may be
incorporated into the public record. Laboratories may not independently publish the results of analyses
for which they are paid to perform under this SOP.

1.0 Sample Distribution and Testing Schedule

Participant laboratory testing for the Mysidopsis bahia survival, growth, and fecundity (chronic) method
will occur between February 22 and March 7, 2000 with final reports due 30 days following termination
of all tests (April 6, 2000).  The testing schedule  is provided below in Table 1. The date ranges on the
schedule indicate the test start dates and test completion dates. Samples will be shipped FedEx Priority
Overnight to arrive at the participant laboratory on the test start date by 10:OOAM. All tests must be
initiated on the day of sample arrival. Testing is  scheduled to occur simultaneously at each participant
laboratory, so adherence to the testing schedule is mandatory for all participant laboratories.

Participant laboratories will receive 4 blind test samples (reflected in the  schedule) that may be whole
volume or ampule samples.  Two samples will arrive on 2/22/00  for test initiation on that day, and two
samples will arrive on 2/29/00 for test initiation on that day.

Each sample aliquot that is prepared and shipped will be assigned a unique sample number. The sample
number will appear clearly and permanently on each container and on an EPA traffic report form that will
accompany each sample. For tests that require additional shipments for sample renewal, the sample
number shall be the same for each initiation and renewal shipment with the addition of a letter (A, B, or
C) after the sample number to designate the sample for use  as initiation (A), renewal 1 (B), or renewal 2
(C).

For whole volume samples,  separate aliquots will be received on test Day 0, Day 2, and Day 4. The first
aliquot (identified with the sample number and the letter "A") shall be used for test initiation on Day 0
and renewal on Day  1.  The second aliquot (identified with the sample number and the letter "B") shall be
used for test renewals on Day 2 and Day 3. The final aliquot (identified with the sample number and the
letter "C") shall be used for test renewal on Day 4, Day 5, and Day 6.  This sample shipment schedule
mimics the typical schedule for chronic monitoring of effluent for compliance.

For ampule samples, three separate ampule containers  (marked with the sample number followed by A, B,
or C) will be received in a single shipment on test Day 0. The container marked "A" shall be
reconstituted on test Day 0 and used for test initiation and renewal on Day 1.  The other aliquots of the
sample shall be refrigerated and stored until use on Day 2 and Day 4, respectively. The container marked

MBC Participant Lab  SOP                      B-63

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"B" shall be reconstituted on test Day 2 and used for renewal on Day 2 and 3. The container marked "C"
shall be reconstituted on Day 4 and used for renewal on Day 4, Day 5 and Day 6. The sample
reconstitution schedule for ampules attempts to mimic the typical sample shipment schedule for chronic
monitoring of effluents for compliance.
Table 1. Schedule for Mysidopsis bahia Survival, Growth, and Fecundity Testing.
Date
(start date - finish date)
2/22/00 - 2/29/00
2/29/00 - 3/7/00
4/6/00
Activity
Conduct Mysid, Mysidopsis bahia, Survival, Growth, and Fecundity Test with samples #1&2
Conduct Mysid, Mysidopsis bahia, Survival, Growth, and Fecundity Test with samples #3&4
Mysid, Mysidopsis bahia, Survival, Growth, and Fecundity Test data due
2.0 Sample Traffic Reporting Tasks

2.1    Sample Receipt Confirmation

An EPA traffic report form (Appendix B) will be received with each sample. The traffic report form will
document important sample collection, shipment, and receipt information. Portions of the form will be
completed by the referee laboratory prior to shipment and will indicate the episode number, sample
number, referee laboratory information, participant laboratory shipping information, sample pre-shipment
information, and the requested analysis.  Immediately upon receipt of the sample, participant laboratories
will be responsible for determining that the sample arrived in satisfactory condition and for documenting
receipt of the sample, post-shipment sample water quality (temperature, pH, dissolved oxygen, and
conductivity or salinity), and any problems on the EPA traffic report form. The participant laboratory
shall complete the traffic report form by recording the required information in the "For Participant Lab
Use Only" box and in the "Post-Shipment" column of the "Sample Collection/Receipt Information" box.

For ampule samples, the pre-shipment and post-shipment measurement of pH, dissolved oxygen, and
salinity shall be omitted. This will avoid possible contamination of the ampule sample prior to
reconstitution. Pre-shipment and post-shipment temperature shall be measured in a temperature check
sample. An additional sample container labeled "temperature check" will be included with each cooler
shipment of ampules. The temperature shall be measured in this container and recorded on the EPA
traffic report form as a surrogate measure of temperature for all ampule samples within that cooler. The
temperature check shall be discarded after temperature measurement, and must not be used in any way for
WET testing.

Immediately upon completion of the EPA traffic report form, the participant laboratory shall fax the
completed form to DynCorp and retain a copy for inclusion in the final data report. Receipt of the faxed
traffic report form by DynCorp will be confirmation of successful sample arrival, so it is important that
the form be completed and faxed immediately.  Forms should be faxed by 11:OOAM (local laboratory
time) to facilitate sample tracking and potential problem resolution. The EPA traffic report form shall be
faxed to:
MBC Participant Lab SOP                      B-64

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                                       Brian Rusignuolo
                                      Sample Coordinator
                                         DynCorp SCC
                                      fax: (703)461-8056
                                     phone: (703)461-2401
2.2    Problem Resolution
Prior to test initiation, notify Brian Rusignuolo of any special shipment receiving instructions (i.e., hold
shipment at airport or FedEx shipment center for pickup). Notification must also be made if any specific
instructions are required for Saturday deliveries.

Participant laboratories should be expecting the arrival of samples based on the provided schedule.  If
samples do not arrive as expected, if samples are damaged in shipment, if samples arrive without
accompanying traffic report forms, if sample numbers are not clearly identified on samples, or if any
other sample shipment or receipt problem is encountered immediately notify Brian Rusignuolo by phone
at (703)461-2401.

Brian must be notified by 11:OOAM (local laboratory time) if samples fail to arrive by the morning FedEx
shipment or if other shipment or sample receipt problems are encountered. DynCorp will track lost
shipments and instruct referee laboratories to resend test samples for arrival the following day if
necessary. If renewal shipments do not arrive on the expected day, DynCorp will provide guidance for
test renewal on a case-by-case basis.  Depending on the volume of sample remaining from previous
shipments, laboratories may be instructed to conduct full renewals with the remaining sample, conduct
partial  renewals with the remaining sample, or omit the sample renewal for that day but carefully record
dissolved oxygen throughout the day and remove excess food and dead organisms from the  test
containers.

3.0 WET Test Analysis

3.1    Sample Preparation

3.1.1  Whole Volume Samples

Whole volume samples will be received in cubitainers with sufficient volume for test conduct and
required water chemistry analysis. No sample preparation or adjustment steps (e.g. pH adjustment or
salinity adjustments) should be conducted prior to test initiation. The whole volume sample shall be
treated as a typical 100% effluent sample received for NPDES compliance monitoring. Test
concentrations of 100%, 50%, 25%, 12.5%, and 6.25% sample shall be prepared from the whole volume
sample for use in the test.  These test concentrations and a dilution water control shall be prepared using
synthetic seawater as the dilution water (prepared according to Section 7 of the methods manuals).

3.1.2  Ampule Samples

Ampule samples will be received as small volume liquid samples in 125ml plastic containers. Prior to
test initiation the ampule samples must be reconstituted according to the instructions in Appendix A to
provide the necessary volume for testing. The reconstituted sample shall then be treated as atypical
100% effluent sample received for NPDES compliance monitoring. Test concentrations of  100%, 50%,


MBC Participant Lab SOP                      B-65

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25%, 12.5%, and 6.25% sample shall be prepared from this reconstituted sample for use in the test. These
test concentrations and a dilution water control shall be prepared using synthetic seawater as the dilution
water (prepared according to Section 7 of the methods manuals).

3.2     Test Conduct

This section of the SOP reiterates testing requirements from Section 3 of the Participant Laboratory
Statement of Work that must be followed for the conduct of the Mysid, Mysldopsls bahia, chronic test
method.  Except where indicated in Sections 3.2.1 and 3.2.2 of this SOP, each test shall be conducted in
accordance with the general guidance and method specific requirements for effluent testing included in
the methods manuals.

3.2.1   General  Testing Requirements

EPA acknowledges that the promulgated WET methods distinguish between requirements (indicated by
the compulsory terms "must" and "shall") and recommendations and guidance (indicated by discretionary
terms "should" and "may"). The latter terms indicate that the analyst has flexibility to optimize successful
test completion and when standardization is necessary to assure the predictability of the methods to
provide reliable results. Additionally, the method manuals allow variations of the methods which are
typically fixed in the permit; therefore, for the purposes of this study, a set of variables will be defined by
EPA (for example, dilution water, salinity, and acute test duration).  Any deviation from defined test
procedures and/or conditions, such as the necessity to change reagents, equipment, test conditions, or
other specified test parameters must be reported to SCC, recorded at the time of modification, noted in
telephone logs of communications, documented in a memorandum, and approved by EPA.

Additional WET test requirements and general requirements listed in the methods manuals of special note
are provided below:

(1)     Personnel that conduct tests must be the same personnel that routinely conduct the WET tests at
        that laboratory facility and who were identified in the prequalification materials.  If these
        individuals cannot be available during any part of the study, the laboratory must contact SCC.
        Personnel conducting the tests must be identified clearly and consistently in records.

(2)     To coordinate testing at participant laboratories, testing of each sample with each method must be
        initiated on the precise day specified in the finalized study schedule.  Samples should be tested
        within 36 hours from the time of sample preparation (determined in this study as the time at
        which individual sample aliquots were divided from the bulk test sample for distribution to
        participant laboratories). Deviation from this schedule must be reported to SCC immediately for
        approval.

(3)     Physical and chemical properties of the test samples must be in the ranges specified in this  SOP,
        the SOW, specific instructions, and the methods manuals. Method specific instructions for any
        adjustments to the test samples prior to sample use (such as reconstitution of ampule samples or
        salinity adjustments) are provided herein. Test samples received at participant laboratories must
        be refrigerated (at 4C  2C) immediately upon receipt and throughout the period of testing.
        The temperature of the refrigeration unit should be routinely or continuously monitored to ensure
        that these sample holding requirements are met.
MBC Participant Lab SOP                       B-66

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(4)     Measurement of test conditions (pH, salinity, total alkalinity, total hardness, and dissolved
        oxygen) shall be performed for each method by the participant laboratories following guidance in
        method manuals. NOTE: Refer to the electronic benchsheet for required and recommended
        information.

(5)     The specified dilution and control waters must be used and prepared according to instructions in
        Section 7 of the methods manuals.

(6)     All WET tests are to be definitive tests with a control and a minimum of five test concentrations
        prepared using a dilution factor of 0.5.

(7)     All tests must be conducted using the number of replicates and number of test containers per
        concentration as specified in Section 3.2.2.

(8)     Test chambers used within a test must be of the same type, size, shape, and material. The
        material must be allowed by the method manuals for the method used.

(9)     Test vessels shall be randomized in accordance with the method manuals.

(10)    Daily observation of mortality and removal of dead organisms for each test is required.

(11)    If test results indicate too great of toxicity (i.e., control mortalities, or complete mortality in all
        concentrations), the laboratories must contact SCC immediately and then investigate possible
        causes, first by checking for transcription and calculation mistakes, and then by investigating
        possible contamination in dilution waters, organism cultures, equipment, or other procedural
        steps.

(12)    If any test that has been initiated fails to be completed for any reason, the laboratory must contact
        SCC immediately for problem resolution and scheduling of additional testing. The incomplete
        test data and the reason for not completing the test must be fully documented in the final report.

(13)    Each laboratory shall be required to report all data obtained during the course of testing,
        including the response of control samples.

(14)    Laboratories must perform all  QA/QC tests listed in Section 4 of the method manuals.
        Laboratories that purchase organisms must supply QA/QC from the test organism supplier and
        follow method manuals for the appropriate QA/QC for purchasing organisms.

(15)    A reference toxicant QC test must be performed for each test method in the month that testing for
        this study occurs. Results of this test must be submitted with the final data package.

(16)    Data and statistical analyses must be submitted in hard copy in the standardized format specified
        in Section 5 of the SOW. All bench  sheets and raw data, including sample tracking and chemistry
        analysis data must be submitted. Data must also be submitted electronically according to an
        electronic template (Microsoft Excel  spreadsheet) that is provided with this SOP.
MBC Participant Lab SOP                       B-67

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(17)    Data analysis must be performed in accordance with the statistical programs specified in the
        methods manuals. Statistical methods and programs used must be reported along with sample calculations.

(18)    An LC50 and IC25 must be reported for each chronic test.  The laboratories must report individual
        toxicity endpoints; laboratories are not allowed to average or perform other data manipulations
        unless required by a methods's instructions.

(1)     Following termination of the mysid chronic test, surviving organisms must be examined by a
        skilled analyst to determine sex and the presence of eggs.  Fecundity endpoints  shall be calculated
        for tests if 50% or more of females in the controls produce eggs.


3.2.2   Method-Specific Requirements

Table 2 provides a summary of test conditions that shall be followed for the conduct of all mysid chronic
tests performed in the WET Interlaboratory Study. This table is extracted from the summary test condition
table in the method manual and modified to fit the scope of this study.  Items that are bold italic in these
tables represent conditions standardized for the purposes of this study where method manuals provide a
range.
MBC Participant Lab SOP                       B-68

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Table 2.  Mysid Shrimp, Mysidopsis bahia, Survival, Growth, and Fecundity Test. Summary of test conditions and test acceptability criteria
for the mysid, Mysldopsls bahia, seven day survival, growth, and fecundity test with effluents and receiving waters	
1. Test type:
2. Salinity:
3. Temperature:
4. Light quality:
5. Light intensity:
6. Photoperiod:
7. Test chamber:
8. Test solution volume:
9. Renewal of test solutions:
10.  Age of test organisms:
11.  No. organisms per test chamber:
12.  No. replicate chambers per concentration:
13.  No. larvae per concentration:
14.  Source of food:
15.  Feeding regime:
16.  Cleaning:
17.  Aeration:
18.  Dilution water:
19.
20.
21.
22.
23.

24.
Test concentrations:
Dilution factor:
Test duration:
Endpoints:
Test acceptability criteria:

 Sample handling and holding requirements:
25. Sample volume required:
Static renewal
25%0 (2%<)
26 1C
Ambient laboratory illumination
10-20 nE/m2/s (50-100 ft-c.) (ambient laboratory levels)
16 h light, 8 h darkness, with phase in/out period
8 oz plastic disposable cups, or 400 mL glass beakers
150 mL per replicate
Daily
7 days
5
8
40
Newly \\atc\\edArtemia nauplii (less than 24 h old)
Feed 150 24 h old nauplii per mysid daily, half after test solution renewal and half after 8-12 h.
Pipette excess food from cups daily immediately before test solution renewal and feeding.
None unless DO falls below 4.0 mg/L, then gently aerate in all cups
25%o (2%o) Bioassay  Grade Forty Fathoms artificial seawater prepared with MILLIPORE
MILLI-Q or equivalent deionized water (see Methods Manual Section 7, Dilution Water)
Five concentrations and a control
0.5
7 days
Survival, growth, and egg development
80% or greater survival, average dry weight 0.20 mg or greater in controls; fecundity may be
used if 50% or more of females in controls produce eggs
Samples treated as effluent samples for NPDES monitoring.  Samples are to be used on the
day specified by the finalized inter lab oratory study testing schedule
3 L per day	
NOTE: Test vessels shall be randomized in accordance with the method manuals.
MBC Participant Lab SOP
                                                                B-69

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3.2.3    Data Analysis

For the mysid chronic test method, the 7 day survival LC50, 7 day survival NOEC, growth IC25, growth
NOEC,  7 day fecundity IC25, and 7 day fecundity NOEC shall be calculated and reported. Data analysis
and statistical procedures should be conducted according to the method manuals. Note: Fecundity should
be calculated if 50% or more of females in controls produce eggs.

3.2.4    Problem Resolution

In the event that problems are encountered during the WET test analysis contact Brian Rusignuolo at
(703)461-2401 for guidance.  Brian will direct your call as appropriate for the resolution of technical
issues.

3.2.5    Sample Disposal

Following the termination of the test, any excess sample shall be disposed of according to standard
laboratory procedures for disposal of effluent samples.

4.0 Data Reporting Deliverables

According to the Participant Laboratory Statement of Work, the submission of three deliverables are
required: 1). Narrative summary of testing, 2). Hardcopy results synopsis and full report, and 3).
Electronic results synopsis.  This section provides instructions for the submission of each of these
deliverables.

4.1  Narrative Summary of Testing

This narrative summary shall clearly identify the laboratory, test method, samples tested, summarized test
results, and any problems associated with the  samples or conduct of the tests. This summary must list any
tests that were initiated but not completed and fully explain the reason for not completing the test.  This
summary must also include a detailed written description of any approved modification to the procedures
provided in this SOW, specific instructions, or the method manuals.  This will include any telephone log
and written correspondence received from the referee laboratory and/or DynCorp during the course of
testing. Lastly, this summary should also provide comments on the performance of the method.

4.2 Hardcopy Results Synopsis and Full Report

At a minimum, this report must consist of the items outlined below in section 5.0, all raw data (biological
and chemical), and laboratory bench sheets. This report must include all pertinent sample information
including copies of all completed traffic report forms, all pertinent test condition and test organism
information, all pertinent quality assurance information including results of the monthly QA/QC reference
toxicant tests, and all summarized and raw results.

4.3  Electronic Results Synopsis

Enclosed with this package is a computer diskette that contains the template for the electronic submission
of results from the Mysid chronic test method. The disk contains a Microsoft Excel 97 spreadsheet file
named MBC	.xls. The MBC indicates that this template is for the Mysidopsis bahia chronic test


MBC Participant Lab SOP                      B-70

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method, and the number following is a unique identifying number for your laboratory. If your laboratory
does not have the hardware or software capabilities to view and enter data into this file, please contact
Brian Rusignuolo at (703)461-2401 to arrange distribution of an alternative version of the electronic
template.   It is recommended to view the electronic benchsheet prior to initiating the test, so the
analyst can verify all the information collected on the laboratory benchsheet will be sufficient to complete
the electronic results.

Notice the following characteristics of the electronic template file:

    (1) The file contains four worksheet pages labeled "Sample #1", "Sample #2", "Sample #3", and
        "Sample #4".  The results from each of the samples that your laboratory tested in this study
        should be entered on a separate worksheet page within the same file.
    (2) Each worksheet page contains seven information boxes (general information, sample
        collection/receipt information, test information, biological data, water quality data, weight data,
        and summarized test results) in which data should be entered. The eighth information box (data
        quality flags) is for SCC use only and will aid in the automated review and quality control check
        of data.
    (3) Cells that are highlighted in pale yellow indicate required information. Cells highlighted in blue
        indicate optional information that may be entered if available.  Cells highlighted in red are for
        SCC use only.
    (4) The file has been protected such that data can only be entered in yellow or blue cells. No other
        cells can be changed.

To complete the electronic results synopsis data deliverable:

    (1) Record information into a separate worksheet page for each sample tested.
    (2) Record requested information or data in all required cells (pale yellow).
    (3) Record requested information or data in optional cells (blue) if data is available.
    (4) Check entered data against hardcopy bench sheets to ensure accuracy.
    (5) Save the file onto the diskette provided.  Also keep a copy of the file for laboratory records (a
        backup in case the diskette crashes when redelivered to DynCorp). Do not change the file  name.
    (6) Submit the diskette with the other data reporting deliverables by the due date to:

                             DynCorp I&ET, Sample Control Center
                             6101 Stevenson Avenue
                             Alexandria, VA 22304
                             Phone:(703)461-2064
                             Fax:(703)461-8056
                             Attention: Robert Brent
5.0     Data Report Format

Final hardcopy data reports should be submitted in the following format:
Note:   Adapted from Section 10 of the methods manuals.

Section 1 - Summary Page
    1.1   Laboratory name
MBC Participant Lab SOP                       B-71

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    1.2   Laboratory address and phone number
    1.3   Name and signature of laboratory QA Officer, certifying that data have been internally
         reviewed and that personnel meticulously followed the methods, and the procedures are deemed
         to be compliant with the methods and acceptable for reporting purposes.
    1.4   Laboratory contact responsible for study
    1.5   Analyst(s) who performed WET tests (full names)
    1.6   Toxicity tests performed
    1.7   Detailed explanations of any difficulties encountered and any approved modifications to the
         techniques specified in this SOW, specific instructions, or the methods manuals.
    1.8   Number of successful tests completed

Section 2 - Sample Information
    2.1   Number of samples received and EPA sample number assigned to each sample.  Copies of all
         completed traffic report forms should be included.
    2.2   Dates of sample receipt
    2.3   Sample temperature when received at laboratory
    2.4   Physical and chemical data of sample contents (as required in appropriate method)
    2.5   Dilution water
         2.5.1   Source and time frame water is used or how maintained
         2.5.2   Collection or preparation date(s), where applicable
         2.5.3   Pretreatment information
         2.5.4   Physical and chemical characteristics (pH, hardness, conductivity, salinity, etc.)
    2.6   Sample storage information
    2.7   Sample preparation for testing information

Section 3 - Test Conditions
    3.1   Toxicity test method used (title, number, source)
    3.2   Endpoint(s)  of test(s)
    3.3   Deviations from reference method(s), if any, and reason(s)
    3.4   Date and time test(s) started, date and time samples were prepared and solutions transferred for
         renewals.
    3.5   Date and time test(s) terminated
    3.6   Type and volume of test chambers
    3.7   Volume of solution used per chamber
    3.8   Number of organisms per test chamber
    3.9   Number of replicate test chambers per treatment
    3.10 Feeding frequency and amount and type of food (be specific with sources, concentrations of
         foods (i.e, algae concentration, YCT solids level, preparation dates))
    3.11 Acclimation of test organisms (temperature mean and range and, where applicable, salinity
         mean and range)
    3.12 Test temperature (mean and range)
    3.13 Test salinity, where applicable (mean and range)
    3.14 Specify if aeration was needed
    3.15 Specify if organisms were dried immediately for weighing or preserved prior to drying
    3.16 Specify how food was prepared and sources of food. Include test results that validate the
         quality of batch food preparations (i.e., Ceriodaphnia dubia tests on YCT preparation).
    3.17 Describe how routine chemistries on new solutions were made (in actual test chamber or in
         beakers after dispensing).


MBC Participant Lab  SOP                      B-72

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    3.18 Describe how randomization was conducted, especially blocking and known parentage. Report
         how brood distinctions were made and male (if any) identification was made.

Section 4 - Test Organisms
    4.1   Scientific name of test species, verification of species documented
    4.2   Age (life stage) of test species (be specific for all species). Age at time of test initiation (for
         example, for C. dubia be sure to clarify the window of age of the neonates as well as the overall
         age of the animals.)
    4.3   Mean length and weight (where applicable)
    4.4   Source and QA/QC test conditions
    4.5   Holding conditions
    4.6   Diseases and treatment (where applicable)
    4.7   Taxonomic key used for species identification

Section 5 - Quality Assurance
    5.1   Reference toxicant used routinely; source; date received; lot number
    5.2   Date and time of most recent reference toxicant test; test results and current control (cusum)
         chart including 20 most recent data points
    5.3   Dilution water used in reference toxicant tests (with characteristics provided)
    5.4   Physical and chemical methods used
    5.5   Reference toxicant results (NOEC, IC25, or LC50 where applicable, LOEC or EC50)

Section 6 - Results
    6.1   Copies of all bench sheets. Be sure to count and notate broods for reproduction test with
         Ceriodaphnia
    6.2   Raw toxicity data in tabular form, including daily records of affected organisms in each
         replicate at each concentration (including controls) and plots of toxicity data
    6.3   Table of endpoints (LC50, IC25, NOEC for each endpoint) and confidence limits (where
         applicable)
    6.4   Statistical methods and software used to calculate endpoints
    6.5   Summary table of physical and chemical data
6.0 Coolers

All coolers will include a prepaid return FedEx label for returning the cooler to the referee laboratory.
Fill out the sender portion of the FedEx label and place it on the cooler. Coolers will be returned by 2
Day shipment, as already marked on the prepared label.  Coolers must be returned to the referee
laboratory within 7 days of sample receipt. Please empty and wipe out the cooler prior to returning it.
MBC Participant Lab SOP                       B-73

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        Appendix A: Instructions for Reconstitution of Liquid Ampule Sample for
          Mysid Shrimp, Mysidopsis bahia, Survival, Growth, and Fecundity Test

For each sample, three containers of liquid reagent will be received (marked with the sample
number followed by an "A", "B", or "C"). The three containers shall be reconstituted as
described below to mimic the sample shipment schedule for effluent samples. The container
marked "A" shall be reconstituted on the day of test initiation (Day 0) and used for renewal on
Day 1.  The container marked "B" shall be reconstituted on Day 2 and used for renewals on Day
2 and Day 3.  The container marked "C" shall be reconstituted on Day 4 and used for renewals
on Day 4, Day 5, and Day 6. Follow the directions below for the reconstitution of each sample
ampule.

1.  Volumetrically add 100 mL of the liquid ampule sample to approximately 1L of synthetic
    seawater. The synthetic seawater should be prepared to a salinity of 25%o (2%o) using
    Bioassay Grade Forty Fathoms artificial sea salts and MILLIPORE MTLLI-Q or equivalent
    deionized water according to Section 7 of the methods manuals.

2.  Mix by swirling or gently shaking.

3.  Bring final volume to 9L (measured using volumetric glassware) with synthetic  seawater
    dilution water.

4.  Mix again by swirling and gently shaking.

5.  Place reconstituted sample in a plastic container of appropriate volume. A container that
    minimizes head space (i.e. cubitainer) is recommended.

6.  This 9L sample is the whole volume reconstituted sample and should be treated  as a typical
    effluent sample.  It should be used directly for the 100% effluent test concentration and
    diluted appropriately to prepare other test concentrations (50%, 25%,  12.5%, and 6.25%).

7.  Use the reconstituted sample for test initiation and Day 1 test renewal. Store sample at 4C.

8.  Perform the Mysid Shrimp, Mysidopsis bahia, Survival, Growth, and Fecundity Test as
    described in the SOW for this study and the methods manuals.

9.  Follow Steps 1 through 6 with each sample container to prepare the reconstituted sample on
    Day 2 and Day 4 for subsequent daily renewals.
MBC Participant Lab SOP                    B-74

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Appendix B: EPA Traffic Report
^EPA
United States
Environmental Protection Agency
Washington, DC 20460
WET Interlaboratory Variability Study Traffic Report
USEPA ENGINEERING AND ANALYSIS DIVISION
SAMPLE CONTROL CENTER
Referee Laboratory Information
Name:
Address:
City:
State:

Sampler name:
EPISODE NO:
SAMPLE NO:
DynCorp - Sample Control Center
Fax completed form immediately 6101 Stevenson Avenue
upon completion, and include Alexandria, VA 22304
hardcopy in final data report to: phone: (703) 461 -21 00
fax: (703)461-8056
Participant Lab Shipping Information
Lab Name:
Address:
City:
State:
Airbill no:
Date shipped:
SAMPLE COLLECTION /
Requested Information
1.
2.
3.
4.
5.
6.
7.
8.
Sample use description
Sample collection/receipt date
Sample collection/receipt time
Sampler / recipient signature
PH
Temperature
Conductivity (freshwater methods) / Salinity
(marine methods)
Dissolved Oxygen concentration
FOR PARTICIPANT LAB USE ONLY
Received by:
Sample condition on receipt:
RECEIPT INFORMATION
Pre-Shipment
I 	 (initiation I 	
renewal 1 I 	 Irenewal 2







Post-Shipment








REQUESTED ANALYSES
9.
I 	 I Pimephales promelas Acute
I 	 I Pimephales promelas Chronic
I 	 I Ceriodaphnia dubia Acute
I 	 I Ceriodaphnia dubia Chronic
I 	 I Selenastrum capricornutum Chronic
I 	 I Menidia beryllina Acute
I 	 I Menidia beryllina Chronic
I 	 I Holmesmysis costata Acute
I 	 I Champia parvula Chronic

I 	 I Cyprinodon variegatus Acute
I 	 I Cyprinodon variegatus Chronic
I 	 I Mysidopsis bahia Chronic


MBC Participant Lab SOP
B-75

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DynCorp
litanisrtiii G Bircrjjnse TasMiijiy
TO:            Participant Laboratories for the Sheepshead Minnow Acute Test Method

FROM:         Robert Brent, WET Study Coordinator

DATE:         February 25, 2000

SUBJECT:      Final Guidance and SOP for the Sheepshead Minnow Acute Test
Enclosed is the final standard operating procedure (SOP) for laboratories participating in the Sheepshead
Minnow acute test method in EPA's WET Interlaboratory Variability Study. This SOP is a supplement to
the statement of work (SOW) that was distributed on July 9, 1999 with the solicitation for this study. The
SOP details the sample distribution and testing schedule and provides important information for
completing participant laboratory tasks, such as instructions for the reconstitution of ampule samples.
Participant laboratories should follow the guidance in the enclosed SOP, the SOW, and the method
manuals.

Also enclosed is the electronic data reporting format disk for the sheepshead minnow  acute method.  A
description and instructions for use of the electronic data report form are provided in the SOP.

Please ensure that the enclosed SOP and disk pertaining to the sheepshead minnow acute test method are
distributed to laboratory staff that will be performing the test method in the study. Also, please see that
laboratory staff read over the SOP  carefully to ensure the proper data is collected and  reported. Please
note that the ampule reconstitution instructions for this method require the addition of SOOmL of
the ampule sample instead of lOOmL that has been used previously for other methods. For this
reason, ampule samples will be provided in SOOmL bottles for this method.
SHMA Participant Lab SOP                     B-76

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                      STANDARD OPERATING PROCEDURE
          Participant Laboratory Support for EPA's WET Interlaboratory Study

                            Cyprinodon variegatus Acute Method
Preamble:
This standard operating procedure document (SOP) is a supplement to the participant laboratory
Statement of Work (SOW).  This SOP details specific information in the SOW regarding the conduct of
the Cyprinodon variegatus acute test method in the WET Interlaboratory Variability Study. All
modifications to the SOP must be approved by DynCorp prior to implementation.

All data deliverables submitted for this interlaboratory study become the property of EPA and may be
incorporated into the public record. Laboratories may not independently publish the results of analyses
for which they are paid to perform under this SOP.
 1.0 Sample Distribution and Testing Schedule

 Participant laboratory testing for the Cyprinodon variegatus acute method will occur between March 7
 and March 18, 2000 with final reports due 30 days following termination of all tests (April 17, 2000).
 The testing schedule is provided below in Table 1. The date ranges on the schedule indicate the test start
 dates and test completion dates. Samples will be shipped FedEx Priority Overnight to arrive at the
 participant laboratory on the test start date by 10:OOAM. All tests must be initiated on the day of sample
 arrival. Testing is scheduled to occur simultaneously at each participant laboratory, so adherence to the
 testing schedule is mandatory for all participant laboratories.

 Participant laboratories will receive 4 blind test samples (reflected in the schedule) that may be whole
 volume or ampule samples.  Two samples will arrive on 3/7/00 for test initiation on that day, and two
 samples will arrive on 3/14/00 for test initiation on that day. Each  sample aliquot that is prepared and
 shipped will be assigned a unique sample number. The sample number will appear clearly and
 permanently on each container and on an EPA traffic report form that will accompany each sample.

 For whole volume samples, one aliquot will be received on test Day 0.  This aliquot shall be used for test
 initiation on Day 0 and test renewal at 48 hours.  For ampule samples, one aliquot will be received on test
 Day 0.  This aliquot shall be reconstituted on Day 0 and shall be  used for test initiation on Day 0 and test
 renewal at 48 hours.

 Table 1. Schedule for Cyprinodon variegatus Acute Testing.
Date
(start date - finish date)
3/7/00 - 3/1 1/00
3/14/00-3/18/00
4/17/00
Activity
Conduct Sheepshead Minnow, Cyprinodon variegatus, Acute Test with samples #1&2
Conduct Sheepshead Minnow, Cyprinodon variegatus, Acute Test with samples #3&4
Sheepshead Minnow, Cyprinodon variegatus, Acute Test data due
SHMA Participant Lab SOP                     B-77

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2.0 Sample Traffic Reporting Tasks

2.1    Sample Receipt Confirmation

An EPA traffic report form (Appendix B) will be received with each sample. The traffic report form will
document important sample collection, shipment, and receipt information. Portions of the form will be
completed by the referee laboratory prior to shipment and will indicate the episode number, sample
number, referee laboratory information, participant laboratory shipping information, sample pre-shipment
information, and the requested analysis.  Immediately upon receipt of the sample, participant laboratories
will be responsible for determining that the sample arrived in satisfactory condition and for documenting
receipt of the sample, post-shipment sample water quality (temperature, pH, dissolved oxygen, and
conductivity or salinity), and any problems on the EPA traffic report form. The participant laboratory
shall complete the traffic report form by recording the required information in the "For Participant Lab
Use Only" box and in the "Post-Shipment" column of the "Sample Collection/Receipt Information" box.

For ampule samples, the pre-shipment and post-shipment measurement of pH, dissolved oxygen, and
conductivity or salinity shall be omitted. This will avoid possible contamination of the ampule sample
prior to reconstitution. Pre-shipment and post-shipment temperature shall be measured in a temperature
check sample. An additional sample container labeled "temperature check" will be included with each
cooler shipment of ampules. The temperature shall be measured in this container and recorded on the
EPA traffic report form as a surrogate measure of temperature for all ampule samples within that cooler.
The temperature check shall be discarded after temperature measurement, and must not be used in any
way for WET testing.

Immediately upon completion of the EPA traffic report form, the participant laboratory shall fax the
completed form to DynCorp and retain a copy for inclusion in the final data report. Receipt of the faxed
traffic report form by DynCorp will be confirmation of successful sample arrival, so it is important that
the form be completed and faxed immediately. Forms should be faxed by 11:OOAM (local laboratory
time) to facilitate sample tracking and potential problem resolution. The EPA traffic report form shall be
faxed to:

                                        Brian Rusignuolo
                                      Sample  Coordinator
                                         DynCorp  SCC
                                      fax: (703)461-8056
                                     phone: (703)461-2401

2.2    Problem Resolution

Prior to test initiation, notify Brian Rusignuolo of any special shipment receiving instructions (i.e., hold
shipment at airport or FedEx shipment center for pickup). Notification must also be made if any  specific
instructions are required for Saturday deliveries.

Participant laboratories should be expecting the arrival of samples based on the provided schedule.  If
samples do not arrive as expected, if samples are damaged in shipment, if samples arrive without
accompanying traffic report forms, if sample numbers are not clearly identified on samples, or if any
other sample shipment or receipt problem is encountered immediately notify Brian Rusignuolo by phone
at (703)461-2401.


SHMA Participant Lab SOP                    B-78

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Brian must be notified by 11:OOAM (local laboratory time) if samples fail to arrive by the morning FedEx
shipment or if other shipment or sample receipt problems are encountered. DynCorp will track lost
shipments and instruct referee laboratories to resend test samples for arrival the following day if
necessary.

3.0 WET Test Analysis

3.1    Sample Preparation

3.1.1   Whole Volume Samples

Whole volume samples will be received in cubitainers with sufficient volume for test conduct and
required water chemistry analysis.  No sample preparation or adjustment steps (e.g. pH or salinity
adjustment) should be conducted prior to test initiation.  The whole volume sample shall be treated as a
typical 100% effluent sample received forNPDES compliance monitoring. Test concentrations of 100%,
50%,  25%, 12.5%, and 6.25% sample shall be prepared from the whole volume sample for use in the test.
These test concentrations and a dilution water control shall be prepared using bioassay grade Forty
Fathoms synthetic seawater as the dilution water (prepared according to Section 7 of the method
manuals).

3.1.2   Ampule Samples

Ampule samples will be received as small volume liquid samples in a 500 ml plastic bottle. Prior to test
initiation the ampule samples must be reconstituted according to the instructions in Appendix A to
provide the necessary volume for testing.  The reconstituted sample shall then be treated as atypical
100% effluent sample received for NPDES compliance monitoring. Test concentrations of 100%, 50%,
25%,  12.5%, and 6.25% sample shall be prepared from this reconstituted sample for use in the test. These
test concentrations and a dilution water control shall be prepared using bioassay grade Forty Fathoms
synthetic seawater as the dilution water (prepared according to Section 7 of the method manuals).

3.2    Test Conduct

This section of the SOP reiterates testing requirements from Section 3 of the Participant Laboratory
Statement of Work that must be followed  for the conduct of the  Sheepshead minnow, Cyprinodon
variegatus, acute test method.  Except where indicated in Sections 3.2.1 and 3.2.2 of this SOP, each test
shall be conducted in accordance with the general guidance and  method specific requirements for effluent
testing included in the methods manuals.

3.2.1   General Testing Requirements

EPA acknowledges that the promulgated WET methods distinguish between requirements (indicated by
the compulsory terms "must" and "shall") and recommendations and guidance (indicated by discretionary
terms  "should" and "may"). The latter terms indicate that the analyst has flexibility to optimize successful
test completion and when standardization  is necessary to assure the predictability of the methods to
provide reliable results. Additionally, the  method manuals allow variations of the methods which are
typically fixed in the permit; therefore, for the purposes of this study, a set of variables will be defined by
EPA (for example, dilution water, salinity, and acute test duration). Any deviation from defined test
procedures and/or conditions, such as the  necessity to change reagents, equipment, test conditions,  or


SHMA Participant Lab SOP                     B-79

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other specified test parameters must be reported to SCC, recorded at the time of modification, noted in
telephone logs of communications, documented in a memorandum, and approved by EPA.

Additional WET test requirements and general requirements listed in the methods manuals of special note
are provided below:

(1)    Personnel that conduct tests must be the same personnel that routinely conduct the WET tests at
       that laboratory facility and who were identified in the prequalification materials.  If these
       individuals cannot be available during any part of the study, the laboratory must contact SCC.
       Personnel conducting the tests must be identified clearly and consistently in records.

(2)    To coordinate testing at participant laboratories, testing of each sample with each method must be
       initiated on the precise day specified in the finalized study schedule.   Samples should be tested
       within 36 hours from the time of sample preparation (determined in this study as the time at
       which individual sample aliquots were divided from the bulk test sample for distribution to
       participant laboratories).  Deviation from this schedule must be reported to SCC immediately for
       approval.

(3)    Physical and chemical properties of the test samples must be in the ranges specified in this SOP,
       the SOW, specific instructions, and the methods manuals. Method specific instructions for any
       adjustments to the test samples prior to sample use (such as reconstitution of ampule samples) are
       provided herein. Test samples received at participant laboratories must be refrigerated (at 4C 
       2C) immediately upon receipt and throughout the period of testing.  The temperature of the
       refrigeration unit should be routinely or continuously monitored to ensure that these sample
       holding requirements are met.

(4)    Measurement of test conditions (pH, salinity, total alkalinity, total hardness, and dissolved
       oxygen) shall be performed for each method by the participant laboratories following guidance in
       method manuals.  NOTE: Refer to the electronic benchsheet for required and recommended
       information.

(5)    The specified dilution and control waters must be  used and prepared according to instructions in
       Section 7 of the methods manuals.

(6)    All WET tests are to be definitive tests with a control and a minimum of five test concentrations
       prepared using a dilution factor of 0.5.

(7)    All tests must be conducted using the number of replicates and number of test containers per
       concentration as specified in Section 3.2.2.

(8)    Test chambers used within a test must be of the same type, size, shape, and material.  The
       material must be allowed by the method manuals for the method used.

(9)    Test vessels shall  be randomized in accordance with the method manuals.

(10)    Daily observation of mortality and removal of dead organisms for each test is  required.
SHMA Participant Lab SOP                     B-80

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(11)   If test results indicate too great of toxicity (i.e., control mortalities, or complete mortality in all
       concentrations), the laboratories must contact SCC immediately and then investigate possible
       causes, first by checking for transcription and calculation mistakes, and then by investigating
       possible contamination in dilution waters, organism cultures, equipment, or other procedural
       steps.

(12)   If any test that has been initiated fails to be completed for any reason, the laboratory must contact
       SCC immediately for problem resolution and scheduling of additional testing. The incomplete
       test data and the reason for not completing the test must be fully documented in the final report.

(13)   Each laboratory shall be required to report all data obtained during the course of testing,
       including the response of control samples.

(14)   Laboratories must perform all QA/QC tests listed in Section 4 of the method manuals.
       Laboratories that  purchase organisms must supply QA/QC from the test organism supplier and
       follow method manuals for the appropriate QA/QC for purchasing organisms.

(15)   A reference toxicant QC test must be performed for each test method in the month that testing for
       this study occurs. Results of this test must be submitted with the final data package.

(16)   Data and statistical analyses must be  submitted in hard copy in the standardized format specified
       in Section 5 of the SOW. All bench sheets and raw data, including sample tracking and chemistry
       analysis data must be submitted. Data must also be submitted electronically according to an
       electronic template (Microsoft Excel spreadsheet) that is provided with this SOP.

(17)   Data analysis must be performed in accordance with the statistical programs specified in the
       methods manuals. Statistical methods and programs used must be reported along with sample
       calculations.

(18)   An IC25 must be reported for each chronic test.  The laboratories must report individual toxicity
       endpoints; laboratories are not allowed to average or perform other data manipulations unless
       required by a method's instructions.

3.2.2   Method-Specific Requirements

Table 2 provides a summary of test conditions that shall be followed for the conduct of all sheepshead
minnow acute tests performed in the WET Interlaboratory Study. This table is extracted from the
summary test condition table in the method manual and modified to fit the scope of this study. Items that
are bold italic in these tables represent conditions standardized for the purposes of this study where
method manuals provide a range.
SHMA Participant Lab SOP                      B-81

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Table 2.  Sheepshead Minnow, Cyprinodon variegatus, Acute Test. Summary of test conditions and test acceptability criteria for sheepshead
minnow, Cyprinodon variegatus, acute toxicity tests with effluents and receiving waters	
1. Test type:
2. Test duration:
3. Temperature:
4. Light quality:
5. Light intensity:
6. Photoperiod:
7. Test chamber size:
8. Test solution volume:
9. Renewal of test solutions:
10. Age of test organisms:
11. No. organisms per test chamber:
12. No. replicate chambers per concentration:
13. No. organisms per concentration:
14. Feeding regime:

15. Test chamber cleaning:
16. Test solution aeration:
17. Dilution water:

18. Test concentrations:
19. Dilution factor
20. Endpoint:
21. Sample handling and holding requirements:

22. Sample volume required:
23. Test acceptability criterion:
24. Salinity:
Static renewal
96 h
25C1C
Ambient laboratory illumination
10-20 nE/m2/s or (50-100 ft-c) (ambient laboratory levels)
16 h light, 8 h darkness
250 ml
200 ml
At48h
1-14 days; 24-h range in age
10
2
20
Artemia nauplii are made available while holding prior to the test; add 0.2 mLArtemia nauplii
concentrate 2 h prior to test solution renewal at 48 h
Cleaning not required
None, unless DO concentration falls below 4.0 mg/L; rate should not exceed 100 bubbles/min
25 %o  2%oBioassay Grade Forty Path oms artificial seawater prepared with MILLIPORE
MILLI-Q or equivalent deionized water (see Methods Manual Section 7, Dilution Water)
Five concentrations and a control
0.5
Mortality (LC50)
Samples treated as effluent samples for NPDES monitoring. Samples are to be used on the day
specified by the finalized interlaboratory study testing schedule
1 Lfor effluents
90% or greater survival in controls
25%o(2%0)	
NOTE: Test vessels shall be randomized in accordance with the method manuals.
SHMA Participant Lab SOP
             B-82

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3.2.3   Data Analysis

For the Sheepshead Minnow acute test method, the 96 hour LC50 shall be calculated and reported. Data
analysis and statistical procedures should be conducted according to the method manuals.

3.2.4   Problem Resolution

In the event that problems are encountered during the WET test analysis contact Brian Rusignuolo at
(703)461-2401 for guidance.  Brian will direct your call as appropriate for the resolution of technical
issues.

3.2.5   Sample Disposal

Following the termination of the test, any excess sample shall be disposed of according to standard
laboratory procedures for disposal of effluent samples.

4.0 Data Reporting Deliverables

According to the Participant  Laboratory Statement of Work, the submission of three deliverables are
required: 1). Narrative summary of testing, 2). Hardcopy results synopsis and full report, and 3).
Electronic results synopsis.  This section provides instructions for the submission of each of these
deliverables.

4.1  Narrative Summary of Testing

This narrative summary shall clearly identify the laboratory, test method, samples tested, summarized test
results, and any problems associated with the samples or conduct of the tests. This summary must list any
tests that were initiated but not completed and fully explain the reason for not completing the test. This
summary must also include a detailed written description of any approved modification to the procedures
provided in this SOW, specific instructions, or the method manuals.  This will include any telephone log
and written correspondence received from the referee laboratory and/or DynCorp during the course of
testing. Lastly, this summary should also provide comments on the performance of the method.

4.2 Hardcopy Results Synopsis and Full Report

At a minimum, this report must consist of the items outlined below in section 5.0, all raw data (biological
and chemical), and laboratory bench sheets. This report must include all pertinent sample information
including copies of all completed traffic report forms, all pertinent test condition and test organism
information, all pertinent quality assurance information including results of the monthly QA/QC reference
toxicant tests, and all summarized and raw results.

4.3  Electronic Results Synopsis.

Enclosed with this package is a computer diskette that contains the template for the electronic submission
of results from the Sheepshead minnow acute test method. The disk contains a Microsoft Excel 97
spreadsheet file named  SHMA	.xls.  The SHMA indicates that this template is for the Sheepshead
minnow acute test method, and the number following is a unique identifying number for your laboratory.
If your laboratory does  not have the hardware or software capabilities to view and enter data into this file,


SHMA Participant Lab SOP                     B-83

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please contact Brian Rusignuolo at (703)461-2401 to arrange distribution of an alternative version of the
electronic template. It is recommended to view the electronic benchsheet prior to initiating the test,
so the analyst can verify all the information collected on the laboratory benchsheet will be sufficient to
complete the electronic results.

Notice the following characteristics of the electronic template file:

    1.  The file contains four worksheet pages labeled "Sample #1", "Sample #2", "Sample #3", and
       "Sample #4". The results from each of the samples that your laboratory tested in this study
       should be entered on a separate worksheet page within the same file.
    2.  Each worksheet page contains six information boxes (general information, sample
       collection/receipt information, test information, biological data, water quality data, and
       summarized test results) in which data should be entered. The seventh information box (data
       quality flags) is for SCC use only and will aid in the automated review and quality control check
       of data.
    3.  Cells that are highlighted in pale yellow indicate required information. Cells highlighted in blue
       indicate optional information that may be entered if available. Cells highlighted in red are for
       SCC use only.
    4.  The file has been protected such that data can only be entered in yellow or blue  cells. No other
       cells can be changed.

To complete the electronic results synopsis data deliverable:

    1.  Record information into a separate worksheet page for each sample tested.
    2.  Record requested information or data in all required cells (pale yellow).
    3.  Record requested information or data in optional cells (blue) if data is available.
    4.  Check entered data against hardcopy bench sheets to ensure accuracy.
    5.  Save the file onto the diskette provided.  Also keep a copy of the file for laboratory records (a
       backup in case the diskette crashes when redelivered to DynCorp).  Do not change the file name.
    6.  Submit the diskette with the other data reporting deliverables by the due date to:

                             DynCorp I&ET, Sample Control Center
                             6101 Stevenson Avenue
                             Alexandria, VA 22304
                             Phone:(703)461-2064
                             Fax:(703)461-8056
                             Attention: Robert Brent

5.0    Data Report Format

Final hardcopy data reports should be submitted in the following format:
Note:  Adapted from Section 10 of the methods manuals.

Section 1 - Summary Page
    1.1   Laboratory name
    1.2   Laboratory address and phone number
SHMA Participant Lab SOP                     B-84

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    1.3   Name and signature of laboratory QA Officer, certifying that data have been internally
         reviewed and that personnel meticulously followed the methods, and the procedures are deemed
         to be compliant with the methods and acceptable for reporting purposes.
    1.4   Laboratory contact responsible for study
    1.5   Analyst(s) who performed WET tests (full names)
    1.6   Toxicity tests performed
    1.7   Detailed explanations of any difficulties encountered and any approved modifications to the
         techniques specified in this SOW, specific instructions, or the methods manuals.
    1.8   Number of successful tests completed

Section 2 - Sample Information
    2.1   Number of samples received and EPA sample number assigned to each sample. Copies of all
         completed traffic report forms should be included.
    2.2   Dates of sample receipt
    2.3   Sample temperature when received at laboratory
    2.4   Physical and chemical data of sample contents (as required in appropriate method)
    2.5   Dilution water
         2.5.1    Source and time frame water is used or how maintained
         2.5.2    Collection or preparation date(s), where applicable
         2.5.3    Pretreatment information
         2.5.4    Physical and chemical characteristics (pH, hardness, conductivity, salinity, etc.)
    2.6   Sample storage information
    2.7   Sample preparation for testing information

Section 3 - Test Conditions
    3.1   Toxicity test method used (title, number, source)
    3.2   Endpoint(s) of test(s)
    3.3   Deviations from reference method(s), if any, and reason(s)
    3.4   Date and time test(s) started, date and time samples were prepared and solutions transferred for
         renewals.
    3.5   Date and time test(s) terminated
    3.6   Type and volume  of test chambers
    3.7   Volume of solution used per chamber
    3.8   Number of organisms per test chamber
    3.9   Number of replicate test chambers per treatment
    3.10 Feeding frequency and amount and type of food (be  specific with sources, concentrations of
         foods (i.e, algae concentration, YCT solids level, preparation dates))
    3.11 Acclimation of test organisms (temperature mean and range and, where applicable, salinity
         mean and range)
    3.12 Test temperature (mean and range)
    3.13 Test salinity, where applicable (mean and range)
    3.14 Specify if aeration was needed
    3.15 Specify if organisms were dried immediately for weighing or preserved prior to drying
    3.16 Specify how food was prepared and sources of food. Include test results that validate the
         quality of batch food preparations (i.e., Ceriodaphnia dubia tests on YCT preparation).
    3.17 Describe how routine chemistries on new solutions were made (in actual test chamber or in
         beakers after dispensing)
SHMA Participant Lab SOP                     B-85

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    3.18  Describe how randomization was conducted, especially blocking and known parentage. Report
         how brood distinctions were made and male (if any) identification was made.

Section 4 - Test Organisms
    4.1   Scientific name of test species, verification of species documented
    4.2   Age (life stage) of test species (be specific for all species). Age at time of test initiation (for
         example, for C. dubia be sure to clarify the window of age of the neonates as well as the overall
         age of the animals.)
    4.3   Mean length and weight (where applicable)
    4.4   Source and QA/QC test conditions
    4.5   Holding conditions
    4.6   Diseases and treatment (where  applicable)
    4.7   Taxonomic key used for species identification

Section 5 - Quality Assurance
    5.1   Reference toxicant used routinely; source; date received; lot number
    5.2   Date and time of most recent reference toxicant test; test results and current control (cusum)
         chart including 20 most recent  data points
    5.3   Dilution water used in reference toxicant tests (with characteristics provided)
    5.4   Physical and chemical methods used
    5.5   Reference toxicant results (NOEC, IC25, or LC50 where applicable, LOEC or EC50)

Section 6 - Results
    6.1   Copies of all bench sheets. Be sure to count and notate broods for reproduction test with
         Ceriodaphnia
    6.2   Raw toxicity data in tabular form, including daily records of affected organisms in each
         replicate at each concentration  (including controls) and plots of toxicity data
    6.3   Table of endpoints (LC50, IC25, NOEC for each endpoint) and confidence limits (where
         applicable)
    6.4   Statistical methods and software used to calculate endpoints
    6.5   Summary table of physical and chemical data

6.0 Coolers

All coolers will include a prepaid return FedEx label for returning the cooler to the referee laboratory.
Fill out the sender portion of the FedEx label and place it on the cooler.  Coolers will be returned by 2
Day shipment, as already marked on the prepared label. Coolers must be returned to the referee
laboratory within 7 days of sample receipt.  Please empty and wipe out the cooler prior to returning it.
SHMA Participant Lab SOP                      B-86

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        Appendix A:  Instructions for Reconstitution of Liquid Ampule Sample for
                 Sheepshead Minnow, Cyprinodon variegatus, Acute Test

For each sample, a single liquid ampule will be received. The container shall be reconstituted
and used to initiate the test. The same reconstituted sample shall be used for test renewal at 48
hours.  Follow the directions below for the reconstitution of the sample ampule.

1.  Volumetrically add 500 mL of the liquid ampule sample to approximately 1L of synthetic
   seawater.  The synthetic seawater should be prepared to a salinity of 25%o (2%o) using
   Bioassay Grade Forty Fathoms artificial sea salts and MILLIPORE MILLI-Q or equivalent
   deionized water and reagent grade chemicals according to Section 7 of the methods manuals.

2.  Mix by swirling or gently shaking.

3.  Bring final volume to 4L (measured using volumetric glassware) with synthetic seawater.

4.  Mix again by swirling and gently shaking.

5.  Place reconstituted sample in a plastic container of appropriate volume.  A container that
   minimizes head space (i.e. cubitainer) is recommended.

6.  This 4L sample is the whole volume reconstituted sample and should be treated as a typical
   effluent sample.  It should be used directly for the 100% effluent test concentration and
   diluted appropriately to prepare other test concentrations (50%, 25%, 12.5%, and 6.25%).

7.  Use the reconstituted sample for test initiation and test renewal at 48 hr.  Store sample at
   4C.

8.  Perform the  Sheepshead Minnow, Cyprinodon variegatus, Acute Test as described in the
   SOW for this study and the methods manuals.
SHMA Participant Lab SOP                   B-87

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Appendix B: EPA Traffic Report
^EPA
United States
Environmental Protection Agency
Washington, DC 20460
WET Interlaboratory Variability Study Traffic Report
USEPA ENGINEERING AND ANALYSIS DIVISION
SAMPLE CONTROL CENTER
Referee Laboratory Information
Name:
Address:
City:
State:

Sampler name:
EPISODE NO:
SAMPLE NO:
DynCorp - Sample Control Center
Fax completed form immediately 6101 Stevenson Avenue
upon completion, and include Alexandria, VA 22304
hardcopy in final data report to: phone: (703) 461 -21 00
fax: (703)461-8056
Participant Lab Shipping Information
Lab Name:
Address:
City:
State:
Airbill no:
Date shipped:
SAMPLE COLLECTION /
Requested Information
1.
2.
3.
4.
5.
6.
7.
8.
Sample use description
Sample collection/receipt date
Sample collection/receipt time
Sampler / recipient signature
PH
Temperature
Conductivity (freshwater methods) / Salinity
(marine methods)
Dissolved Oxygen concentration
FOR PARTICIPANT LAB USE ONLY
Received by:
Sample condition on receipt:
RECEIPT INFORMATION
Pre-Shipment
I 	 (initiation I 	
renewal 1 I 	 Irenewal 2







Post-Shipment








REQUESTED ANALYSES
9.
I 	 I Pimephales promelas Acute
I 	 I Pimephales promelas Chronic
I 	 I Ceriodaphnia dubia Acute
I 	 I Ceriodaphnia dubia Chronic
I 	 I Selenastrum capricornutum Chronic
I 	 I Menidia beryllina Acute
I 	 I Menidia beryllina Chronic
I 	 I Holmesmysis costata Acute
I 	 I Champia parvula Chronic

I 	 I Cyprinodon variegatus Acute
I 	 I Cyprinodon variegatus Chronic
I 	 I Mysidopsis bahia Chronic


SHMA Participant Lab SOP
B-88

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DynCorp
litanisrtiii G Bircrjjnse TasMiijiy
TO:            Participant Laboratories for the Sheepshead Minnow Chronic Test Method

FROM:         Robert Brent, WET Study Coordinator

DATE:         March 9, 2000

SUBJECT:      Final Guidance and SOP for the Sheepshead Minnow Chronic Test
Enclosed is the final standard operating procedure (SOP) for laboratories participating in the Sheepshead
Minnow chronic test method in EPA's WET Interlaboratory Variability Study. This SOP is a supplement
to the statement of work (SOW) that was distributed on July 9, 1999 with the solicitation for this study.
The SOP details the sample distribution and testing schedule and provides important information for
completing participant laboratory tasks, such as instructions for the reconstitution of ampule samples.
Participant laboratories should follow the guidance in the enclosed SOP, the SOW, and the method
manuals.

Also enclosed is the electronic data reporting format disk for the sheepshead minnow chronic method. A
description and instructions for use of the electronic data report form are provided in the SOP.

Please ensure that the enclosed SOP and disk pertaining to the sheepshead minnow chronic test method
are distributed to laboratory staff that will be performing the test method in the study. Also, please see
that laboratory staff read over the SOP carefully to ensure the proper data is collected and reported.
Please note that the ampule reconstitution instructions for this method require the addition of
SOOmL of the ampule sample instead of lOOmL that has been  used previously for other methods.
For this reason, ampule samples will be provided in SOOmL bottles for this method.
SHMC Participant Lab SOP                     B-89

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                     STANDARD OPERATING PROCEDURE
          Participant Laboratory Support for EPA's WET Interlaboratory Study

                 Cyprinodon variegatus Larval  Survival and Growth Method
Preamble:
This standard operating procedure document (SOP) is a supplement to the participant laboratory
Statement of Work (SOW).  This SOP details specific information in the SOW regarding the conduct of
the Cyprinodon variegatus larval survival and growth test method in the WET Interlaboratory Variability
Study.  All modifications to the SOP must be approved by DynCorp prior to implementation.

All data deliverables submitted for this interlaboratory study become the property of EPA and may be
incorporated into the public record. Laboratories may not independently publish the results of analyses
for which they are paid to perform under this SOP.

1.0 Sample Distribution and Testing Schedule

Participant laboratory testing for the Cyprinodon variegatus survival and growth method will occur
between March 21 and April 4, 2000 with final reports due 30 days following termination of all tests
(May 4, 2000).  The schedule for the testing is provided below.  The date ranges on the schedule indicate
the test start dates and test completion dates.  Samples will be shipped FedEx Priority Overnight to arrive
at the participant laboratory on the test start date by 10:OOAM. All tests must be initiated on the day of
sample arrival.  Testing is scheduled to occur simultaneously at each participant laboratory, so adherence
to the testing schedule is mandatory for all participant laboratories.

Laboratories participating in the base study design will receive 4 blind test samples (reflected in the
schedule) that may be whole volume or ampule samples. Two samples will arrive on 3/21/00 for test
initiation on that day, and two samples will arrive on 3/28/00 for test initiation on that day.

Each sample aliquot that is prepared and shipped will be assigned a unique sample number. The sample
number will appear clearly and permanently on each container and on an EPA traffic report form that will
accompany each sample. For tests that require additional shipments for sample renewal, the sample
number shall be the same for each initiation and renewal shipment with the addition of a letter (A, B, or
C) after the sample number to designate the sample for use  as initiation (A), renewal 1 (B), or renewal 2
(C).

For whole volume samples, separate aliquots will be received on test Day 0, Day 2, and Day 4. The first
aliquot (identified with the sample number and the letter "A") shall be used for test initiation on Day 0
and renewal on Day 1.  The second aliquot (identified with the sample  number and the letter "B") shall be
used for test renewals on Day 2 and Day 3. The final aliquot (identified with the sample number and the
letter "C") shall be used for test renewal on Day 4, Day 5, and Day 6. This sample shipment schedule
mimics the typical schedule for chronic monitoring of effluent for compliance.

For ampule samples , three separate ampule containers (marked with the  sample number followed by A,
B, or C) will be received in a single shipment on test Day 0. The container marked "A" shall be
reconstituted on test Day 0 and used for test initiation on Day 0 and renewal on Day 1. The other aliquots
of the sample shall be refrigerated and stored until use on Day 2 and Day 4, respectively. The container

SHMC Participant Lab SOP                     B-90

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marked "B" shall be reconstituted on test Day 2 and used for renewal on Day 2 and 3. The container
marked "C" shall be reconstituted on Day 4 and used for renewal on Day 4, Day 5 and Day 6. The
sample reconstitution schedule for ampules attempts to mimic the typical sample shipment schedule for
chronic monitoring of effluents for compliance.
Table 1. Schedule for Cyprinodon variegatus Survival and Growth Testing.
Date
(start date - finish date)
3/21/00 - 3/28/00
3/28/00 - 4/4/00
5/4/00
Activity
Conduct Sheepshead minnow, Cyprinodon variegatus, Larval Survival and Growth Test with samples #1&2
Conduct Sheepshead minnow, Cyprinodon variegatus, Larval Survival and Growth Test with samples #3&4
Sheepshead minnow, Cyprinodon variegatus, Larval Survival and Growth Test data due
2.0 Sample Traffic Reporting Tasks

2.1    Sample Receipt Confirmation

An EPA traffic report form (Appendix B) will be received with each sample. The traffic report form will
document important sample collection, shipment, and receipt information.  Portions of the form will be
completed by the referee laboratory prior to shipment and will indicate the episode number, sample
number, referee laboratory information, participant laboratory shipping information, sample pre-shipment
information, and the requested analysis.  Immediately upon receipt of the sample, participant laboratories
will be responsible for determining that the sample arrived in satisfactory condition and for documenting
receipt of the sample, post-shipment sample water quality (temperature, pH, dissolved oxygen, and
salinity), and any problems on the EPA traffic report form.  The participant laboratory shall complete the
traffic report form by recording the required information in the "For Participant Lab Use Only" box and
in the "Post-Shipment" column of the "Sample Collection/Receipt Information" box.

For ampule samples, the pre-shipment and post-shipment measurement of pH, dissolved oxygen, and
salinity shall be omitted. This will avoid possible contamination of the ampule sample prior to
reconstitution. Pre-shipment and post-shipment temperature shall be measured in a temperature check
sample. An additional sample container labeled "temperature check" will be included with each cooler
shipment of ampules. The temperature shall be measured in this container and recorded on the EPA
traffic report form as a surrogate measure of temperature for all ampule samples within that cooler. The
temperature check shall be discarded after temperature measurement, and must not be used in any way for
WET testing.

Immediately upon completion of the EPA traffic report form, the participant laboratory shall fax the
completed form to DynCorp and retain a copy for inclusion in the final data report. Receipt of the faxed
traffic report form by DynCorp will be confirmation of successful sample arrival, so it is important that
the form be completed and faxed immediately. Forms should be faxed by  11:OOAM (local laboratory
time) to facilitate sample tracking and potential problem  resolution. The EPA traffic report form shall be
faxed to:
SHMC Participant Lab SOP                     B-91

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                                       Brian Rusignuolo
                                      Sample Coordinator
                                         DynCorp SCC
                                      fax: (703)461-8056
                                     phone: (703)461-2401
2.2    Problem Resolution
Prior to test initiation, notify Brian Rusignuolo of any special shipment receiving instructions (i.e., hold
shipment at airport or FedEx shipment center for pickup). Notification must also be made if any specific
instructions are required for Saturday deliveries.

Participant laboratories should be expecting the arrival of samples based on the provided schedule.  If
samples do not arrive as expected, if samples are damaged in shipment, if samples arrive without
accompanying traffic report forms, if sample numbers are not clearly identified on samples, or if any
other sample shipment or receipt problem is encountered immediately notify Brian Rusignuolo by phone
at (703)461-2401.

Brian must be notified by 11:OOAM (local laboratory time) if samples fail to arrive by the morning FedEx
shipment or if other shipment or sample receipt problems are encountered. DynCorp will track lost
shipments and instruct referee laboratories to resend test samples for arrival the following day if
necessary. If renewal shipments do not arrive on the expected day, DynCorp will provide guidance for
test renewal on a case-by-case basis.  Depending on the volume of sample remaining from previous
shipments, laboratories may be instructed to conduct full renewals with the remaining sample, conduct
partial renewals with the remaining sample, or omit the sample renewal for that day but carefully record
dissolved oxygen throughout the day and remove excess food and dead organisms from the test
containers.

3.0 WET Test Analysis

3.1    Sample Preparation

3.1.1   Whole Volume Samples

Whole volume samples will be received in cubitainers with sufficient volume for test conduct and
required water chemistry analysis. Samples will be received at the proper salinity range, so no sample
preparation or adjustment steps (e.g. pH adjustment or salinity adjustment) should be conducted prior to
test initiation.  The whole volume sample shall be treated as a typical 100% effluent sample received for
NPDES compliance monitoring. Test concentrations of 100%, 50%, 25%, 12.5%, and 6.25% sample
shall be prepared from the whole volume sample for use in the test.  These test concentrations and a
dilution water control shall be prepared using synthetic seawater as the dilution water (prepared according
to Section 7 of the methods manuals).

3.1.2   Ampule Samples

Ampule samples will be received as liquid samples in 500ml plastic bottles. Prior to test initiation the
ampule samples must be reconstituted according to the instructions in Appendix A to provide the
necessary volume for testing. The reconstituted sample shall then be treated as a typical 100% effluent


SHMC Participant Lab SOP                     B-92

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sample received for NPDES compliance monitoring. Test concentrations of 100%, 50%, 25%, 12.5%,
and 6.25% sample shall be prepared from this reconstituted sample for use in the test. These test
concentrations and a dilution water control shall be prepared using synthetic seawater as the dilution
water (prepared according to Section 7 of the methods manuals).

3.2    Test Conduct

This section of the SOP reiterates testing requirements from Section 3 of the Participant Laboratory
Statement of Work that must be followed for the conduct of the Sheepshead minnow, Cyprinodon
variegatus, larval survival and growth test method. Except where indicated in Sections 3.2.1 and 3.2.2 of
this SOP, each test shall be conducted in accordance with the general guidance and method specific
requirements for effluent testing included in the methods manuals.

3.2.1   General Testing Requirements

EPA acknowledges that the promulgated WET methods distinguish between requirements (indicated by
the compulsory terms "must" and "shall") and recommendations and guidance (indicated by discretionary
terms "should" and "may"). The latter terms indicate that the analyst has flexibility to optimize successful
test completion and when standardization is necessary to assure the predictability of the methods to
provide reliable results. Additionally, the method manuals allow variations of the methods which are
typically fixed in the permit; therefore, for the purposes of this study, a set of variables will be defined by
EPA (for example, dilution water, salinity, and acute test duration). Any deviation from defined test
procedures and/or conditions, such as the necessity to change reagents, equipment, test conditions, or
other specified test parameters must be reported to SCC, recorded at the time of modification, noted in
telephone logs of communications, documented in a memorandum, and approved by EPA.

Additional WET test requirements and general requirements listed in the methods manuals of special note
are provided below:

(1)    Personnel that conduct tests must be the same personnel that routinely conduct the WET tests at
       that laboratory facility and who were identified  in the prequalification materials.  If these
       individuals cannot be available during any part of the study, the laboratory must contact SCC.
       Personnel conducting the tests must be identified clearly and consistently in records.

(2)    To coordinate testing at participant laboratories, testing of each sample with each method must be
       initiated on the precise  day specified in the finalized study schedule. Samples should be tested
       within 36 hours from the time of sample preparation (determined in this study as the time at
       which individual sample aliquots were divided from the bulk test sample  for distribution to
       participant laboratories).  Deviation from this schedule must be reported to SCC immediately for
       approval.

(3)    Physical and chemical properties of the test samples must be in the ranges specified in this SOP,
       the SOW, specific instructions, and the methods manuals. Method specific instructions for any
       adjustments to the test samples prior to sample use (such as reconstitution of ampule samples or
       salinity adjustments) are provided herein.  Test samples received at participant laboratories must
       be refrigerated (at 4C   2C) immediately upon receipt and throughout the period of testing.
       The temperature of the  refrigeration unit should be routinely or continuously monitored to ensure
       that these sample holding requirements are met.


SHMC Participant Lab SOP                     B-93

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(4)     Measurement of test conditions (pH, salinity, total alkalinity, total hardness, and dissolved
       oxygen) shall be performed for each method by the participant laboratories following guidance in
       method manuals.  NOTE: Refer to the electronic benchsheet for required and recommended
       information.

(5)     The specified dilution and control waters must be used and prepared according to instructions in
       Section 7 of the methods manuals.

(6)     All WET tests are to be definitive tests with a control and a minimum of five test concentrations
       prepared using a dilution factor of 0.5.

(7)     All tests must be conducted using the number of replicates and number of test containers per
       concentration as specified in Section 3.2.2.

(8)     Test chambers used within a test must be of the same type, size, shape, and material.  The
       material must be allowed by the method manuals for the method used.

(9)     Test vessels shall be randomized in accordance with the method manuals.

(10)   Daily observation of mortality and removal of dead organisms for each test is required.

(11)   If test results indicate too great of toxicity (i.e., control mortalities, or complete mortality in all
       concentrations), the laboratories must contact SCC immediately and then investigate possible
       causes, first by checking for transcription and calculation mistakes, and then by investigating
       possible contamination in dilution waters, organism cultures, equipment, or other procedural
       steps.

(12)   If any test that has been initiated fails to be completed for any reason, the laboratory must contact
       SCC immediately for problem resolution and scheduling of additional testing. The incomplete
       test data and  the reason for not completing the test must be fully documented in the final report.

(13)   Each laboratory shall be required to report all data obtained during the course of testing,
       including the response of control samples.

(14)   Laboratories  must perform all QA/QC tests listed in Section 4 of the method manuals.
       Laboratories  that purchase organisms must supply QA/QC from the test organism supplier and
       follow method manuals for the appropriate QA/QC for purchasing organisms.

(15)   A reference toxicant QC test must be performed for each test method in the month that testing for
       this study occurs. Results of this test must be submitted with the final data package.

(16)   Data and statistical analyses must be submitted in hard copy in the standardized format specified
       in Section 5 of the SOW. All bench sheets and raw data, including sample tracking and chemistry
       analysis data must be submitted.  Data must also be submitted electronically according to an
       electronic template (Microsoft Excel spreadsheet) that is provided with this SOP.
SHMC Participant Lab SOP                      B-94

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(17)   Data analysis must be performed in accordance with the statistical programs specified in the
       methods manuals. Statistical methods and programs used must be reported along with sample calculations.

(18)   An IC25 must be reported for each chronic test.  The laboratories must report individual toxicity
       endpoints; laboratories are not allowed to average or perform other data manipulations unless
       required by a methods's instructions.

3.2.2   Method-Specific Requirements

Table 2 provides a summary of test conditions that shall be followed for the conduct of all Sheepshead
minnow larval survival and growth tests performed in the WET Interlaboratory Study. This table is
extracted from the summary test condition table in the method manual and modified to fit the scope of
this study.  Items that are bold italic in these tables represent conditions standardized for the purposes of
this study where method manuals provide a range.
SHMC Participant Lab SOP                      B-95

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Table 2.  Sheepshead Minnow, Cyprinodon variegatus, Larval Survival And Growth Test. Summary of test conditions and test acceptability
criteria for the sheepshead minnow, Cyprinodon variegatus, larval survival and growth test with effluents and receiving waters	
1. Test type:
2. Salinity:
3. Temperature:
4. Light quality:
5. Light intensity:
6. Photoperiod:
7. Test chamber size:
8. Test solution volume:
9. Renewal of test solutions:
10. Age of test organisms:
11. No. larvae per test chamber:
12. No. replicate chambers per concentration:
13. No. larvae per concentration:
14. Source of food:
15. Feeding regime:

16. Cleaning:
17. Aeration:

18. Dilution water:

19. Test concentrations:
20. Dilution factor:
21. Test duration:
22. Endpoints:
23. Test acceptability criteria:
24. Sample handling and holding requirements:

25. Sample volume required:	
Static renewal
25%0(2%0)
251C
Ambient laboratory illumination
10-20 nE/m2/s (50-100 ft-c) (ambient laboratory levels)
16 h light, 8 h darkness
600 mL beaker
500 mL/replicate (loading and DO restrictions must be met)
Daily
Newly hatched larvae (less than 24 h old; 24-h range in age)
10
4
40
Newly \\atc\\edArtemia nauplii, (less than 24-h old)
Feed once a day 0.10 g wet weight Artemia nauplii per replicate on Days 0-2; Feed 0.15 g wet
weight Artemia nauplii per replicate on Days 3-6
Siphon daily, immediately before test solution renewal and feeding
None, unless DO falls below 4.0 mg/L, then aerate all chambers. Rate should be less than 100
bubbles/min
25%o (2%o) Bioassay Grade Forty Fathoms artificial seawater prepared with MILLIPORE
MILLI-Q or equivalent deionized water (see Methods Manual Section 7, Dilution Water)
Five concentrations and a control
0.5
7 days
Survival and growth (weight)
80% or greater survival in controls; average dry weight per surviving organism in control
chambers should be 0.60  mg or greater, if unpreserved, or 0.50 mg or greater after no more than 7
days in 4% formalin or 70% ethanol
Samples treated as effluent samples for NPDES monitoring. Samples are to be used on the day
specified by the finalized interlaboratory study testing schedule
6 L per day	
NOTE: Test vessels shall be randomized in accordance with the method manuals.
SHMC Participant Lab SOP
             B-96

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3.2.3   Data Analysis

For the Sheepshead minnow larval survival and growth test method, the 7 day survival LC50, 7 day
survival NOEC, growth IC25, and growth NOEC shall be calculated and reported. Data analysis and
statistical procedures should be conducted according to the method manuals.

3.2.4   Problem Resolution

In the event that problems are encountered during the WET test analysis contact Brian Rusignuolo at
(703)461-2401 for guidance. Brian will direct your call as appropriate for the resolution of technical
issues.

3.2.5   Sample Disposal

Following the termination of the test, any excess sample shall be disposed of according to standard
laboratory procedures for disposal of effluent samples.

4.0 Additional Information on Data Reporting Deliverables

According to the Participant Laboratory Statement of Work, the submission of three deliverables are
required: 1). Narrative summary of testing, 2). Hardcopy results synopsis and full report, and 3).
Electronic results synopsis.  This section provides instructions for the submission of each of these
deliverables.

4.1  Narrative Summary of Testing

This narrative summary shall clearly identify the laboratory, test method, samples tested, summarized test
results, and any problems associated with the samples or conduct of the tests. This summary must list any
tests that were initiated but not completed  and fully explain the reason for not completing the test. This
summary must also include  a detailed written description of any approved modification to the procedures
provided in this SOW, specific instructions, or the method manuals. This will include any telephone log
and written correspondence received from the referee laboratory and/or DynCorp during the course of
testing. Lastly, this summary should also provide comments on the performance of the method.

4.2 Hardcopy Results Synopsis and Full Report

At a minimum, this report must consist of the items outlined below in section 5.0, all raw data (biological
and chemical), and laboratory bench sheets. This report must include all pertinent sample information
including copies  of all completed traffic report forms, all pertinent test condition and test organism
information, all pertinent quality assurance information including results of the monthly QA/QC reference
toxicant tests, and all summarized and raw results.

4.3  Electronic Results Synopsis

Enclosed with this package  is a computer diskette that contains the template for the electronic submission
of results from the  Sheepshead minnow larval survival and growth test method.  The disk contains a
Microsoft Excel 97 spreadsheet file named SHMC	.xls. The SHMC indicates that this template is for
the  Sheepshead minnow chronic test method, and the number following is a unique identifying number


SHMC Participant Lab SOP                     B-97

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for your laboratory. If your laboratory does not have the hardware or software capabilities to view and
enter data into this file, please contact Brian Rusignuolo at (703)461-2401 to arrange distribution of an
alternative version of the electronic template.  It is recommended to view the electronic benchsheet
prior to initiating the test, so the analyst can verify all the information collected on the laboratory
benchsheet will be sufficient to complete the electronic results.

Notice the following characteristics of the electronic template file:

    1.  The file contains four worksheet pages labeled "Sample #1", "Sample #2", "Sample #3", and
       "Sample #4".  The results from each of the samples that your laboratory tested in this study
       should be entered on a separate worksheet page within the same file.
    2.  Each worksheet page contains seven information boxes (general information,  sample
       collection/receipt information, test information, biological data, water quality data, weight data,
       and summarized test results) in which data should be entered. The eighth information box (data
       quality flags) is for SCC use only and will aid in the automated review and quality control check
       of data.
    3.  Cells that are highlighted in pale yellow indicate required information. Cells highlighted in blue
       indicate optional information that may be entered if available.  Cells highlighted in red are for
       SCC use only.
    4.  The file has been protected such that data can only be entered in yellow or blue cells. No other
       cells can be changed.

To complete the electronic results synopsis data deliverable:

    1.  Record information into a separate worksheet page for each sample tested.
    2.  Record requested information or data in all required cells (pale yellow).
    3.  Record requested information or data in optional cells (blue) if data is available.
    4.  Check entered data against hardcopy bench sheets to ensure accuracy.
    5.  Save the file onto the diskette provided. Also  keep a copy of the file for laboratory records (a
       backup in case the diskette crashes when redelivered to DynCorp).  Do not change the file name.
    6.  Submit the diskette with the other data reporting deliverables by the due date to:

                             DynCorp I&ET, Sample Control Center
                             6101 Stevenson Avenue
                             Alexandria, VA 22304
                             Phone:(703)461-2064
                             Fax:(703)461-8056
                             Attention: Robert Brent

5.0    Data Report Format

Final hardcopy data reports should be submitted in the following format:
Note:  Adapted from Section 10 of the methods manuals.

Section 1 - Summary Page
    1.1   Laboratory name
    1.2   Laboratory address and phone number
SHMC Participant Lab SOP                     B-98

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    1.3   Name and signature of laboratory QA Officer, certifying that data have been internally
         reviewed and that personnel meticulously followed the methods, and the procedures are deemed
         to be compliant with the methods and acceptable for reporting purposes.
    1.4   Laboratory contact responsible for study
    1.5   Analyst(s) who performed WET tests (full names)
    1.6   Toxicity tests performed
    1.7   Detailed explanations of any difficulties encountered and any approved modifications to the
         techniques specified in this SOW, specific instructions, or the methods manuals.
    1.8   Number of successful tests completed

Section 2 - Sample Information
    2.1   Number of samples received and EPA sample number assigned to each sample.  Copies of all
         completed traffic report forms should be included.
    2.2   Dates of sample receipt
    2.3   Sample temperature when received at laboratory
    2.4   Physical and chemical data of sample contents (as required in appropriate method)
    2.5   Dilution water
         2.5.1    Source and time frame water is used or how maintained
         2.5.2    Collection or preparation date(s), where applicable
         2.5.3    Pretreatment information
         2.5.4    Physical and chemical characteristics (pH, hardness, conductivity, salinity, etc.)
    2.6   Sample storage information
    2.7   Sample preparation for testing information

Section 3 - Test Conditions
    3.1   Toxicity test method used (title, number, source)
    3.2   Endpoint(s)  of test(s)
    3.3   Deviations from reference method(s), if any, and reason(s)
    3.4   Date and time test(s) started, date and time samples were prepared and solutions transferred for
         renewals.
    3.5   Date and time test(s) terminated
    3.6   Type and volume  of test chambers
    3.7   Volume of solution used per chamber
    3.8   Number of organisms per test chamber
    3.9   Number of replicate test chambers per treatment
    3.10 Feeding frequency and amount and type of food (be  specific with sources, concentrations of
         foods (i.e, algae concentration, YCT solids level, preparation dates))
    3.11 Acclimation of test organisms (temperature mean and range and, where applicable, salinity
         mean and range)
    3.12 Test temperature (mean and range)
    3.13 Test salinity, where applicable (mean and range)
    3.14 Specify if aeration was needed
    3.15 Specify if organisms were dried immediately for weighing or preserved prior to drying
    3.16 Specify how food was prepared and sources of food. Include test results that validate the
         quality of batch food preparations (i.e., Ceriodaphnia dubia tests on YCT preparation).
    3.17 Describe how routine chemistries on new solutions were made (in actual test chamber or in
         beakers after dispensing).
SHMC Participant Lab SOP                     B-99

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    3.18  Describe how randomization was conducted, especially blocking and known parentage. Report
         how brood distinctions were made and male (if any) identification was made.

Section 4 - Test Organisms
    4.1   Scientific name of test species, verification of species documented
    4.2   Age (life stage) of test species (be specific for all species). Age at time of test initiation (for
         example, for C. dubia be sure to clarify the window of age of the neonates as well as the overall
         age of the animals.)
    4.3   Mean length and weight (where applicable)
    4.4   Source and QA/QC test conditions
    4.5   Holding conditions
    4.6   Diseases and treatment (where  applicable)
    4.7   Taxonomic key used for species identification

Section 5 - Quality Assurance
    5.1   Reference toxicant used routinely; source; date received; lot number
    5.2   Date and time of most recent reference toxicant test; test results and current control (cusum)
         chart including 20 most recent  data points
    5.3   Dilution water used in reference toxicant tests (with characteristics provided)
    5.4   Physical and chemical methods used
    5.5   Reference toxicant results (NOEC, IC25, or LC50 where applicable, LOEC or EC50)

Section 6 - Results
    6.1   Copies of all bench sheets. Be sure to count and notate broods for reproduction test with
         Ceriodaphnia
    6.2   Raw toxicity data in tabular form, including daily records of affected organisms in each
         replicate at each concentration  (including controls) and plots of toxicity data
    6.3   Table of endpoints (LC50, IC25, NOEC for each endpoint) and confidence limits (where
         applicable)
    6.4   Statistical methods and software used to calculate endpoints
    6.5   Summary table of physical and chemical data
6.0 Coolers

All coolers will include a prepaid return FedEx label for returning the cooler to the referee laboratory.
Fill out the sender portion of the FedEx label and place it on the cooler.  Coolers will be returned by 2
Day shipment, as already marked on the prepared label. Coolers must be returned to the referee
laboratory within 7 days of sample receipt. Please empty and wipe out the cooler prior to returning it.
SHMC Participant Lab SOP                     B-100

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       Appendix A:  Instructions for Reconstitution of Liquid Ampule Sample for
      Sheepshead Minnow, Cyprinodon variegatus, Larval Survival and Growth Test

For each sample, three ampules containing liquid will be received (marked with the sample
number followed by an "A", "B", or "C"). The three containers shall be reconstituted as
described below to mimic the sample shipment schedule for effluent samples.  The container
marked "A" shall be reconstituted on the day of test initiation (Day 0) and used for renewal on
Day 1. The container marked "B" shall be reconstituted on Day 2 and used for renewals on Day
2 and Day 3. The container marked "C" shall be reconstituted on Day 4 and used for renewals
on Day 4, Day 5, and Day 6. Follow the directions below for the reconstitution of each sample
ampule.

1.  Volumetrically add 500 mL of the liquid ampule sample to approximately  1L of synthetic
    seawater.  The synthetic seawater should be prepared to a salinity of 25%o (2%o) using
    Bioassay Grade Forty Fathoms artificial  sea salts and MILLIPORE MILLI-Q or equivalent
    deionized water according to Section 7 of the methods manuals.

2.  Mix by swirling or gently shaking.

3.  Bring final volume to 21L (measured using volumetric glassware) with synthetic seawater
    dilution water.

4.  Mix again by swirling and gently shaking.

5.  Place reconstituted sample in a plastic container of appropriate volume.  A container that
    minimizes head space (i.e. cubitainer) is  recommended.

6.  This 21L sample is the whole volume reconstituted sample and should be treated as a typical
    effluent sample. It should be used directly for the 100% effluent test concentration and
    diluted appropriately to prepare other test concentrations (50%, 25%, 12.5%, and 6.25%).

7.  Use the reconstituted sample for test initiation and Day 1 test renewal.  Store sample at 4C.

8.  Perform the Sheepshead Minnow, Cyprinodon variegatus, Larval Survival and Growth Test
    as described in the SOW for this study and the methods manuals.

9.  Follow Steps 1 through 6 with each sample container to prepare the reconstituted sample on
    Day 2 and Day 4 for subsequent daily renewals.
SHMC Participant Lab SOP                  B-101

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Appendix B: EPA Traffic Report
United States
PDA Environmental Protection Agency
v ti-AA Washington, DC 20460
EPISODE NO:
SAMPLE NO:
DynCorp - Sample Control Center
WET Interlaboratory Variability Study Traffic Report Fax completed form immediately 6101 Stevenson Avenue
USEPA ENGINEERING AND ANALYSIS DIVISION upon completion, and include Alexandria, VA 22304
SAMPLE CONTROL CENTER hardcopy in final data report to: phone: (703) 461 -21 00
fax: (703)461-8056
Referee Laboratory Information Participant Lab Shipping Information
Name: Lab Name:
Address: Address:
City: City:
State: State:
Airbill no:
Sampler name: Date shipped:
FOR PARTICIPANT LAB USE ONLY
Received by:
Sample condition on receipt:
SAMPLE COLLECTION / RECEIPT INFORMATION
Requested Information
1.
2.
3.
4.
5.
6.
7.
8.
Sample use description
Sample collection/receipt date
Sample collection/receipt time
Sampler / recipient signature
PH
Temperature
Conductivity (freshwater methods) / Salinity
(marine methods)
Dissolved Oxygen concentration
Pre-Shipment
I 	 (initiation I 	 I renewal 1 I 	 Irenewal 2







Post-Shipment








REQUESTED ANALYSES
9.
I  I Pimephales promelas Acute
I  I Pimephales promelas Chronic
I  I Ceriodaphnia dubia Acute
I  I Ceriodaphnia dubia Chronic
I  I Selenastrum capricornutum Chronic
I  I Menidia beryllina Acute
I  I Menidia beryllina Chronic
I  I Holmesmysis costata Acute
I  I Champia parvula Chronic

I  I Cyprinodon variegatus Acute
I  I Cyprinodon variegatus Chronic
I  I Mysidopsis bahia Chronic


SHMC Participant Lab SOP
B-102

-------
DynCorp
litanisrtiii G Bircrjjnse TasMiijiy
TO:            Participant Laboratories for the Menldla beryllina Acute Test Method

FROM:         Robert Brent, WET Study Coordinator

DATE:         October 25, 1999

SUBJECT:      Final Guidance and SOP for the Menidia beryllina Acute Test
Enclosed is the final standard operating procedure (SOP) for laboratories participating in the Menidia
beryllina acute test method in EPA's WET Interlaboratory Variability Study. This SOP is a supplement
to the statement of work (SOW) that was distributed on July 9, 1999 with the solicitation for this study.
The SOP details the sample distribution and testing schedule and provides important information for
completing participant laboratory tasks, such as instructions for the reconstitution of ampule samples.
Participant laboratories should follow the guidance in the enclosed SOP, the SOW, and the method
manuals.

Also enclosed is the electronic data reporting format disk for the Menidia beryllina acute method. A
description and instructions for use of the electronic data report form are provided in the SOP.

Please ensure that the enclosed SOP and disk pertaining to the Menidia beryllina acute test method are
distributed to laboratory staff that will be performing the test method in the study.
ISMA Participant Lab SOP                     B-103

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                      STANDARD OPERATING PROCEDURE
          Participant Laboratory Support for EPA's WET Interlaboratory Study

                               Menidia beryllina Acute Method
Preamble:
This standard operating procedure document (SOP) is a supplement to the participant laboratory
Statement of Work (SOW).  This SOP details specific information in the SOW regarding the conduct of
the Menidia beryllina acute test method in the WET Interlaboratory Variability Study. All modifications
to the SOP must be approved by DynCorp prior to implementation.

All data deliverables submitted for this interlaboratory study become the property of EPA and may be
incorporated into the public record. Laboratories may not independently publish the results of analyses
for which they are paid to perform under this SOP.

1.0 Sample Distribution and Testing Schedule

Participant laboratory testing for the Menidia beryllina acute method will occur between November 2 and
November 13, 19990 with final reports due 30 days following termination of all tests (December 13,
1999). The schedule for the testing is provided below. The date ranges on the schedule indicate the test
start dates and test completion dates. Samples will be shipped FedEx Priority Overnight to arrive at the
participant laboratory on the test start date by 10:OOAM.  All tests must be initiated on the day of sample
arrival. Testing is scheduled to occur simultaneously at each participant laboratory, so adherence to the
testing schedule is mandatory for all participant laboratories.

Laboratories participating in the base study design will receive 4 blind test samples (reflected in the
schedule) that may be whole volume or ampule samples. Two samples will arrive on 11/2/99 for test
initiation on that day, and two samples will arrive on 11/9/99 for test initiation on that day.

Each sample aliquot that is prepared and shipped will be assigned a unique sample number. The sample
number will appear clearly and permanently on each container and on an EPA traffic report form that will
accompany each sample. For whole volume samples, one aliquot will be received on test Day 0. This
aliquot shall be used for test initiation on Day 0 and test renewal at 48 hours. For ampule samples, one
aliquot will be received on test Day 0. This aliquot shall be reconstituted on Day  0, and the reconstituted
sample shall be used for test initiation on Day 0 and test renewal at 48 hours.

Table 1. Schedule for Menidia beryllina Acute Testing.
Date
(start date - finish date)
11/2/99- 11/6/99
11/9/99- 11/13/99
12/13/99
Activity
Conduct Inland Silverside, Menidia beryllina, Acute Test with samples #1&2
Conduct Inland Silverside, Menidia beryllina, Acute Test with samples #3&4
Inland Silverside, Menidia beryllina, Acute Test data due
ISMA Participant Lab SOP                     B-104

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2.0 Sample Traffic Reporting Tasks

2.1    Sample Receipt Confirmation

An EPA traffic report form (Appendix B) will be received with each sample. The traffic report form will
document important sample collection, shipment, and receipt information. Portions of the form will be
completed by the referee laboratory prior to shipment and will indicate the episode number, sample
number, referee laboratory information, participant laboratory shipping information, sample pre-shipment
information, and the requested analysis.  Immediately upon receipt of the sample, participant laboratories
will be responsible for determining that the sample arrived in satisfactory condition and for documenting
receipt of the sample, post-shipment sample water quality (temperature, pH, dissolved oxygen, and
conductivity or salinity), and any problems on the EPA traffic report form. The participant laboratory
shall complete the traffic report form by recording the required information in the "For Participant Lab
Use Only" box and in the "Post-Shipment" column of the "Sample Collection/Receipt Information" box.

For ampule samples, the pre-shipment and post-shipment measurement of pH, dissolved oxygen, and
conductivity or salinity shall be omitted. This will avoid possible contamination of the ampule sample
prior to reconstitution. Pre-shipment and post-shipment temperature shall be measured in a temperature
check sample. An additional sample container labeled "temperature check" will be included with each
cooler shipment of ampules. The temperature shall be measured in this container and recorded on the
EPA traffic report form as a surrogate measure of temperature for all ampule samples within that cooler.
The temperature check shall be discarded after temperature measurement, and must not be used in any
way for WET testing.

Immediately upon completion of the EPA traffic report form, the participant laboratory shall fax the
completed form to DynCorp and retain a copy for inclusion in the final data report. Receipt of the faxed
traffic report form by DynCorp will be confirmation of successful sample arrival, so it is important that
the form be completed and faxed immediately. Forms should be faxed by 11:OOAM (local laboratory
time) to facilitate sample tracking and potential problem resolution. The EPA traffic report form shall be
faxed to:

                                        Brian Rusignuolo
                                      Sample  Coordinator
                                         DynCorp  SCC
                                      fax: (703)461-8056
                                     phone: (703)461-2401

2.2    Problem Resolution

Prior to test initiation, notify Brian Rusignuolo of any special shipment receiving instructions (i.e., hold
shipment at airport or FedEx shipment center for pickup). Notification must also be made if any  specific
instructions are required for Saturday deliveries.

Participant laboratories should be expecting the arrival of samples based on the provided schedule.  If
samples do not arrive as expected, if samples are damaged in shipment, if samples arrive without
accompanying traffic report forms, if sample numbers are not clearly identified on samples, or if any
other sample shipment or receipt problem is encountered immediately notify Brian Rusignuolo by phone
at (703)461-2401.


ISMA Participant Lab SOP                     B-105

-------
Brian must be notified by 11:00AM (local laboratory time) if samples fail to arrive by the morning
FedEx shipment or if other shipment or sample receipt problems are encountered. DynCorp will track
lost shipments and instruct referee laboratories to resend test samples for arrival the following day if
necessary.

3.0 WET Test  Analysis

3.1    Sample  Preparation

3.1.1   Whole Volume Samples

Whole volume samples will be received in cubitainers with sufficient volume for test conduct and
required water chemistry analysis. No sample preparation or adjustment steps (e.g. pH adjustment)
should be conducted prior to test initiation. The whole volume sample shall be treated as a typical 100%
effluent sample  received for NPDES compliance monitoring. Test concentrations of 100%, 50%, 25%,
12.5%, and 6.25% sample shall be prepared from the whole volume sample for use in the test. These test
concentrations and a dilution water control shall be prepared using synthetic seawater as the dilution
water (prepared according to Section 7 of the method manuals).

3.1.2   Ampule Samples

Ampule samples will be received as small volume liquid samples in 125ml plastic containers. Prior to
test initiation the ampule samples must be reconstituted according to the instructions in Appendix A to
provide the necessary volume for testing. The reconstituted sample shall then be treated as atypical
100% effluent sample received for NPDES compliance monitoring. Test concentrations of 100%, 50%,
25%,  12.5%, and 6.25% sample shall be prepared from this reconstituted sample for use  in the test.  These
test concentrations and a dilution water control shall be prepared using synthetic seawater as the dilution
water (prepared according to Section 7 of the method manuals).

3.2    Test Conduct

This section of the SOP reiterates testing requirements from Section 3 of the Participant Laboratory
Statement of Work that must be followed for the conduct of the Inland Silverside, Menidia beryllina,
acute test method. Except where indicated in Sections 3.2.1 and 3.2.2 of this SOP, each test shall be
conducted in accordance with the general guidance and method specific requirements for effluent testing
included  in the methods manuals.

3.2.1   General Testing Requirements

EPA acknowledges that the promulgated WET methods distinguish between requirements (indicated by
the compulsory  terms "must" and "shall") and recommendations and guidance (indicated by discretionary
terms "should" and "may"). The latter terms indicate that the analyst has flexibility to optimize  successful
test completion  and when standardization is necessary to assure the predictability of the methods to
provide reliable results. Additionally, the method manuals allow variations of the methods which  are
typically fixed in the permit; therefore, for the purposes of this study, a set of variables will be defined by
EPA (for example, dilution water, salinity, and acute test duration). Any deviation from defined test
procedures and/or conditions, such as the necessity to change reagents, equipment, test conditions, or
ISMA Participant Lab SOP                     B-106

-------
other specified test parameters must be reported to SCC, recorded at the time of modification, noted in
telephone logs of communications, documented in a memorandum, and approved by EPA.

Additional WET test requirements and general requirements listed in the methods manuals of special note
are provided below:

(1)    Personnel that conduct tests must be the same personnel that routinely conduct the WET tests at
       that laboratory facility and who were identified in the prequalification materials. If these
       individuals cannot be available during any part of the study, the laboratory must contact SCC.
       Personnel conducting the tests must be identified clearly and consistently in records.

(2)    To coordinate testing at participant laboratories, testing of each sample with each method must be
       initiated on the precise day specified in the finalized study schedule.  Samples should be tested
       within 36 hours from the time of sample preparation (determined in this study as the time at
       which individual sample aliquots were divided from the bulk test sample for distribution to
       participant laboratories).  Deviation from this schedule must be reported to SCC immediately for
       approval.

(3)    Physical and chemical properties of the test samples must be in the ranges specified in this SOP,
       the SOW, specific instructions, and the methods manuals. Method specific instructions for any
       adjustments to the test samples prior to sample use (such as reconstitution of ampule samples or
       salinity adjustments) are provided herein.  Test samples received  at participant laboratories must
       be refrigerated (at 4C  2C) immediately upon receipt and throughout the period of testing.
       The temperature of the refrigeration unit should be routinely or continuously monitored to ensure
       that these sample holding requirements are met.

(4)    Measurement of test conditions (pH, conductivity or salinity, total alkalinity, total hardness, and
       dissolved oxygen) shall be performed for each method by the participant laboratories following
       guidance in method manuals.

(5)    The specified dilution and control waters must be used and prepared according to instructions in
       Section 7 of the methods manuals.

(6)    All WET tests are to be definitive tests with a control and a minimum of five test concentrations
       prepared using a dilution factor of 0.5.

(7)    All tests must be conducted using the number of replicates and number of test containers per
       concentration as specified in Section 3.2.2.

(8)    Test chambers used within a test must be of the same type, size, shape, and material.  The
       material must be allowed by the method manuals for the method used.

(9)    Test vessels shall be randomized in accordance with the method manuals.

(10)   Daily observation of mortality and removal of dead organisms  for each test is required.

(11)   If test results indicate too great of toxicity (i.e.,  control mortalities, or complete mortality in all
       concentrations), the laboratories must contact SCC immediately and then investigate possible


ISMA Participant Lab SOP                      B-107

-------
        causes, first by checking for transcription and calculation mistakes, and then by investigating
        possible contamination in dilution waters, organism cultures, equipment, or other procedural
        steps.

(12)    If any test that has been initiated fails to be completed for any reason, the laboratory must contact
        SCC immediately for problem resolution  and scheduling of additional testing. The incomplete
        test data and the reason for not completing the test must be fully documented in the final report.

(13)    Each laboratory shall be required to report all data obtained during the course of testing,
        including the response of control samples.

(14)    Laboratories must perform all QA/QC tests listed in Section 4 of the method manuals.
        Laboratories that purchase organisms must supply QA/QC from the test organism supplier and
        follow method manuals for the appropriate QA/QC for purchasing organisms.

(15)    A reference toxicant QC test must be performed for each test method in the month that testing for
        this study occurs. Results of this test must be submitted with the final data package.

(16)    Data and statistical analyses must be submitted in hard copy in the standardized format specified
        in Section 5 of the SOW. All bench sheets and raw data, including sample tracking and chemistry
        analysis data must be submitted. Data must also be submitted electronically according to an
        electronic template (Microsoft Excel spreadsheet) that is provided with this SOP.

(17)    Data analysis must be performed in accordance with the statistical programs specified in the
        methods manuals. Statistical methods and programs used must be reported along with sample
        calculations.

(18)    An IC25 must be reported for each chronic test.  The laboratories must report individual toxicity
        endpoints; laboratories are not allowed to average  or perform other data manipulations unless
        required by a methods's instructions.

3.2.2   Method-Specific Requirements

Table 2 provides a summary of test conditions that shall be followed for the conduct of all Inland
Silverside acute tests performed in the WET Interlaboratory Study. This table is extracted from the
summary test condition table in the method manual and modified to fit the scope of this study. Items that
are bold italic in this table represent conditions standardized for the purposes of this study where method
manuals provide a range.
ISMA Participant Lab SOP                      B-108

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Table 2.  Inland Silverside, Menidia beryllina, Acute Test. Summary of test conditions and test acceptability criteria for inland silverside, Menidia
beryllina, acute toxicity test with effluents and receiving waters	
1. Test type:
2. Test duration:
3. Temperature:
4. Light quality:
5. Light intensity:
6. Photoperiod:
7. Test chamber size:
8. Test solution volume:
9. Renewal of test solutions:
10. Age of test organisms:
11. No. organisms per test chamber:
12. No. replicate chambers per concentration:
13. No. organisms per concentration:
14. Feeding regime:

15. Test chamber cleaning:
16. Test solution aeration:
17. Dilution water:

18. Test concentrations:
19. Dilution factor:
20. Endpoint:
21. Sample handling and holding requirements:

22. Sample volume required:
23. Test acceptability criterion:
24. Salinity:
Static-renewal
96 h
25C1C
Ambient laboratory illumination
10-20 ^E/mVs (50-100 ft-c) (ambient laboratory levels)
16 h light, 8 h darkness
250 ml
200 ml
At48h
9-14 days; 24-h range in age
10
2
20
Artemia nauplii are made available while holding prior to the test; add 0.2 mLArtemia nauplii
concentrate 2 h prior to test solution renewal at 48 h
Cleaning not required
None, unless DO concentration falls below 4.0 mg/L; rate should not exceed 100 bubbles/min
25%o (2%o) Bioassay Grade Forty Fathoms artificial seawater prepared with MILLIPORE
MILLI-Q or equivalent deionized water (see Methods Manual Section 7, Dilution Water)
Five concentrations and a control
0.5
Mortality (LC50)
Samples treated as effluent samples for NPDES monitoring. Samples are to be used on the day
specified by the finalized interlaboratory study testing schedule
1 Lfor effluents
90% or greater survival in controls
25%0(2%0)	
NOTE: Test vessels shall be randomized in accordance with the method manuals.
     ISMA Participant Lab SOP
          B-109

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3.2.3    Data Analysis

For the Inland Silverside acute test method, the 96 hour LC50 and NOEC shall be calculated and reported.
Data analysis and statistical procedures should be conducted according to the method manuals.

3.2.4    Problem Resolution

In the event that problems are encountered during the WET test analysis contact Brian Rusignuolo at
(703)461-2401 for guidance.  Brian will direct your call as appropriate for the resolution of technical
issues.

3.2.5    Sample Disposal

Following the termination of the test, any excess sample shall be disposed of according to standard
laboratory procedures for disposal of effluent samples.

4.0 Additional Information  on Data Reporting Deliverables

According to the Participant Laboratory Statement of Work, the submission of three deliverables are
required: 1). Narrative summary of testing, 2). Hardcopy results synopsis and full report, and 3).
Electronic results synopsis.  Deliverables #1 and 2 shall be submitted according to the requirements
specified in the SOW. This section provides additional instructions for the submission of the electronic
results synopsis.

Enclosed with this package  is  a computer diskette that contains the template for the electronic submission
of results from the Inland Silverside minnow acute test method. The disk contains a Microsoft Excel 97
spreadsheet file named ISA	.xls. The ISA indicates that this template is for the Inland Silverside
minnow acute test method, and the number following is a unique  identifying number for your laboratory.
If your laboratory does not have the hardware or software capabilities to view and enter data into this file,
please contact Brian Rusignuolo at (703)461-2401 to arrange distribution of an alternative version of the
electronic template.

Notice the following characteristics of the electronic template file:

    1.   The file contains four worksheet pages labeled "Sample #1", "Sample #2", "Sample #3", and
        "Sample #4".  The results from each of the samples that your laboratory tested in this  study
        should be entered on a separate worksheet page within the same file.
    2.   Each worksheet page  contains six information boxes (general information, sample
        collection/receipt information, test information, biological data, water quality data, and
        summarized test results) in which data should be entered.  The seventh information box (data
        quality flags) is for SCC use only and will aid in the automated review and quality control check
        of data.
    3.   Cells that are highlighted in pale yellow indicate required information.  Cells highlighted in blue
        indicate optional information that may be entered if available.  Cells highlighted in red are for
        SCC use only.
    4.   The file has been protected such that data can only be entered in yellow or blue cells.  No other
        cells can be changed.
ISMA Participant Lab SOP                     B-110

-------
To complete the electronic results synopsis data deliverable:

    1.  Record information into a separate worksheet page for each sample tested.
    2.  Record requested information or data in all required cells (pale yellow).
    3.  Record requested information or data in optional cells (blue) if data is available.
    4.  Check entered data against hardcopy bench sheets to ensure accuracy.
    5.  Save the file onto the diskette provided. Do not change the file name.
    6.  Submit the diskette with the other data reporting deliverables by the due date to:

                              DynCorp I&ET, Sample Control Center
                              6101 Stevenson Avenue
                              Alexandria, VA 22304
                              Phone:(703)461-2064
                              Fax:(703)461-8056
                              Attention: Robert Brent

5.0 Coolers

All coolers will include a prepaid return FedEx label for returning the cooler to the referee laboratory.
Fill out the sender portion of the FedEx label and place it on the cooler.  Coolers will be returned by 2
Day shipment, as already marked on the prepared label.  Coolers must be returned to the referee
laboratory within 7 days of sample receipt. Please empty and wipe out the cooler prior to returning it.
ISMA Participant Lab SOP                      B-111

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        Appendix A: Instructions for Reconstitution of Liquid Ampule Sample for
                     Inland Silverside, Menidia beryllina, Acute Test

For each sample, a single liquid ampule will be received. The container shall be reconstituted
and used to initiate the test.  The same reconstituted sample shall be used for test renewal at 48
hours.  Follow the directions below for the reconstitution of the sample ampule.

1.  Volumetrically add  100 mL of the liquid ampule sample to approximately 1L of of synthetic
    seawater.  The synthetic seawater should be prepared to a salinity of 25%o (2%o) using
    Bioassay Grade Forty Fathoms artificial sea salts and MILLIPORE MILLI-Q or
    equivalent deionized water and reagent grade chemicals according to Section 7 of the
    methods manuals.

2.  Mix by swirling or gently shaking.

3.  Bring final volume to 4L (measured using volumetric glassware) with synthetic seawater.

4.  Mix again by swirling and gently shaking.

5.  Place reconstituted sample in a plastic container of appropriate volume. A container that
    minimizes head space (i.e. cubitainer) is recommended.

6.  This 4L sample is the whole volume reconstituted sample and should be treated as a typical
    effluent sample.  It should be used directly for the 100% effluent test concentration and
    diluted appropriately to prepare other test concentrations (50%, 25%, 12.5%, and 6.25%).

7.  Use the reconstituted sample for test initiation and test renewal at 48 hr. Store sample at
    4C.

8.  Perform the Inland Silverside, Menidia beryllina, Acute Test as described in the SOW for
    this study and the methods manuals.
ISMA Participant Lab SOP                    B-112

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Appendix B: EPA Traffic Report
^EPA
United States
Environmental Protection Agency
Washington, DC 20460
WET Interlaboratory Variability Study Traffic Report
USEPA ENGINEERING AND ANALYSIS DIVISION
SAMPLE CONTROL CENTER
Referee Laboratory Information
Name:
Address:
City:
State:

Sampler name:
EPISODE NO:
SAMPLE NO:
DynCorp - Sample Control Center
Fax completed form immediately 6101 Stevenson Avenue
upon completion, and include Alexandria, VA 22304
hardcopy in final data report to: phone: (703) 461 -21 00
fax: (703)461-8056
Participant Lab Shipping Information
Lab Name:
Address:
City:
State:
Airbill no:
Date shipped:
SAMPLE COLLECTION /
Requested Information
1.
2.
3.
4.
5.
6.
7.
8.
Sample use description
Sample collection/receipt date
Sample collection/receipt time
Sampler / recipient signature
PH
Temperature
Conductivity (freshwater methods) / Salinity
(marine methods)
Dissolved Oxygen concentration
FOR PARTICIPANT LAB USE ONLY
Received by:
Sample condition on receipt:
RECEIPT INFORMATION
Pre-Shipment
I 	 (initiation I 	
renewal 1 I 	 Irenewal 2







Post-Shipment








REQUESTED ANALYSES
9.
I 	 I Pimephales promelas Acute
I 	 I Pimephales promelas Chronic
I 	 I Ceriodaphnia dubia Acute
I 	 I Ceriodaphnia dubia Chronic
I 	 I Selenastrum capricornutum Chronic
I 	 I Menidia beryllina Acute
I 	 I Menidia beryllina Chronic
I 	 I Holmesmysis costata Acute
I 	 I Champia parvula Chronic

I 	 I Cyprinodon variegatus Acute
I 	 I Cyprinodon variegatus Chronic
I 	 I Mysidopsis bahia Chronic


ISMA Participant Lab SOP
B-113

-------
DynCorp
litanisrtiii G Bircrjjnse TasMiijiy
TO:           Participant Laboratories for the Menldla beryllina Chronic Test Method

FROM:        Robert Brent, WET Study Coordinator

DATE:        October 13, 1999

SUBJECT:     Final Guidance and SOP for the Menidia beryllina Chronic Test
Enclosed is the final standard operating procedure (SOP) for laboratories participating in the Menidia
beryllina chronic test method in EPA's WET Interlaboratory Variability Study. This SOP is a
supplement to the statement of work (SOW) that was distributed on July 9, 1999 with the solicitation for
this study. The SOP details the sample distribution and testing schedule and provides important
information for completing participant laboratory tasks, such as instructions for the reconstitution of
ampule samples.  Participant laboratories should follow the guidance in the enclosed SOP, the SOW, and
the method manuals.

Also enclosed is the electronic data reporting format disk for the Menidia beryllina chronic method. A
description and instructions for use of the electronic data report form are provided in the SOP.

Please ensure that the enclosed SOP and disk pertaining to the Menidia beryllina chronic test method are
distributed to laboratory staff that will be performing the test method in the study.
ISMC Participant Lab SOP
B-114

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                     STANDARD OPERATING PROCEDURE
          Participant Laboratory Support for EPA's WET Interlaboratory Study

                   Menidia beryllina Larval Survival and Growth Method
Preamble:
This standard operating procedure document (SOP) is a supplement to the participant laboratory
Statement of Work (SOW).  This SOP details specific information in the SOW regarding the conduct of
the Menidia beryllina larval survival and growth test method in the WET Interlaboratory Variability
Study.  All modifications to the SOP must be approved by DynCorp prior to implementation.

All data deliverables submitted for this interlaboratory study become the property of EPA and may be
incorporated into the public record. Laboratories may not independently publish the results of analyses
for which they are paid to perform under this SOP.

1.0 Sample Distribution and Testing Schedule

Participant laboratory testing for the Menidia beryllina survival and growth method will occur between
October 19 and November 2, 1999 with final reports due 30 days following termination of all tests
(December 2, 1999). The schedule for the testing is provided below.  The date ranges on the schedule
indicate the test start dates and test completion dates. Samples will be shipped FedEx Priority Overnight
to arrive at the participant laboratory on the test start date by 10:00AM. All tests must be initiated on the
day of sample arrival.  Testing is scheduled to occur simultaneously at each participant laboratory, so
adherence to the testing schedule is mandatory for all participant laboratories.

Each participant laboratory will receive 4 blind test samples (reflected in the schedule) that may be whole
volume or ampule samples.  Two samples will arrive on 10/19/99 for test initiation on that day, and two
samples will arrive on  10/26/99 for test initiation on that day.

Each sample aliquot that is prepared and shipped will be assigned a unique sample number. The sample
number will appear clearly and permanently on each container and on an EPA traffic report form that will
accompany each sample. For tests that require additional shipments for sample renewal, the sample
number shall be the same for each initiation and renewal shipment with the addition of a letter (A, B, or
C) after the sample number to designate the sample for use as initiation (A), renewal 1  (B), or renewal 2
(C).

For whole volume samples, separate aliquots will  be received on test Day 0, Day 2, and Day 4. The first
aliquot (identified with the sample number and the letter "A") shall be used for test initiation on Day 0
and test renewal on Day 1. The second aliquot (identified with the sample number and the letter "B")
shall be used for test renewals on Day 2 and Day 3.  The final aliquot (identified with the sample number
and the letter "C") shall be used for test renewal on Day 4, Day 5, and Day 6. This sample shipment
schedule mimics the typical schedule for chronic monitoring of effluent for compliance.

For ampule samples, three separate ampule containers  (marked with the sample number followed by A, B,
or C) will be received in a single shipment on test Day 0. The container marked "A" shall be
reconstituted on test Day 0 and used for test initiation and renewal on Day  1.  The other aliquots of the
sample shall be  refrigerated and stored until use on Day 2 and Day 4, respectively.  The container marked

ISMC Participant Lab SOP                     B-115

-------
"B" shall be reconstituted on test Day 2 and used for renewal on Day 2 and 3. The container marked "C"
shall be reconstituted shall be reconstituted on Day 4 and used for renewal on Day 4, Day 5 and Day 6.
The sample reconstitution schedule for ampules also attempts to mimic the typical sample shipment
schedule for chronic monitoring of effluents for compliance.
Table 1. Schedule for Menidia beryllina Survival and Growth Testing.
Date
(start date - finish date)
10/19/99 - 10/26/99
10/26/99 - 1 1/2/99
12/2/99
Activity
Conduct Inland Silverside, Menidia beryllina, Larval Survival and Growth Test with samples #1&2
Conduct Inland Silverside, Menidia beryllina, Larval Survival and Growth Test with samples #3&4
Inland Silverside, Menidia beryllina, Larval Survival and Growth Test data due
2.0 Sample Traffic Reporting Tasks

2.1    Sample Receipt Confirmation

An EPA traffic report form (Appendix B) will be received with each sample. The traffic report form will
document important sample collection, shipment, and receipt information. Portions of the form will be
completed by the referee laboratory prior to shipment and will indicate the episode number, sample
number, referee laboratory information, participant laboratory shipping information, sample pre-shipment
information, and the requested analysis.  Immediately upon receipt of the sample, participant laboratories
will be responsible for determining that the sample arrived in satisfactory condition and for documenting
receipt of the sample, post-shipment sample water quality (temperature, pH, dissolved oxygen, and
conductivity or salinity), and any problems on the EPA traffic report form. The participant laboratory
shall complete the traffic report form by recording the required information in the "For Participant Lab
Use Only" box and in the "Post-Shipment" column of the "Sample Collection/Receipt Information" box.

For ampule samples, the pre-shipment and post-shipment measurement of pH, dissolved oxygen, and
conductivity or salinity shall be omitted. This will avoid possible contamination of the ampule sample
prior to reconstitution. Pre-shipment and post-shipment temperature shall be measured in a temperature
check sample. An additional sample container labeled "temperature check" will be included with each
cooler shipment of ampules. The temperature shall be measured in this container and recorded on the
EPA traffic report form as a surrogate measure of temperature for all ampule samples within that cooler.
The temperature check shall be discarded after temperature measurement, and must not be used in any
way for WET testing.

Immediately upon completion of the EPA traffic report form, the participant laboratory shall fax the
completed form to DynCorp and retain a copy for inclusion in the final data report. Receipt of the faxed
traffic report form by DynCorp will be confirmation of successful sample arrival, so it is important that
the form be completed and faxed immediately. Forms should be faxed by 11:OOAM (local laboratory
time) to facilitate sample tracking and potential problem resolution. The EPA traffic report form shall be
faxed to:
ISMC Participant Lab SOP                     B-116

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                                       Brian Rusignuolo
                                      Sample Coordinator
                                         DynCorp SCC
                                      fax: (703)461-8056
                                     phone: (703)461-2401
2.2    Problem Resolution
Prior to test initiation, notify Brian Rusignuolo of any special shipment receiving instructions (i.e., hold
shipment at airport or FedEx shipment center for pickup). Notification must also be made if any specific
instructions are required for Saturday deliveries.

Participant laboratories should be expecting the arrival of samples based on the provided schedule.  If
samples do not arrive as expected, if samples are damaged in shipment, if samples arrive without
accompanying traffic report forms, if sample numbers are not clearly identified on samples, or if any
other sample shipment or receipt problem is encountered immediately notify Brian Rusignuolo by phone
at (703)461-2401.

Brian must be notified by 11:OOAM  (local laboratory time) if samples fail to arrive by the morning FedEx
shipment or if other shipment or sample receipt problems are encountered. DynCorp will track lost
shipments and instruct referee laboratories to resend test samples for arrival the following day if
necessary. If renewal shipments do not arrive on the expected day, DynCorp will provide guidance for
test renewal on a case-by-case basis.  Depending on the volume of sample remaining from previous
shipments, laboratories may be instructed to conduct full renewals with the remaining sample, conduct
partial  renewals with the remaining sample, or omit the sample renewal for that day but carefully record
dissolved oxygen throughout the day and remove excess food and dead organisms from the  test
containers.

3.0 WET Test Analysis

3.1    Sample Preparation

3.1.1  Whole Volume Samples

Whole volume samples will be received in cubitainers with sufficient volume for test conduct and
required water chemistry analysis. No sample preparation or adjustment steps (e.g. pH adjustment or
salinity adjustment) should be conducted prior to test initiation. The whole volume sample  shall be
treated as a typical 100% effluent sample received for NPDES compliance monitoring. Test
concentrations of 100%, 50%, 25%,  12.5%, and 6.25% sample shall be prepared from the whole volume
sample for use in the test.  These test concentrations and a dilution water control shall be prepared using
synthetic seawater as the dilution water (prepared according to Section 7 of the methods manuals).

3.1.2  Ampule Samples

Ampule samples will be received as  small volume liquid samples in 125ml plastic containers. Prior to
test initiation the ampule samples must be reconstituted according to the instructions in Appendix A to
provide the necessary volume for testing. The reconstituted sample shall then be treated as atypical
100% effluent sample received for NPDES compliance monitoring. Test concentrations of  100%, 50%,


ISMC Participant Lab SOP                     B-117

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25%, 12.5%, and 6.25% sample shall be prepared from this reconstituted sample for use in the test. These
test concentrations and a dilution water control shall be prepared using synthetic seawater as the dilution
water (prepared according to Section 7 of the methods manuals).

3.2     Test Conduct

This section of the SOP reiterates testing requirements from Section 3 of the Participant Laboratory
Statement of Work that must be followed for the conduct of the Inland Silverside minnow, Menidia
beryllina, larval survival and growth test method. Except where indicated in Sections 3.2.1 and 3.2.2 of
this SOP, each test shall be conducted in accordance with the general guidance and method specific
requirements for effluent testing included in the methods manuals.

3.2.1    General Testing Requirements

EPA acknowledges that the promulgated WET methods distinguish between requirements (indicated by
the compulsory terms "must" and "shall") and recommendations and guidance (indicated by discretionary
terms "should" and "may"). The latter terms indicate that the analyst has flexibility to optimize successful
test completion and when standardization is necessary to assure the predictability of the methods to
provide reliable results. Additionally, the method manuals allow variations of the methods which are
typically fixed in the permit; therefore, for the purposes of this study, a set of variables will be defined by
EPA (for example, dilution water, salinity, and acute test duration). Any deviation from defined test
procedures and/or conditions, such as the necessity to change reagents, equipment, test conditions, or
other specified test parameters must be reported to SCC, recorded at the time of modification, noted in
telephone logs of communications, documented in a memorandum, and approved by EPA.

Additional WET test requirements and general requirements listed in the methods manuals of special note
are provided below:

(1)     Personnel that conduct tests must be the same personnel that routinely conduct the WET tests at
        that laboratory facility and who  were identified in the prequalification materials. If these
        individuals cannot be available during any part of the study, the laboratory must contact SCC.
        Personnel conducting the tests must be identified clearly and consistently in records.

(2)     To coordinate testing at participant laboratories, testing of each sample with each method must be
        initiated on the precise  day specified in the finalized study schedule.  Samples should be tested
        within 36 hours from the time of sample preparation (determined in this study as the time at
        which individual sample aliquots were divided from the bulk test sample  for distribution to
        participant laboratories).  Deviation from this schedule must be reported to SCC immediately for
        approval.

(3)     Physical and chemical properties of the test samples must be in the ranges specified in this SOP,
        the SOW, specific instructions, and the methods manuals. Method specific instructions for any
        adjustments to the test samples prior to sample use (such as reconstitution of ampule samples or
        salinity adjustments) are provided herein. Test samples received at participant laboratories must
        be refrigerated (at 4C   2C) immediately upon receipt and throughout the period of testing.
        The temperature of the  refrigeration unit should be routinely or continuously monitored to ensure
        that these sample holding requirements are met.
ISMC Participant Lab SOP                      B-118

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(4)     Measurement of test conditions (pH, conductivity or salinity, total alkalinity, total hardness, and
        dissolved oxygen) shall be performed for each method by the participant laboratories following
        guidance in method manuals.

(5)     The specified dilution and control waters must be used and prepared according to instructions in
        Section 7 of the methods manuals.

(6)     All WET tests are to be definitive tests with a control and a minimum of five test concentrations
        prepared using a dilution factor of 0.5.

(7)     All tests must be conducted using the number of replicates and number of test containers per
        concentration as specified in Section 3.2.2.

(8)     Test chambers used within a test must be of the same type, size, shape, and material. The
        material must be allowed by the method manuals for the method used.

(9)     Test vessels shall be randomized in accordance with the method manuals.

(10)    Daily observation of mortality and removal of dead organisms for each test is required.

(11)    If test results indicate too great of toxicity (i.e., control mortalities, or complete mortality in all
        concentrations), the laboratories must contact SCC immediately and then investigate possible
        causes, first by checking for transcription and calculation mistakes, and then by investigating
        possible contamination in dilution waters, organism cultures, equipment, or other procedural
        steps.

(12)    If any test that has been initiated fails to be completed for any reason, the laboratory must contact
        SCC immediately for problem resolution and scheduling of additional testing. The incomplete
        test data and the reason for not completing the test must be fully documented in the  final report.

(13)    Each laboratory shall be required to report all data obtained during the course of testing,
        including the response of control samples.

(14)    Laboratories must perform all  QA/QC tests listed in Section 4 of the method manuals.
        Laboratories that purchase organisms must supply QA/QC from the test organism supplier and
        follow method manuals for the appropriate QA/QC for purchasing organisms.

(15)    A reference toxicant QC test must be performed for each test method in the month that testing for
        this study occurs. Results of this test must be submitted with the final data package.

(16)    Data and statistical analyses must be submitted in hard copy in the standardized format specified
        in Section 5 of the SOW. All bench sheets and raw data, including sample tracking and chemistry
        analysis data must be submitted. Data must also be submitted electronically according to an
        electronic template (Microsoft Excel spreadsheet) that is provided with this SOP.

(17)    Data analysis must be performed in accordance with the statistical programs specified in the
        methods manuals. Statistical methods and programs used must be reported along with sample
        calculations.
ISMC Participant Lab SOP                     B-119

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(18)   An IC25 must be reported for each chronic test. The laboratories must report individual toxicity
       endpoints; laboratories are not allowed to average or perform other data manipulations unless
       required by a methods's instructions.

3.2.2  Method-Specific Requirements

Table 2 provides a summary of test conditions that shall be followed for the conduct of all Inland
Silverside minnow larval survival and growth tests performed in the WET Interlaboratory Study. This
table is extracted from the summary test condition table in the method manual and modified to fit the
scope of this study. Items that are bold italic in this table represent conditions standardized for the
purposes of this study where method manuals provide a range.
ISMC Participant Lab SOP                     B-120

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Table 2. Inland Silverside, Menidia beryllina, Larval
the inland silverside, Menidia beryllina, larval survival
 Survival and Growth Test. Summary of test conditions and test acceptability criteria for
and growth test with effluents and receiving waters	
1.  Test type:
2.  Salinity:
3.  Temperature:
4.  Light quality:
5.  Light intensity:
6.  Photoperiod:
7.  Test chamber size:
8.  Test solution volume:
9.  Renewal of test solutions:
10. Age of test organisms:
11. No. larvae per test chamber:
12. No. replicate chambers per concentration:
13. No. larvae per concentration:
14. Source of food:

15. Feeding regime:

16. Cleaning:
17. Aeration:

18. Dilution water:

19. Test concentrations:
20. Dilution factor:
21. Test duration:
22. Endpoints:
23. Test acceptability criteria:
24. Sample handling and holding requirements:

25. Sample volume required:
  Static renewal
  25%0(2%<)
  251C
  Ambient laboratory illumination
  10-20 uE/m2/s (50-100 ft-c) (Ambient laboratory levels)
  16 h light, 8 h darkness
  1 L containers
  750 mL/replicate (loading and DO restrictions must be met)
  Daily
  7-11 days post hatch; 24-h range in age
  10
  4
  40
  Newly \\atc\\edArtemia nauplii (survival of 7-9 days old Menidia beryllina larvae improved by
  feeding 24 h oldArtemia)
  Feed 0.10 g wet weight^4rte/w/a nauplii per replicate on days 0-2; Feed 0.15 g wet weight Artemia
  nauplii per replicate on days 3-6
  Siphon daily, immediately before test solution renewal and feeding
  None, unless DO concentration falls below 4.0 mg/L, then aerate all chambers. Rate should be
  less than 100 bubbles/min.
  25%o (2%o) Bioassay Grade Forty Fathoms artificial seawater prepared with MILLIPORE
  MILLI-Q or equivalent deionized water (see Methods Manual Section 7, Dilution Water)
  Five concentrations and a control
  0.5
  7 days
  Survival and growth (weight)
  80% or greater survival in controls, 0.50 mg average dry weight of control larvae when larvae
  dried immediately after test termination, or 0.43 mg or greater average dry weight of control
  larvae, preserved not more than 7 days in 4% formalin or 70% ethanol
  Samples treated as effluent samples for NPDES monitoring. Samples are to be used on the day
  specified by the finalized interlaboratory study testing schedule
  6 L/day
NOTE: Test vessels shall be randomized in accordance with the method manuals.
ISMC Participant Lab SOP
              B-121

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3.2.3    Data Analysis

For the Inland Silverside larval survival and growth test method, the 7 day survival LC50, 7 day survival
NOEC,  growth IC25, and growth NOEC shall be calculated and reported. Data analysis and statistical
procedures should be conducted according to the method manuals.

3.2.4    Problem Resolution

In the event that problems are encountered during the WET test analysis contact Brian Rusignuolo at
(703)461-2401 for guidance.  Brian will direct your call as appropriate for the resolution of technical
issues.

3.2.5    Sample Disposal

Following the termination of the test, any excess sample shall be disposed of according to standard
laboratory procedures for disposal of effluent samples.

4.0 Additional Information  on Data Reporting Deliverables

According to the Participant Laboratory Statement of Work, the submission of three deliverables are
required: 1). Narrative summary of testing, 2). Hardcopy results synopsis and full report, and 3).
Electronic results synopsis.  Deliverables #1 and 2 shall be submitted according to the requirements
specified in the SOW. This section provides additional instructions for the submission of the electronic
results synopsis.

Enclosed with this package is  a computer diskette that contains the template for the electronic submission
of results from the Inland Silverside minnow larval survival and growth test method. The disk contains a
Microsoft Excel 97 spreadsheet file named ISC	.xls. The ISC indicates that this template is for the
Inland Silverside minnow chronic test method, and the number following is a unique identifying number
for your laboratory. If your laboratory does not have the hardware or software capabilities to view and
enter data into this file, please contact Brian Rusignuolo at (703)461-2401 to arrange distribution of an
alternative version of the electronic template.

Notice the following  characteristics of the electronic template file:

    1.   The file contains four worksheet pages labeled "Sample #1", "Sample #2", "Sample #3", and
        "Sample #4". The results from each of the samples that your laboratory tested in this study
        should be entered on a separate worksheet page within the same file.
    2.   Each worksheet page  contains seven information boxes (general information, sample
        collection/receipt information, test information, biological data, water quality data, weight data,
        and summarized test results) in which data should be entered. The eighth information box (data
        quality flags) is for SCC use only and will aid in the automated review and quality control check
        of data.
    3.   Cells that are highlighted in pale yellow indicate required information.  Cells highlighted in blue
        indicate optional information that may be entered if available. Cells highlighted in red are for
        SCC use only.
    4.   The file has been protected such that data can only be entered in yellow or blue cells.  No other
        cells can be changed.


ISMC Participant Lab  SOP                     B-122

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To complete the electronic results synopsis data deliverable:

    1.  Record information into a separate worksheet page for each sample tested.
    2.  Record requested information or data in all required cells (pale yellow).
    3.  Record requested information or data in optional cells (blue) if data is available.
    4.  Check entered data against hardcopy bench sheets to ensure accuracy.
    5.  Save the file onto the diskette provided. Do not change the file name.
    6.  Submit the diskette with the other data reporting deliverables by the due date to:

                              DynCorp I&ET, Sample Control Center
                              6101 Stevenson Avenue
                              Alexandria, VA 22304
                              Phone:(703)461-2064
                              Fax:(703)461-8056
                              Attention: Robert Brent

5.0 Coolers

All coolers will include a prepaid return FedEx label for returning the cooler to the referee laboratory.
Fill out the sender portion of the FedEx label and place it on the cooler.  Coolers will be returned by 2
Day shipment, as already marked on the prepared label.  Coolers must be returned to the referee
laboratory within 7 days of sample receipt. Please empty and wipe out the cooler prior to returning it.
ISMC Participant Lab SOP                     B-123

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        Appendix A:  Instructions for Reconstitution of Liquid Ampule Sample for
          Inland Silverside, Menidia beryllina, Larval Survival and Growth Test

For each sample, three liquid ampules will be received (marked with the sample number
followed by an "A", "B", or "C"). The three containers shall be reconstituted as described below
to mimic the sample shipment schedule for effluent samples. The container marked "A" shall be
reconstituted on the day of test initiation (Day 0) and used for renewal on Day 1. The container
marked "B" shall be reconstituted on Day 2 and used for renewals on Day 2 and Day 3. The
container marked "C" shall be reconstituted on Day 4 and used for renewals on Day 4, Day 5,
and Day 6. Follow the directions below for the reconstitution of each sample ampule.

1.     Volumetrically add 100 mL of the liquid ampule sample to approximately 1L of synthetic
       seawater. The synthetic seawater should be prepared to a salinity  of 25%o (2%o) using
       Bioassay Grade Forty Fathoms artificial sea salts and MILLIPORE MTLLI-Q or
       equivalent deionized water according to Section 7 of the methods  manuals.

2.     Mix by swirling or gently shaking.

3.     Bring final volume to 21L (measured using volumetric glassware) with synthetic
       seawater dilution water.

4.     Mix again by swirling and gently shaking.

5.     Place reconstituted sample in a plastic container of appropriate volume. A container that
       minimizes head space (i.e. cubitainer) is recommended.

6.     This 21L sample is the whole volume reconstituted sample and should be treated as a
       typical effluent sample.  It should be used directly for the 100% effluent test
       concentration and diluted appropriately to prepare other test concentrations (50%, 25%,
       12.5%, and 6.25%).

7.     Use the reconstituted sample for test initiation and Day 1 test renewal.  Store sample at
       4C.

8.     Perform the Inland Silverside, Menidia beryllina, Larval Survival  and Growth Test as
       described in the SOW for this study and the methods manuals.

9.     Follow Steps 1  through 6 with each sample container to prepare the reconstituted sample
       on Day 2 and Day 4 for subsequent daily renewals.
ISMC Participant Lab SOP                   B-124

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Appendix B: EPA Traffic Report
^EPA
United States
Environmental Protection Agency
Washington, DC 20460
WET Interlaboratory Variability Study Traffic Report
USEPA ENGINEERING AND ANALYSIS DIVISION
SAMPLE CONTROL CENTER
Referee Laboratory Information
Name:
Address:
City:
State:

Sampler name:
EPISODE NO:
SAMPLE NO:
DynCorp - Sample Control Center
Fax completed form immediately 6101 Stevenson Avenue
upon completion, and include Alexandria, VA 22304
hardcopy in final data report to: phone: (703) 461 -21 00
fax: (703)461-8056
Participant Lab Shipping Information
Lab Name:
Address:
City:
State:
Airbill no:
Date shipped:
SAMPLE COLLECTION /
Requested Information
1.
2.
3.
4.
5.
6.
7.
8.
Sample use description
Sample collection/receipt date
Sample collection/receipt time
Sampler / recipient signature
PH
Temperature
Conductivity (freshwater methods) / Salinity
(marine methods)
Dissolved Oxygen concentration
FOR PARTICIPANT LAB USE ONLY
Received by:
Sample condition on receipt:
RECEIPT INFORMATION
Pre-Shipment
I 	 (initiation I 	
renewal 1 I 	 Irenewal 2







Post-Shipment








REQUESTED ANALYSES
9.
I 	 I Pimephales promelas Acute
I 	 I Pimephales promelas Chronic
I 	 I Ceriodaphnia dubia Acute
I 	 I Ceriodaphnia dubia Chronic
I 	 I Selenastrum capricornutum Chronic
I 	 I Menidia beryllina Acute
I 	 I Menidia beryllina Chronic
I 	 I Holmesmysis costata Acute
I 	 I Champia parvula Chronic

I 	 I Cyprinodon variegatus Acute
I 	 I Cyprinodon variegatus Chronic
I 	 I Mysidopsis bahia Chronic


ISMC Participant Lab SOP
B-125

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              Appendix C:




List of Referee and Participant Laboratories

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Referee Laboratories Involved in the WET Variability Study

EA Engineering, Science, and Technology, Inc.
Ogden Environmental and Energy Services, Inc.
MEC Analytical, Inc.
EnviroSystems, Inc.

Participant Laboratories Involved in the WET Variability Study

Analytical Environmental Testing, Inc.
Analytical Services, Inc.
Aqua Survey, Inc.
AQUA-Science
Aquatech Environmental Services, Inc.
Aquatic Bioassay Consulting Laboratories, Inc.
Aquatic Consulting & Testing, Inc.
Beckmar Environmental Laboratory
Bio-Aquatic Testing, Inc.
Biological Monitoring, Inc.
Block Environmental Services, Inc.
Burlington Research, Inc.
C-K Associates, Inc.
Central Virginia Laboratories & Consultants, Inc.
CH2M Hill
Chadwick & Associates, Inc.
City & County of Honolulu, Water Quality Laboratory
City of Phoenix Water Services Department Laboratory
City of San Diego,  Marine Biology Laboratory
City of San Jose, Environmental Services Department Laboratory
Coastal Bioanalysts, Inc.
Cosper Environmental Services, Inc.
County Sanitation Districts of Los Angeles, San Jose Creek Water Quality Laboratory
Eastman Chemical  Company
EnviroData Group, LLC
Environmental Science & Engineering, Inc.
EnviroScience, Inc.
EnviroSystems, Inc.
ETT Environmental, Inc.
EVS Environment Consultants
Global Environmental  Consulting, LLC
Hampton Roads Sanitation District
Hydrosphere Research
King County Environmental Lab
Law Engineering & Environmental Services,  Inc.

                                          C-l

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Participant Laboratories Involved in the WET Variability Study (continued)

MEC Analytical Systems, Inc.- Carlsbad, CA
MEC Analytical Systems, Inc.- Tiburon, CA
Metro Wastewater Reclamation District - Denver
Metropolitan Water Reclamation District of Greater Chicago
NEORSD Analytical Services
New England Bioassay, Inc.
Northshore Sanitation District
Ogden Environmental & Energy Services Company, Inc. - Fife, WA
Ogden Environmental & Energy Services Company, Inc. - San Diego, CA
Pacific EcoRisk
Pima County Wastewater Management Department - Ina Road Water Pollution Control Facility
QC Laboratories, Inc
Research, Environmental & Industrial (REI) Consultants, Inc.
Shealy Environmental Services, Inc.
Tetra Tech, Inc.
The ADVENT Group, Inc.
The SeaCrest Group
Toxikon Corporation
ToxScan, Inc.
Whole Effluent Toxicity Testing Laboratories, Inc.
                                         C-2

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      Appendix D:
Preliminary Testing Results

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D.I     Preliminary Testing for Ceriodaphnia Acute and Chronic Test Methods
D. 1.1 Reference Toxicant Sample Type

The reference toxicant sample type was composed of moderately hard synthetic freshwater prepared
according to Section 7 of the WET method manuals (USEPA, 1994a) and spiked with KC1.  The spiking
level for this sample type was targeted to produce a test result (LC50 for the Ceriodaphnia acute test and
IC25 for the Ceriodaphnia chronic test) of 50% sample.  Spiking levels for Part 2 preliminary testing
were selected based on historical testing at the referee laboratory for the Ceriodaphnia acute test method
and from an initial Part 2 range-finding test for the  Ceriodaphnia chronic test method.  Tables D1 and D2
show the results from preliminary testing for Ceriodaphnia acute and chronic test methods, respectively.
For the Ceriodaphnia acute test method, Part 2 testing resulted in an LC50 of 424 mg KC1/L. Based on
this result, the spiking level for Part 4 testing was increased to 850 mg KC1/L (approximately 424 / the
target result of 50% sample). Part 4 Ceriodaphnia  acute testing produced an LC50 of 574 mg KC1/L or
67.6% sample.  The spiking  level for the interlaboratory testing phase (1000 mg  KC1/L) was based on an
average of the LC50 results obtained in Part 2 and Part 4 testing (approximately the average result of 500
/ the target result of 50% sample).  Referee laboratory testing of this interlaboratory sample yielded
LC50s of 40.6% and 34.4%  sample.

Table Dl. Results from Ceriodaphnia acute preliminary testing.
Sample type
Reference
toxicant
Effluent
Receiving
water
Part3
2
4
IL
2
3
4
IL
2
3
4
IL
Concentrations tested
(mg KC1/L)
37.5, 75, 150, 300, 600
53, 106, 212.5, 425, 850
62.5, 125, 250, 500, 1000
131,262.5,525, 1050,2100
131,262.5,525, 1050,2100
167.5, 335, 670, 1340, 2680
167.5, 335, 670, 1340, 2680
106, 212, 424, 848, 1696
106, 212, 424, 848, 1696
125, 250, 500, 1000, 2000
112.5,225,450,900, 1800
NOAEC
(mg KC1/L)
300
425
250
525
525
670
335
424
424
250
450
LC50
(mg KC1/L)
424
574
406
651
689
948
670
526
487
365
554
LC50
(% sample)
70.7
67.6
40.6 & 34.4b
31.0
32.8
35.4
25.0
31.0
28.7
18.3
30.8
a Preliminary testing Parts 1-4 and referee laboratory testing during the interlaboratory testing phase (IL). Part 1 preliminary
testing was conducted using only the chronic method, and Part 3 was conducted using only the acute method.
b The referee laboratory tested two reference toxicant samples due to a sample distribution error (see Section 6.4 in the main
body of this report).
                                               D-l

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Part 2 preliminary testing for the Ceriodaphnia chronic test method produced IC25 values of 323 mg
KC1/L and 138 mg KC1/L in two separate tests. The referee laboratory selected a Part 4 spiking level of
650 mg KC1/L so that the IC25 would potentially fall between 50% (if the true IC25 was closer to 323 mg
KC1/L) and 25% sample (if the true IC25 was closer to 138 mg KC1/L).  Results of this test revealed an
IC25 of 132 mg KC1/L.  The referee laboratory then repeated the Part 4 test with a slightly lower spiking
level. This test produced an IC25 of 134 mg KC1/L.  Since three consecutive tests produced IC25 values
between 132 and 138 mg KC1/L, the referee laboratory selected a final spiking level of 270 mg KC1/L
(the average result of 135 / the target result of 50% sample) to achieve the target result of 50% sample in
interlaboratory testing.  Unfortunately, this spiking level did not produce the desired effect during
interlaboratory testing.  The resulting sample was marginally toxic and produced toxic results in  only
some laboratories  (see Section 5.3 in the main body of this report).
Table D2. Results from Ceriodaphnia chronic preliminary testing.
Sample
type
Reference
toxicant
Effluent
Receiving
water
Part3
2b
2
4
4C
IL
ld
2
4
IL
ld
2
4
4C
IL
Concentrations tested
(mg KC1/L)
56, 100, 180, 320, 560
43.75, 87.5, 175, 350, 700
40.5,81, 162.5,325,650
31.25,62.5, 125,250,500
17, 34, 67.5, 135, 270
6.25, 12.5, 25, 50, 100
87.5, 175, 350, 700, 1400
106.3, 212.5, 425, 850, 1700
131,263,525, 1050,2100
6.25, 12.5, 25, 50, 100
75, 150, 300, 600, 1200
92.5, 185, 370, 740, 1480
62.5, 125, 250, 500, 1000
75, 150, 300, 600, 1200
Survival
NOEC
(mg KC1/L)
320
175
325
125
270
100
350
425
263
100
300
185
250
300
Reproduction
NOEC
(mg KC1/L)
320
87.5
81
125
270
100
350
425
263
100
300
92.5
250
300
IC25
(mg KC1/L)
323
138
132
134
>270
-
424
538
389
-
368
114
342
372
IC25
(% sample)
57.7
19.8
20.3
26.9
>100
>100
30.3
31.6
18.5
>100
30.7
7.7
34.2
31.3
a Preliminary testing Parts 1-4 and referee laboratory testing during the interlaboratory testing phase (IL).  Part 1 preliminary
testing was conducted using only the chronic method, and Part 3 was conducted using only the acute method.
b An initial Part 2 range-finding test was conducted due to a lack of historical referee laboratory data for this method and
toxicant.
c Part 4 testing was repeated to confirm spiking levels.
d Part 1 testing was conducted on unspiked samples. The units for test concentrations in Part 1 were percent sample.
                                                D-2

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D.I.2 Effluent Sample Type

The effluent sample type was composed of a municipal wastewater treatment plant effluent spiked with
KC1.  The referee laboratory (EA Engineering, Science and Technology, Inc.) collected the effluent from
a municipal wastewater treatment plant that is designed to treat 180 mgd, is able to handle peak flows of
400 mgd, and currently treats 140 to 150 mgd. The facility employs tertiary treatment for biological
nutrient removal including single-stage nitrification/denitrification, sand filtration,
chlorination/dechlorination, and anaerobic digestion.  The effluent source was selected based on historical
consistency in chemical and toxicological testing conducted by the referee  laboratory. This same effluent
source was used for all freshwater methods, the Mysidopsis chronic test method, and the sheepshead acute
and chronic test methods. Water chemistry of the effluent on each sample collection date is shown in
Table D3. More detailed chemical analyses (including total dissolved solids, total suspended solids, total
organic  carbon, biological oxygen demand, and chemical oxygen demand)  were performed at the
beginning and end of preliminary testing to better characterize the sample source. Historically, the
effluent has demonstrated low to no acute or chronic toxicity to freshwater organisms. Part 1 preliminary
testing confirmed this consistency (Table D2).

For Part 2 of preliminary testing, the effluent sample was spiked with KC1 to provide a consistently toxic
sample.  The spiking level for this sample type was targeted to produce a test result (LC50 for the
Ceriodaphnia acute test and IC25 for the Ceriodaphnia chronic test) of 25% sample. Part 2 preliminary
testing for the Ceriodaphnia acute test method produced an LC50 of 651 mg KC1 /L. This sample was
held for 7 days at 4C and then retested for Part 3 preliminary testing. After holding, the sample
produced an LC50 of 689 mg KC1/L, which represents only a 5.8% change from the original Part 2 test
result. The spiking level for Part 4 preliminary testing (2680 mg KC1/L) was based on an average of the
LC50 results obtained in Part 2 and Part 3 testing (the average result of 670 /the target result of 25%
sample). Part 4 testing produced an LC50  of 948 mg KC1/L or 35.4% sample. The same spiking level
was used for the interlaboratory testing phase.  Referee laboratory testing of the interlaboratory sample
produced an LC50 of 25.0% sample.

For the Ceriodaphnia chronic test method, Part 1 testing of the unspiked effluent resulted in no toxicity
(IC25 > 100% sample). Spiking of the effluent sample in Part 2 testing produced an IC25 of 424 mg
KC1/L.  Based on this result, the spiking level was increased to 1700 mg KC1/L (approximately 424 / the
target result of 25% sample) for Part 4 testing. Part 4 testing produced an IC25 of 538 mg KC1/L or
31.6% sample.  Based on this result, the spiking level used for interlaboratory testing was further
increased to 2100 mg KC1/L (approximately 538 / the target result of 25% sample). The interlaboratory
sample yielded an IC25 of 18.5% sample in referee laboratory testing.
                                              D-3

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Table D3.  Water chemistry of effluent sample source for freshwater test methods.
Parameters
Alkalinity
(mg/L as CaCO3)
Hardness (mg/L as
CaC03)
Conductivity (wS/cm)
pH
Temperature (C)
Total residual chlorine
(mg/L)
Dissolved oxygen
(mg/L)
Total ammonia (mg/L)
Total dissolved solids
(mg/L)
Total suspended solids
(mg/L)
Total organic carbon
(mg/L)
Biological oxygen
demand (mg/L)
Chemical oxygen
demand (mg/L)
Sampling date3
08/11/99
76
148
828
7.4
28.1
<0.01
7.5
1.17
420
7.0
21.4
3.8
59.9
08/19/99
62
160
933
7.1
26
<0.01
7.2
0.284
-
-
-
-
-
08/24/99
68
160
894
7.2
25.7
<0.01
7.8
0.122
-
-
-
-
-
09/07/99
78
172
909
7.2
23.9
<0.01
7.6
0.191
-
-
-
-
-
09/20/99
68
156
728
7.5
23.3
<0.01
8.1
-
-
-
-
-
-
11/29/99
54
152
695
6.8
12.9
<0.01
8.9
0.0371
-
-
-
-
-
12/07/99
38
148
675
6.6
17.6
-
7.7
0.024
-
-
-
-
-
12/10/99
70
128
766
7.3
17.0
-
8.9
0.079
-
-
-
-
-
01/06/00
-
-
798
7.1
15.6
<0.01
9.1
-
-
-
-
-
-
01/24/00
76
156
959
7.7
11.9
-
9.5
-
-
-
-
-
-
02/08/00
82
172
933
7.4
10.9
<0.01
10.3
1.88
626
<2.5
26.6
4.8
77.2
1"-" indicates that the parameter was not tested on the given sampling date.
                                                                  D-4

-------
D. 1.3  Receiving Water Sample Type

The receiving water sample type was composed of a natural surface freshwater spiked with KC1.  The
referee laboratory (EA Engineering, Science and Technology, Inc.) collected the receiving water from the
Gunpowder River, in Baltimore County, Maryland. Initial samples were collected from a location near
Bunker Hill Road.  Results of preliminary testing indicated that these unspiked samples occasionally
showed toxicity to fathead minnows and Selenastrum capricornutum.  To avoid the potential problems
associated with intermittent ambient toxicity, subsequent freshwater samples were collected from a new
location (near Falls Road), upstream from potential sources of non-point source runoff. The results from
toxicity tests conducted with the Falls Road samples indicated no acute or chronic toxicity to
Ceriodaphnia dubia (Table D2) or other freshwater species. Water chemistry of the receiving water is
shown in Table D4 for each sample collection date. More detailed chemical analyses (including total
dissolved solids, total suspended solids, total organic carbon, biological oxygen demand, and chemical
oxygen demand) were performed at the beginning and end of preliminary testing to better characterize the
sample source.

The receiving water sample was spiked with KC1 to provide a consistently toxic sample. The spiking
level for this sample type was targeted to provide a test result (LC50 for the Ceriodaphnia acute test and
IC25 for the Ceriodaphnia chronic test) of 25% sample.  Part 2 preliminary testing for the  Ceriodaphnia
acute test method produced an LC50 of 526 mg KC1/L.  This sample was held for 7 days at 4C and then
retested for Part 3  preliminary testing. After holding, the sample produced an LC50 of 487 mg KC1/L,
which represents only a 7.4% change from the original Part 2 test result. For Part 4 preliminary testing,
the referee laboratory increased the spiking level to 2000 mg KC1/L to better achieve the target result of
25% sample.  Part 4 testing produced an LC50 of 365 mg KC1/L or 18.3% sample. The spiking level
used for interlaboratory testing (1800 mg KC1/L) was based on an average of the LC50 results obtained in
Part 2, 3, and 4 testing (approximately the average result of 460 / the target result of 25% sample).
Referee laboratory testing of this sample produced an LC50 of 30.8% sample during the interlaboratory
testing phase.

For the Ceriodaphnia chronic test method, Part  1 testing of the unspiked receiving water resulted in no
toxicity (IC25 > 100% sample).  Spiking  of the receiving water sample in Part 2 testing produced an IC25
of 368 mg KC1/L. Based on this result, the spiking level was increased to 1480 mg KC1/L for Part 4
testing (approximately 368 / the target result of 25% sample). Part 4 testing produced an IC25 of 114 mg
KC1/L or 7.7% sample.  Due to the discrepancy between the results of Part 2 and Part 4 testing, Part 4
testing was repeated. Repeated Part 4 testing produced an IC25 of 342 mg KC1/L, which was consistent
with Part 2 testing results.  Final spiking levels were based on these tests and set at 1200 mg KC1/L for
the interlaboratory testing phase. Referee laboratory testing of the interlaboratory sample produced an
IC25 of 31.3% sample.
                                              D-5

-------
Table D4.  Water chemistry of receiving water sample source for freshwater test methods.
Parameters
Alkalinity
(mg/L as CaCO3)
Hardness (mg/L as CaCO3)
Conductivity CuS/cm)
pH
Temperature (C)
Total residual chlorine
(mg/L)
Dissolved oxygen (mg/L)
Total ammonia (mg/L)
Total dissolved solids
(mg/L)
Total suspended solids
(mg/L)
Total organic carbon
(mg/L)
Biological oxygen demand
(mg/L)
Chemical oxygen demand
(mg/L)
Sampling date3
06/24/99
30
80
183
7.7
14.1
<0.01
10.4
0.056
94.5
5.5
1.3
<1.0
12.8
07/07/99
28
60
154
7.9
15.0
<0.01
9.6
0.096
-
-
-
-
-
07/14/99
30
60
148
6.8
16.3
<0.01
10.3
-
-
-
-
-
-
07/28/99
24
60
159
7.9
18.3
<0.01
9.8
-
-
-
-
-
-
08/05/99
28
48
161
7.9
16.6
<0.01
10.7
0.092
-
-
-
-
-
08/20/99
34
44
155
7.3
16.5
<0.01
10.9
0.307
-
-
-
-
-
09/21/99
34
60
157
7.5
14.6
<0.01
10.0
-
-
-
-
-
-
12/21/99
26
36
139
8.3
7.8
-
12.6
-
-
-
-
-
-
01/31/00
28
56
124
7.8
2.1
-
14.6
-
-
-
-
-
-
 "-" indicates that the parameter was not tested on the given sampling date.
                                                                 D-6

-------
D.2     Preliminary Testing for Fathead Acute and Chronic Test Methods
D.2.1   Reference Toxicant Sample Type

The reference toxicant sample type was composed of moderately hard synthetic freshwater prepared
according to Section 7 of the WET method manuals (USEPA, 1994a) and spiked with KC1. The spiking
level for this sample type was targeted to produce a test result (LC50 for the fathead acute test and IC25
for the fathead chronic test) of 50% sample. Tables D5 and D6 show the results from preliminary testing
using fathead acute  and chronic test methods, respectively. Spiking levels selected for Part 2 preliminary
testing resulted in an LC50 of 915 mg KC1/L for the fathead acute test method. Based  on this result, the
spiking level for Part 4 testing was increased to 1830 mg KC1/L (915 / the target result of 50% sample).
Part 4 fathead acute testing produced an LC50 of 1167 mg KC1/L (63.8% sample).  Spiking levels were
further increased to  2200 mg KC1/L for the interlaboratory testing phase to better achieve the target result
of 50% sample.  Referee laboratory testing of the interlaboratory sample produced an LC50 of 42.4%
sample.

Part 2 preliminary testing using the fathead chronic test method produced an IC25  of 545 mg KC1/L.
Based on this result, the spiking level for Part 4 testing was decreased to 1090 mg KC1 /L (545 / the target
result of 50% sample). Part 4 testing yielded an IC25 of 610 mg KC1/L. Based on Part 4 testing results,
the spiking level for the interlaboratory sample was further increased to 1220 mg KC1/L (610 / the target
result of 50% sample). Referee laboratory testing of the interlaboratory sample yielded an  IC25 of 63.3%
sample.

Table D5. Results  from fathead acute preliminary testing.
Sample type
Reference toxicant
Effluent
Receiving water
Part3
2
4
IL
2
3
4
IL
2
o
5
4
IL
Concentrations tested
(mg KC1/L)
104.9,209.8,419.5,839, 1678
114.5,229,457.5,915, 1830
138,275,550, 1100,2200
300, 600, 1200, 2400, 4800
300, 600, 1200, 2400, 4800
350, 700, 1400, 2800, 5600
333, 666, 1332, 2664, 5328
250, 500, 1000, 2000, 4000
250, 500, 1000, 2000, 4000
300, 600, 1200, 2400, 4800
313,625, 1250,2500,5000
NOAEC
(mg KC1/L)
419.5
915
550
600
600
700
666
1000
500
600
625
LC50
(mg KC1/L)
915
1167
924
1308
1356
990
1028
1270
1168
1256
985
LC50
(% sample)
54.5
63.8
42.4
27.3
28.3
17.7
19.3
31.7
29.2
26.2
19.7
a Preliminary testing Parts 1-4 and referee laboratory testing during the interlaboratory testing phase (IL). Part 1 preliminary
testing was conducted using only the chronic method, and Part 3 was conducted using only the acute method.
                                               D-7

-------
Table D6.  Results from fathead chronic preliminary testing.
Sample type
Reference
toxicant
Effluent
Receiving
water
Part3
2
4
IL
lb
2
4
IL
i b, c
jb, d
2
4
IL
Concentrations tested
(mg/L KC1)
88.4, 176.8, 353.5, 707, 1414
68, 136, 272.5, 545, 1090
76, 153, 305, 610, 1220
6.25, 12.5, 25, 50, 100
144,287.5,575, 1150,2300
181.3, 362.5, 725, 1450, 2900
225, 450, 900, 1800, 3600
6.25, 12.5, 25, 50, 100
6.25, 12.5, 25, 50, 100
136, 272.5, 545, 1090, 2180
140,280,560, 1120,2240
150, 300, 600, 1200, 2400
Survival
NOEC
(mg/L KC1)
353.5
545
610
100
575
725
900
100
100
545
560
600
Reproduction
NOEC
(mg/L KC1)
353.5
545
610
100
575
725
900
50
100
545
560
600
IC25
(mg/L
KC1)
545
610
772
-
721
901
968
-
-
566
606
708
IC25
(% sample)
38.5
56.0
63.3
>100
31.4
31.1
26.9
5.6
>100
26.0
27.0
29.5
a Preliminary testing Parts 1-4 and referee laboratory testing during the interlaboratory testing phase (IL). Part 1 preliminary
testing was conducted using only the chronic method, and Part 3 was conducted using only the acute method.
b Part 1 testing was conducted on unspiked samples. The units for test concentrations in Part 1 were percent sample.
c Receiving water sample was collected from Bunker Hill Road site.
d Receiving water sample was collected from Falls Road site.
D.2.2   Effluent Sample Type

The effluent sample source used for the fathead acute and chronic test methods was the same as described
in Section D. 1.2.  This effluent sample was spiked with KC1 to provide a consistently toxic sample. The
spiking level for this sample type was targeted to produce a test result (LC50 for the fathead acute test and
IC25 for the fathead chronic test) of 25% sample. Part 2 preliminary testing for the fathead acute test
method produced an LC50 of 1308 mg KC1/L. This sample was held for 7 days at 4C and then retested
for Part 3 preliminary testing.  After holding, the sample produced an LC50 of 1356 mg KC1/L, which
represents only a 3.7% change from the original Part 2 test result.  For Part 4 preliminary testing, the
referee laboratory increased the spiking level to 5600 mg KC1/L to better achieve the target effect level of
25% sample. This test produced an LC50 of 990 mg KC1/L or 17.7% sample; however, less than 90%
(65%) survival was experienced in the control.  Since Part 4 testing was unreliable, the spiking level for
interlaboratory testing was based on an average of Part 2 and Part 3 testing results and set at 5328 mg
KC1/L (the average result of 1332 / the target result of 25% sample).  This interlaboratory sample yielded
an LC50 of 19.3% sample in referee laboratory testing.
                                               D-8

-------
For the fathead chronic test method, Part 1 testing of the unspiked effluent resulted in no toxicity (IC25 >
100% sample). Spiking of the effluent sample in Part 2 testing produced an IC25 of 721 mg KC1/L.
Based on this result, the spiking level was increased to 2900 mg KC1/L for Part 4 testing (approximately
721 /the target result of 25% sample).  Part 4 testing produced an IC25 of 901 mg KC1/L or 31.1%
sample. Based on Part 4 testing, the spiking level for interlaboratory testing was further increased to 3600
mg KC1/L (approximately  901 / the target result of 25% sample).  This interlaboratory sample yielded an
IC25 of 26.9% sample in referee laboratory testing.

D.2.3   Receiving Water Sample Type

The receiving water sample source used for the fathead acute and chronic test methods was the same as
described in Section D. 1.3. The receiving water sample was spiked with KC1 to provide a consistently
toxic sample. The spiking  level for this sample type was targeted to provide a test result (LC50 for the
fathead acute test and IC25 for the fathead chronic test) of 25% sample.  Part 2 preliminary testing for the
fathead acute test method produced an LC50 of 1270 mg KC1/L. This sample was held  for 7 days at 4C
and then retested for Part 3 preliminary testing. After holding, the sample produced an LC50 of 1168 mg
KC1/L, which represented only a 8.7% change from the original Part 2 test result. The spiking level for
Part 4 preliminary testing (4800 mg KC1/L) was based on an average of the LC50 results obtained in Part
2 and Part 3 testing (approximately the average result of 1219 / the target result of 25%  sample). Part 4
testing produced an LC50 of 1256 mg KC1/L or 26.2% sample. Based on this result, the spiking level
was further increased to 5000 mg KC1/L (approximately 1256 / the target result of 25%  sample) for the
interlaboratory testing phase. Referee laboratory testing of the interlaboratory sample yielded an LC50 of
19.7% sample.

For the fathead chronic test method, Part 1 testing of the unspiked receiving water collected from the
Bunker Hill Road site indicated toxicity (IC25 of 5.6% sample).  Following this test, the referee
laboratory moved the receiving water collection site farther upstream to the Falls Road site.  Part 1 testing
of receiving water from the new location revealed no toxicity (IC25 >100% sample).  Part 2 testing of the
spiked receiving water produced an IC25 of 566 mg KC1/L. Based on this result, the spiking level was
increased to 2240 mg KC1/L for Part 4 testing (approximately 566 / the target result of 25% sample). Part
4 testing produced an IC25 of 606 mg KC1/L or 27.0% sample. Based on this result, the final spiking
level for interlaboratory testing was increased to 2400 mg KC1/L (approximately 606 / the target result of
25% sample).  This interlaboratory sample yielded an IC25 of 29.5% sample in referee laboratory testing.
                                              D-9

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D.3    Preliminary Testing for the Selenastrum Chronic Test Method

D.3.1  Reference Toxicant Sample Type

The reference toxicant sample type for the Selenastrum chronic test method was composed of deionized
water spiked with KC1. The spiking level for this sample type was targeted to produce an IC50 of 38%
sample (see Section D.9). Table D7 shows the results from preliminary testing for the Selenastrum
chronic test method. Spiking levels for Part 2 preliminary testing were based on the results of an initial
Part 2 range-finding test. The range-finding test resulted in an IC50 of 925 mg KC1/L.  Spiking levels
selected for Part 2 testing resulted in an IC50 of 2925 mg KC1/L for the Selenastrum chronic test method.
Based on this result, the spiking level for Part 4 testing was increased to 7888 mg KC1/L. Part 4 testing
with EDTA produced an IC50 of 1713 mg KC1/L (or 57.1% sample) and Part 4 testing without EDTA
produced an IC50 of 2943 mg KC1/L (or 98.1% sample). Due to the discrepancy between Part 4  results
with EDTA and Part 2 results with EDTA, Part 4 testing was repeated. In additional Part 4 testing, IC50s
of 1808 mg KC1/L (or 22.9% sample) and 462 mg KC1/L (or 5.9% sample) were produced with EDTA
and without EDTA, respectively.  The spiking level for interlaboratory testing was set at 5655 mg KC1/L
(average result of 2149 / the target result of 38% sample) based on an average of Part 2 and Part 4 testing
with EDTA. Referee laboratory testing of the interlaboratory sample yielded IC50s of 35.5% sample and
37.6% sample with and without EDTA, respectively.

D.3.2  Effluent Sample Type

The effluent sample source used for the Selenastrum chronic test method was the same as described in
Section D.I.2. Part 1 testing of the unspiked effluent resulted in no toxicity (IC50 > 100% sample). The
effluent sample was then spiked with KC1 to provide a consistently toxic sample. The spiking level for
this sample was targeted to produce a test result (IC50) of 38% sample. Part 2 preliminary testing for the
Selenastrum chronic test method produced an IC50 of 4383 mg KC1/L. This sample was held for 7 days
at 4C and then retested for Part 3 preliminary testing. After holding, the sample produced an IC50 of
5143 mg KC1/L, which represents a 17% difference from the initial Part 2 test result. Based on Part 2
testing results, the spiking level for Part 4 testing was set at 11540 mg KC1/L (approximately 4383 / the
target result of 38% sample).  Part 4 testing resulted in an IC50 of 4609 mg KC1/L or 39.9% sample with
EDTA and an IC50 of 4821 mg KC1/L or 41.8% sample without EDTA. Since Part 4 testing results were
very close to the 38% sample target, the same spiking level was maintained for interlaboratory testing.
Referee laboratory results during interlaboratory testing were 29.9% sample with EDTA and 19.8%
sample without EDTA.
                                             D-10

-------
Table D7.  Results from Selenastrum chronic preliminary testing.
Sample type
Reference toxicant
Effluent
Receiving water
Part3
2b
2
4w/EDTAc
4 w/EDTA
4w/oEDTA
4 w/o EDTAC
IL w/ EDTA
IL w/o EDTA
ld
2 w/EDTA
3 w/EDTA
4 w/EDTA
4 w/o EDTA
IL w/ EDTA
IL w/o EDTA
ld-e
I4f
2 w/EDTA
2w/EDTAg
2 w/ EDTAg
3 w/ EDTA
4 w/ EDTA
4 w/o EDTA
IL w/ EDTA
IL w/o EDTA
Concentrations tested
(mg KC1/L)
100, 1000, 10000, 30000, 60000
375, 750, 1500, 3000, 6000
188, 375, 750, 1500, 3000
493, 986, 1972, 3944, 7888
188, 375, 750, 1500, 3000
493, 986, 1972, 3944, 7888
353, 707, 1414, 2828, 5655
353, 707, 1414, 2828, 5655
6.25, 12.5, 25, 50, 100
938, 1875, 3750, 7500, 15000
938, 1875, 3750, 7500, 15000
721, 1443,2885,5770, 11540
721, 1443,2885,5770, 11540
721, 1443,2885,5770, 11540
721, 1443,2885,5770, 11540
6.25, 12.5, 25, 50, 100
50, 100
225, 450, 900, 1800, 3600
1000, 2000, 4000, 8000, 16000
500, 1000, 2000, 4000, 8000
500, 1000, 2000, 4000, 8000
807, 1614, 3228, 6456, 12912
807, 1614, 3228, 6456, 12912
732, 1464,2928,5857, 11713
732, 1464,2928,5857, 11713
NOEC
(mg KC1/L)
100
375
375
986
187.5
<493
1414
1414
100
937.5
937.5
2885
2885
2885
1443
100
100
3600
1000
<500
1000
3228
1624
2928
2928
IC50
(mg KC1/L)
925
2925
1713
1808
2943
462
2007
2126
-
4383
5143
4609
4821
3502
2319
-
-
>3600
4885
4399
4928
4069
2098
3093
4201
IC50
(% sample)
1.5
48.7
57.1
22.9
98.1
5.9
35.5
37.6
>100
29.2
34.3
39.9
41.8
29.9
19.8
97
>100
>100
30.5
55.0
61.6
31.5
16.2
26.8
36.4
a Preliminary testing Parts 1-4 and referee laboratory testing during the interlaboratory testing phase (IL).
b An initial Part 2 rangefinding test was conducted due to a lack of historical referee laboratory data for this method and toxicant.
c Part 4 testing was repeated to confirm spiking levels.
d Part 1 testing was conducted on unspiked samples. The units for test concentrations in Part 1 were percent sample.
e Receiving water sample was collected from Bunker Hill Road site.
f Receiving water sample was collected from Falls Road site.
g Part 2 testing was repeated to confirm spiking levels.
                                                      D-ll

-------
D.3.3  Receiving Water Sample Type

The receiving water sample source used for the Selenastrum chronic test method was the same as
described in Section D.I.3.  Part 1 testing on the unspiked receiving water collected from the Bunker Hill
Road site indicated toxicity (IC50 of 97% sample). Following this test, the referee laboratory moved the
receiving water collection site farther upstream to the Falls Road site. Part 1 testing of receiving water
from this site revealed no toxicity (IC50 >100% sample).  The receiving water sample was then spiked
with KC1 to provide a consistently toxic sample.  The spiking level for this sample was targeted to
produce a test result (IC50) of 38% sample. Part 2 preliminary testing for the Selenastrum  chronic test
method produced an IC50 of >3600 mg KC1/L.  Since the test result was outside of the concentration
range tested, Part 2 testing was repeated. Two additional Part 2 tests produced ICSOs of 4885 and 4399
mg KC1/L.  The sample from the latter Part 2 test was held for 7 days at 4C and then retested for Part 3
preliminary testing.  After holding, the sample produced an IC50 of 4928 mg KC1/L, which represents
only a  12% difference from the original Part 2 test result.  Based on an average of results from the second
Part 2 test and Part 3 testing, the spiking level for Part 4 testing was set at 12912 mg KC1/L (average
result of 4906 / the target result of 38% sample).  Part 4 testing produced ICSOs of 4069 mg KC1/L (or
31.5% sample) and 2098 mg KC1/L (or 16.2% sample) with and without EDTA, respectively.  The
spiking level for interlaboratory testing was based on an average of results from Part 2 and Part 4 testing
and set at 11713 mg KC1/L (average result of 4451 / the target result of 38% sample). Referee laboratory
results during interlaboratory testing yielded IC50s of 26.8% and 36.4% sample for tests conducted with
and without EDTA, respectively.

D.4    Preliminary Testing for the Mysidopsis Chronic Test Method

D.4.1  Reference Toxicant Sample Type

The reference toxicant sample type was composed of synthetic seawater (prepared using bioassay grade
Forty Fathoms artificial sea salts added to deionized water) spiked with KC1. The spiking level for this
sample was targeted to produce a test result (IC25) of 50% sample.  Spiking levels for Part 2 preliminary
testing were selected based on historical testing at the referee laboratory for the Mysidopsis chronic test
method.  Table D8 shows the results from preliminary testing for the Mysidopsis chronic test method.
Part 2 preliminary testing resulted in an IC25  of 426 mg KC1/L.  Based on this result, the spiking level for
Part 4 testing was increased to 900 mg KC1/L (approximately 426 / the target result of 50% sample).  Part
4 testing resulted in an IC25 of 530 mg KC1/L (58.9% sample). Based on this result, the spiking level for
interlaboratory testing was further increased to 1200 mg KC1/L.  Referee laboratory testing of the
interlaboratory sample resulted in an IC25 of 36.4% sample.
                                             D-12

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D.4.2   Effluent Sample Type

The effluent sample source used for the Mysidopsis chronic test method was the same as described in
Section D. 1.2. The salinity of the effluent was adjusted to 25 ppt prior to the conduct of marine tests.
Part 1 testing on the unspiked effluent resulted in an IC25 of 64.4% sample. The effluent sample was
then spiked with KC1 to provide a consistently toxic sample.  The spiking level for this sample type was
targeted to produce a test result (IC25) of 25% sample. Part 2 preliminary testing for the Mysidopsis
chronic test method produced an IC25 of 486 mg KC1/L. This sample was held for 7 days at 4C and
then retested for Part 3 preliminary testing.  After holding, the sample produced an IC25 of 420 mg
KC1/L, which represented a 14% difference from the original Part 2 test result. Based on the result of Part
2 testing, the  spiking level for Part 4 testing was increased to 1960 mg KC1/L  (approximately 486 / the
target result of 25% sample).  Part 4 testing produced an IC25 of 521 mg KC1/L (26.6% sample).  Based
on an average of results from Part 2 and Part 4 testing, the spiking level for interlaboratory testing was
increased to 2000 mg KC1/L (approximately the average result of 504 / the target result of 25% sample).
Referee laboratory testing of the interlaboratory sample resulted in an IC25 of 29.9% sample.
Table D8.  Results from Mysidopsis chronic preliminary testing.
Sample type
Reference
toxicant
Effluent
Receiving
water
Part3
2
4
IL
fb
2
3
4
IL
fb
2
3
4
IL
Concentrations tested
(mg KC1/L)
45.25, 90.5, 181, 362, 724
56.3, 113,225,450,900
75, 150, 300, 600, 1200
6.25, 12.5, 25, 50, 100
90, 180, 360, 720, 1440
90, 180, 360, 720, 1440
122.5, 245, 490, 980, 1960
125, 250, 500, 1000, 2000
6.25, 12.5, 25, 50, 100
90, 180, 360, 720, 1440
90, 180, 360, 720, 1440
122.5, 245, 490, 980, 1960
150, 300, 600, 1200, 2400
Survival
NOEC
(mg KC1/L)
362
450
300
100
360
360
490
500
100
360
720
490
300
Growth
NOEC
(mg KC1/L)
362
450
300
50
360
360
490
500
50
360
360
490
300
IC25
(mg KC1/L)
426
530
437
-
486
420
521
598
-
486
634
608
564
IC25
(% sample)
58.8
58.9
36.4
64.4
33.7
29.2
26.6
29.9
88
33.8
44.0
31.0
23.5
a Preliminary testing Parts 1-4 and referee laboratory testing during the interlaboratory testing phase (IL).
b Part 1 testing was conducted on unspiked samples. The units for test concentrations in Part 1 were percent sample.
                                              D-13

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D.4.3  Receiving Water Sample Type

The receiving water sample type was composed of natural seawater spiked with KC1.  The referee
laboratory (EA Engineering, Science and Technology, Inc.) collected receiving water from Manasquan
Inlet, in Manasquan, Monmouth County, New Jersey.  Seawater from this location has historically been
non-toxic to the test species, and is currently used by EPA's Division of Environmental Science and
Assessment as dilution water for toxicity testing. Water chemistry of this receiving water on each sample
collection date is shown in Table D9.

Table D9.  Water chemistry of receiving water sample source for Mysidopsis chronic and
sheepshead acute and chronic test methods.
Parameters
Alkalinity (mg/L as CaCO3)
pH
Temperature (C)
Total residual chlorine (mg/L)
Dissolved oxygen (mg/L)
Salinity (ppt)
Copper Cwg/L)
Total ammonia (mg/L)
Total dissolved solids (mg/L)
Total suspended solids (mg/L)
Total organic carbon (mg/L)
Biological oxygen demand (mg/L)
Chemical oxygen demand (mg/L)
Sampling date3
11/03/99
98
8.0
3.5
<0.01
-
28.3
<10
0.980
33,300
10.5
<1.0
2.1
854
11/23/99
110
8.2
3.9
-
10.9
31.7
-
-
-
-
-
-
-
12/29/99
-
8.3
2.5
-
9.2
35
-
-
-
-
-
-
-
01/13/00
-
7.5
10.6
-
10.1
31.9
-
-
-
-
-
-
-
a"-" indicates that the parameter was not tested on the given sampling date.

The receiving water sample was filtered and adjusted to a salinity of 25 ppt prior to toxicity testing.  Part
1 testing on the unspiked receiving water sample indicated moderate toxicity (IC25 of 88% sample). The
receiving water sample was then spiked with KC1 to provide a consistently toxic sample. The spiking
level for this sample type was targeted to produce a test result (IC25) of 25% sample.  Part 2 preliminary
testing for the Mysidopsis chronic test method produced an IC25 of 486 mg KC1/L. This sample was held
for 7 days at 4C and then retested for Part 3 preliminary testing.  After holding, the sample produced an
IC25 of 634 mg KC1/L, which represented a 30% change from the initial Part 2 test result. For Part 4
preliminary testing, the spiking level was increased to 1960 mg  KC1/L (approximately 486 / the target
result of 25% sample) based on Part 2 testing results. Part 4 testing resulted in an IC25 of 608 mg KC1/L.
Based on this result, the spiking level was further increased to 2400 mg KC1/L (approximately 608 / the
                                             D-14

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target result of 25% sample) for the interlaboratory testing phase.  Referee laboratory testing of the
interlaboratory sample resulted in an IC25 of 23.5% sample.

D.5    Preliminary Testing for Sheepshead Acute and Chronic Test Methods

D.5.1  Reference Toxicant Sample Type

The reference toxicant sample type for the sheepshead acute and chronic test methods was composed of
synthetic seawater (prepared using bioassay grade Forty Fathoms artificial sea salts added to deionized
water) spiked with KC1.  The spiking level for this sample type was targeted to produce a test result
(LC50 for the sheepshead acute test and IC25  for the sheepshead chronic test) of 50% sample. Tables
DIG and Dl 1 show the preliminary testing results for sheepshead acute and chronic test methods,
respectively.  Spiking levels selected for Part 2 testing resulted in an LC50 of 1580 mg KC1/L for the
sheepshead acute test method. Based on this result, the spiking level for Part 4 testing was increased to
3160 mg KC1/L (1580 / the target result of 50% sample). Part 4 sheepshead acute testing produced an
LC50 of 1157 mg KC1/L or 36.6% sample.  The same spiking level was used for the interlaboratory
testing phase. Referee laboratory testing of the interlaboratory sample yielded an LC50 of 40.6% sample.

Part 2 preliminary testing for the sheepshead chronic method produced an IC25 of 1257 mg KC1/L. Based
on this result, the spiking level for Part 4 testing was increased to 2600 mg KC1/L (approximately 1257 /
the target result of 50% sample).  Results of this test revealed an IC25 of 1528 mg KC1/L. Based on Part
4 testing results, the spiking level for interlaboratory testing was further increased to 3000 mg KC1/L
(approximately 1528  /the target result of 50% sample).  Referee laboratory testing of the interlaboratory
sample yielded an IC25 of 54.3% sample.

D.5.2  Effluent Sample  Type

The effluent sample source used for the sheepshead acute and chronic test methods was the same as
described in Section D. 1.2. This effluent sample was adjusted to a salinity of 25 ppt and spiked with KC1
to provide a consistently toxic sample.  The spiking level for this sample type was targeted to produce a
test result (LC50 for the sheepshead acute test and IC25 for the sheepshead chronic test) of 25% sample.
Part 2 preliminary testing for the sheepshead acute test method produced an LC50 of 1329 mg KC1/L.
This sample was held for 7 days at 4C and then retested for Part 3 preliminary testing.  After holding, the
sample produced an LC50 of 1694 mg KC1/L, which represents a 27% change from the original Part 2
test result. For Part 4 preliminary testing, the referee laboratory increased the spiking level to 5200 mg
KC1/L. This test produced an LC50 of 1172 mg KC1/L or 22.5% sample. The same spiking level was
used for the interlaboratory testing phase. Referee laboratory testing of the interlaboratory sample yielded
an LC50 of 35.4% sample.
                                             D-15

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Table D10. Results from sheepshead acute preliminary testing.
Sample
type
Reference
toxicant
Effluent
Receiving
water
Part3
2
4
IL
2
3
4
IL
2
3
4
IL
Concentrations tested
(mg KC1/L)
155,310,620, 1240,2480
198,395,790, 1580,3160
198,395,790, 1580,3160
310,620, 1240,2480,4960
310,620, 1240,2480,4960
325, 650, 1300, 2600, 5200
325, 650, 1300, 2600, 5200
310,620, 1240,2480,4960
310,620, 1240,2480,4960
398,795, 1590,3180,6360
350, 700, 1400, 2800, 5600
NOAEC
(mg KC1/L)
1240
790
790
620
1240
650
1300
1240
620
795
700
LC50
(mg KC1/L)
1580
1157
1283
1329
1694
1172
1840
1580
1488
1247
1450
LC50
(% survival)
63.7
36.6
40.6
26.8
34.2
22.5
35.4
31.9
30.0
19.6
25.9
a Preliminary testing Parts 1-4 and referee laboratory testing during the interlaboratory testing phase (IL). Part 1 preliminary
testing was conducted using only the chronic method, and Part 3 was conducted using only the acute method.
Table Dll. Results from sheepshead chronic preliminary testing.
Sample
type
Reference
toxicant
Effluent
Receiving
water
Part3
2
4
IL
lb
2
4
IL
lb
2
4
IL
Concentrations tested
(mg KC1/L)
98, 195, 390, 780, 1560
163, 325, 650, 1300, 2600
188, 375, 750, 1500, 3000
6.25, 12.5, 25, 50, 100
250, 500, 1000, 2000, 4000
275,550, 1100,2200,4400
300, 600, 1200, 2400, 4800
6.25, 12.5, 25, 50, 100
195,390,780, 1560,3120
253, 505, 1010, 2020, 4040
275,550, 1100,2200,4400
Survival
NOEC
(mg KC1/L)
780
1300
1500
100
1000
1100
1200
100
780
1010
1100
Growth
NOEC
(mg KC1/L)
780
1300
1500
100
500
1100
1200
100
780
505
1100
IC25
(mg KC1/L)
1257
1528
1629
-
1158
1276
1406
-
1028
1172
1210
IC25
(% sample)
80.6
58.8
54.3
>100
28.9
29.0
29.3
>100
33.0
29.0
27.5
a Preliminary testing Parts 1-4 and referee laboratory testing during the interlaboratory testing phase (IL). Part 1 preliminary
testing was conducted using only the chronic method, and Part 3 was conducted using only the acute method.
b Part 1 testing was conducted on unspiked samples.  The units for test concentrations in Part 1 were percent sample.
                                                    D-16

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For the sheepshead chronic test method, Part 1 testing on the unspiked effluent resulted in no toxicity
(IC25 > 100% sample). Spiking of the effluent sample in Part 2 testing produced an IC25 of 1158 mg
KC1/L. Based on this result, the spiking level was increased to 4400 mg KC1/L for Part 4 preliminary
testing (approximately 1158 / the target result of 25% sample). Part 4 testing produced an IC25 of 1276
mg KC1/L or 29.0% sample. Based on the average of results from Part 2 and Part 4 testing, the spiking
level for interlaboratory testing was further increased to 4800 mg/L (approximately the average result of
1217 / the target result of 25% sample). Referee laboratory testing of the interlaboratory sample yielded
an IC25 of 29.3% sample.

D. 5.3  Receiving Water Sample Type

The receiving water sample source used for the sheepshead acute and chronic test methods was the same
as described in Section D.4.3.  The receiving water sample was filtered, adjusted to a salinity of 25 ppt,
and spiked with KC1 to provide a consistently toxic sample. The spiking level for this sample type was
targeted to provide a test result (LC50 for the sheepshead acute test and IC25 for the sheepshead chronic
test) of 25% sample. Part 2 preliminary testing for the sheepshead acute test method produced an LC50
of 1580 mg KC1/L.  This sample was held for 7 days at 4C and then retested for Part 3 preliminary
testing.  After holding, the sample produced an LC50 of 1488 mg KC1/L, which represented only a 5.8%
change from the original Part 2 test result. Based on the results of Part 2 testing, the spiking level for Part
4 testing was set at 6360 mg KC1/L (approximately 1580 / the target result of 25% sample).  Part 4 testing
resulted in an LC50 of 1247 mg KC1/L or 19.6% sample. Based on an average of Part 2 and Part 4
preliminary testing results, the spiking level was reduced to 5600 mg KC1/L (approximately the average
result of 1414 / the target result of 25% sample) for the interlaboratory testing phase. Referee laboratory
testing of the interlaboratory sample yielded an LC50 of 25.9% sample.

For the sheepshead chronic test method, Part 1 testing of the unspiked receiving water revealed no
toxicity (IC25  >100%  sample). Part 2 testing of the spiked receiving water produced an IC25 of 1028 mg
KC1/L. Based on this result, the spiking level was increased to 4040 mg KC1/L for Part 4 testing
(approximately 1028 / the target result of 25% sample).  Part 4 testing produced an IC25 of 1172 mg
KC1/L or 29.0% sample. Based on an average of test results from Part 2 and Part 4 testing, the spiking
level was set at 4400 mg KC1/L (the average result of 1100 / the target result of 25% sample) for the
interlaboratory testing  phase. Referee laboratory testing of the interlaboratory sample yielded an IC25 of
27.5% sample.
                                             D-17

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D.6     Preliminary Testing for Silverside Acute and Chronic Test Methods
D.6.1   Reference Toxicant Sample Type

The reference toxicant sample type for the silverside acute and chronic test methods was composed of
synthetic seawater (prepared using bioassay grade Forty Fathoms artificial sea salts added to deionized
water) spiked with copper sulfate (CuSO4). The spiking level for this sample type was targeted to
produce a test result (LC50 for the silverside acute test and IC25 for the silverside chronic test) of 50%
sample. Tables D12 and D13 show the results of preliminary testing for silverside acute and chronic test
methods, respectively. Spiking levels selected for Part 2 testing resulted in an LC50 of >0.25 mg Cu/L
for the silverside acute test method.  Based on this result, the spiking level for Part 4 testing was increased
to 1 mg Cu/L. Part 4 sheepshead acute testing produced an LC50 of 0.29 mg Cu/L or 29% sample. The
same spiking level was used during the interlaboratory testing phase; however, test results on the
interlaboratory sample were >100% sample due to an error in the preparation of this sample type (see
Section 5.3 in the main body of this report).

Part 2 preliminary testing for the silverside chronic method produced an IC25 of >0.3 mg Cu/L. Based on
this result, the spiking level for Part 4 testing was increased to 1 mg Cu/L. Part 4 testing produced an
IC25 of 0.189 mg Cu/L or 18.9% sample.  The same spiking level was used during the interlaboratory
testing phase, and referee laboratory testing of this sample yielded an IC25 of 15.2% sample.

Table D12.  Results from silverside acute preliminary testing.
Sample type
Reference toxicant
Effluent
Receiving water
Part3
2
4
IL
2
3
4
IL
2
3
4
IL
Concentrations tested
(mg Cu/L)
0.016,0.031,0.063,0.125,0.25
0.063,0.125,0.25,0.5, 1.000
0.063,0.125,0.25,0.5, 1.000
0.031,0.063,0.125,0.25,0.5
0.031,0.063,0.125,0.25,0.5
0.058, 0.115, 0.231, 0.461, 0.922
0.058, 0.115, 0.231, 0.461, 0.922
0.031,0.063,0.125,0.25,0.5
0.031,0.063,0.125,0.25,0.5
0.035,0.071,0.141,0.283,0.565
0.035,0.071,0.141,0.283,0.565
LC50
(mg Cu/L)
>0.25
0.29
>1
0.234
0.347
0.171
0.296
0.141
0.146
0.154
0.276
LC50
(% sample)
>100
29.0
>100
46.9
69.5
18.5
32.1
28.2
29.2
27.3
48.9
a Preliminary testing Parts 1-4 and referee laboratory testing during the interlaboratory testing phase (IL). Part 1 preliminary
testing was conducted using only the chronic method, and Part 3 was conducted using only the acute method.
                                              D-18

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Table D13. Results from silverside chronic preliminary testing.
Sample type
Reference
toxicant
Effluent
Receiving water
Part3
2
4
IL
lb
2
4
IL
lb
2
4
IL
Concentrations tested
(mg Cu/L)
0.019,0.038,0.075,0.15,0.3
0.063,0.125,0.25,0.5, 1.0
0.063,0.125,0.25,0.5, 1.0
6.25, 12.5, 25, 50, 100
0.038,0.075,0.15,0.3,0.6
0.075,0.15,0.3,0.6, 1.2
0.05,0.1,0.2,0.4,0.8
6.25, 12.5, 25, 50, 100
0.038,0.075,0.15,0.3,0.6
0.031, 0.062, 0.124, 0.247, 0.494
0.031, 0.062, 0.124, 0.247, 0.494
Survival
NOEC
(mgCu/L)
0.15
0.125
0.125
50
0.15
0.15
0.1
100
0.75
0.124
0.062
Growth
NOEC
(mg Cu/L)
0.15
0.125
0.125
25
0.15
0.15
0.1
100
0.75
0.124
0.062
IC25
(mgCu/L)
>0.3
0.189
0.152
-
0.227
0.171
0.23
-
0.155
0.149
0.103
IC25
(% sample)
>100
18.9
15.2
43.9
37.8
14.3
28.8
>100
25.9
30.2
20.9
a Preliminary testing Parts 1-4 and referee laboratory testing during the interlaboratory testing phase (IL). Part 1 preliminary
testing was conducted using only the chronic method, and Part 3 was conducted using only the acute method.
b Part 1 testing was conducted on unspiked samples. The units for test concentrations in Part 1 were percent sample.
D.6.2   Effluent Sample Type

The effluent sample type used for the silverside acute and chronic test methods was composed of an
industrial wastewater effluent spiked with CuSO4. The referee laboratory (Ogden Environmental and
Energy Services, Inc.) collected the effluent from an industrial wastewater treatment facility designed to
treat oil refinery waste.  This effluent source was selected based on historical consistency in chemical and
toxicological testing conducted by the referee laboratory. Water chemistry from the effluent source is
listed in Table D14.

The effluent sample was adjusted to a salinity of 25 ppt and then spiked with CuSO4 to provide a
consistently toxic sample that was appropriate for the silverside acute and chronic test method.  The
spiking level for this sample type was targeted to produce a test result (LC50 for the silverside acute test
and IC25 for the silverside chronic test) of 25% sample. Part 2 preliminary testing for the silverside acute
test method produced an LC50 of 0.234 mg Cu/L. This sample was held for 7 days at 4C and then
retested for Part 3 preliminary testing.  After holding, the sample produced an LC50 of 0.347 mg Cu/L,
which represents a 48% change from the original Part 2 test result.  Based on Part 2 results, the referee
laboratory increased the spiking level in Part 4 testing to 0.922 mg Cu/L (approximately 0.234 / the target
result of 25% sample).  Part 4 testing resulted in an LC50 of 0.171 mg Cu/L or 18.5% sample. The same
                                               D-19

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spiking level was used during the interlaboratory testing phase, and referee laboratory testing of this
sample yielded an LC50 of 32.1% sample.

For the silverside chronic test method, Part 1 testing of the unspiked effluent resulted in an IC25 of 43.9%
sample. Spiking of the effluent sample in Part 2 testing produced an IC25 of 0.227 mg Cu/L. Based on
this result, the spiking level was increased to 1.2 mg Cu/L for Part 4 testing. Part 4 testing produced an
IC25 of 0.171 mg Cu/L or  14.3% sample.  Based on an average of Part 2 and Part 4 testing results, the
spiking level for interlaboratory testing was set at 0.8 mg Cu/L (approximately the average result of 0.199
/ the target result of 25% sample). Referee laboratory testing of the interlaboratory sample produced an
IC25 of 28.8% sample.

Table D14. Water chemistry of effluent sample source for  silverside acute and chronic test
methods.
Parameters
Alkalinity (mg/L as CaCO3)
Hardness (mg/L as CaCO3)
Conductivity (^S/cm)
pH
Temperature (C)
Total residual chlorine (mg/L)
Dissolved oxygen (mg/L)
Salinity (ppt)
Copper Cwg/L)
Total ammonia (mg/L)
Total dissolved solids (mg/L)
Total suspended solids (mg/L)
Total organic carbon (mg/L)
Biological oxygen demand (mg/L)
Chemical oxygen demand (mg/L)
Sampling date3
08/02/99
217
287
1457
7.37
16.7
0.02
7.9
0
22
14.0
975
4
13.2
15
41
08/23/99
202
289
1424
7.08
23.5
0.03
4.2
0
-
-
-
-
-
-
-
08/26/99
214
282
1476
7.31
18.5
0.02
3.9
0
-
-
-
-
-
-
-
08/28/99
208
288
1486
7.35
22.0
0.02
8.9
0
-
-
-
-
-
-
-
09/17/99
182
292
1320
7.16
19.2
<0.01
8.7
0
-
-
-
-
-
-
-
 "-" indicates that the parameter was not tested on the given sampling date.
D.6.3  Receiving Water Sample Type

The receiving water sample type used for the silverside acute and chronic test methods was composed of a
natural seawater spiked with CuSO4. The referee laboratory (Ogden Environmental and Energy Services,
Inc.) collected natural seawater from the Scripps Institution of Oceanography (Scripps) seawater system
in La Jolla, CA. Scripps pumps seawater from a fixed collection site 320 m offshore of La Jolla, CA,
                                             D-20

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filters the seawater through a sand filter, and incorporates the seawater into a flow-through system for use
in supplying aquariums housed at Scripps. The referee laboratory routinely uses natural seawater from
Scripps' seawater system for in-house organism culturing and dilution water.  The referee laboratory
transported water from Scripps to the laboratory where it was incorporated into the laboratory's flow-
through natural seawater system that includes two 2,200-gallon storage tanks, an in-line 20-(im filter, and
an in-line heater/chiller unit.  Table D15 shows the water chemistry of the receiving water sample
collected for preliminary testing.  Prior to testing, receiving water was filtered through a 0.2-(im filter and
adjusted to a salinity of 25 ppt with the addition of deionized water.

Table D15. Water chemistry of the receiving water sample source for silverside acute and chronic
test methods.
Parameters
Alkalinity (mg/L as CaCO3)
Hardness (mg/L as CaCO3)
Conductivity OS/cm)
pH
Temperature (C)
Total residual Chlorine (mg/L)
Dissolved oxygen (mg/L)
Salinity (ppt)
Copper (Mg/L)
Total ammonia (mg/L)
Total dissolved solids (mg/L)
Total suspended solids (mg/L)
Total organic carbon (mg/L)
Biological oxygen demand (mg/L)
Chemical oxygen demand (mg/L)
Sampling date
08/02/99
75
>2000
53,100
8.08
9.9
0.01
7.2
34
5.4
<0.1
28,000
<1.0
0.5
2
26
The receiving water sample was then spiked with CuSO4 to provide a consistently toxic sample.  The
spiking level for this sample type was targeted to produce a test result (LC50 for the silverside acute test
and IC25 for the silverside chronic) of 25% sample. Part 2 preliminary testing forthe silverside acute test
method produced an LC50 of 0.141 mg Cu/L. This sample was held for 7 days at 4C and then retested
                                              D-21

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for Part 3 preliminary testing. After holding, the sample produced an LC50 of 0.146 mg Cu/L, which
represented only a 3.5% change from the initial Part 2 test result.  Based on Part 2 testing results, the
spiking level for Part 4 preliminary testing was increased to 0.565 mg Cu/L(approximately 0.141 / the
target result of 25% sample). Part 4 testing produced an LC50 of 0.154 mg Cu/L or 27.3% sample.  The
same spiking level was used during the interlaboratory testing phase, and referee laboratory testing of this
sample yielded an LC50 of 48.9% sample.

For the silverside chronic test method, Part 1 testing of the unspiked effluent resulted in no toxicity (IC25
> 100% sample).  Spiking of the receiving water sample in Part 2 testing produced an IC25 of 0.155 mg
Cu/L. Based on this result, the spiking level was decreased slightly to 0.494 mg Cu/L for Part 4 testing.
Part 4 testing produced an IC25 of 0.149 mg Cu/L or 30.2% sample. The same spiking level was used
during the interlaboratory testing phase, and referee laboratory testing of this sample yielded an IC25 of
20.9% sample.

D.7    Preliminary Testing for the Champia Chronic Test Method

The  referee laboratory supporting the Champia chronic test method (EnviroSystems, Inc.) originally was
instructed to conduct preliminary testing as described in Section 4 (in the main body of this report).  On
January 28, 2000, interlaboratory testing for the Champia chronic test method was canceled  due to a lack
of participant laboratory support (see Section 2.1 in the main body of this report). With this  cancellation,
the objectives of any uncompleted preliminary tests were adjusted to better direct the use of preliminary
test data toward single-laboratory testing rather than preparation for interlaboratory testing.  As a result,
preliminary testing occurred during two time periods; testing from July to September 1999 was conducted
in preparation for interlaboratory testing, and testing from March to May 2000 was  conducted to provide
additional single-laboratory data for the Champia chronic test method.

D.7.1  Reference Toxicant Sample Type

The  reference toxicant sample type was composed of natural seawater spiked with CuSO4. Since natural
seawater is recommended for the Champia chronic test method, the same natural seawater source was
used as the matrix for the reference toxicant sample, dilution water in all tests, and the receiving water
sample matrix.  This natural seawater source is described in more detail in Section D.7.3. For use as the
reference toxicant sample matrix and dilution water, the natural seawater was filtered through a 0.45-(im
membrane filter and steam sterilized at 150C for 30 minutes.  Seawater used for the receiving water
sample matrix was unfiltered and unsterilized.

Table D16 shows the results from preliminary testing for the Champia chronic test method.  Spiking
levels selected for Part 2 testing of the reference toxicant sample type resulted in an IC25 of 0.155 /u,g
Cu/L. Part 4 preliminary testing using the same spiking levels produced an IC25 of 0.265 //g Cu/L.
When the reference toxicant sample was retested in the spring of 2000, this sample type resulted in an
                                             D-22

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IC25 of 0.263 jWg Cu/L.  For the three tests conducted on the reference toxicant sample type, a mean IC25
of 0.228 ^g Cu/L and a CV of 27.6% was calculated.
Table D16. Results from Champia chronic preliminary testing.
Sample
type
Reference
toxicant
Effluent
Receiving
water
Part3
2
4
A
1
1
1
3
4
A
A
1
2
3
4
A
A
A
Test
date
7/27/99
8/17/99
5/16/00
7/28/99
8/3/99
8/10/99
8/4/99
9/14/99
5/9/00
5/9/00
7/28/99
7/27/99
8/3/99
8/18/99
5/31/00
5/23/00
5/23/00
Sample
description
filtered, sterilized
natural seawater
spiked with Cu
unspiked municipal
effluent adjusted to
salinity of 30ppt
unspiked natural
seawater (unfiltered
and unsterilized)
spiked natural
seawater (unfiltered
and unsterilized)
unspiked natural
seawater (unfiltered
and unsterilized)
Units
MgCu/L
MgCu/L
MgCu/L
percent
percent
percent
percent
percent
percent
percent
percent
MgCu/L
MgCu/L
MgCu/L
MgCu/L
percent
percent
Concentrations tested
0.5, 1.0,5, 10, 15
0.5, 1,5, 10, 15
0.15,0.5, 1,5, 10
0.2, 0.7, 2.0, 7.0, 10.0
0.156,0.312,0.625, 1.25,
2.5, 5, 10
0.156,0.312,0.625, 1.25,
2.5, 5, 10
0.2, 0.7, 2.0, 7.0, 10.0
0.05,0.1,0.2,0.7,2,7, 10
0.05,0.1,0.2,0.7,2,7, 10
0.05,0.1,0.2,0.7,2,7, 10
100b
0.625, 1.25,2.5,5, 10,20
0.625, 1.25,2.5,5, 10,20
0.625, 1.25, 2.5, 5, 10, 20,
40
0.15,0.5, 1,5, 10
6.25, 12.5, 25, 50, 100
6.25, 12.5, 25, 50, 100
NOEC
(units)
0.5
<0.5
0.15
0.2
0.156
O.156
O.2
0.050
2.0
0.70
NA
0.625
0.625
0.625
1.0
6.25
100
IC25
(units)
0.155
0.265
0.263
0.172
0.240
0.119
0.162
0.064
0.407
0.852
NA
0.699
0.438
0.866
1.45
7.53C
90.4
a Preliminary testing Parts 1-4 and additional testing (A) requested following cancellation of interlaboratory testing.
b Tested as a single concentration (100%) receiving water. No toxicity was indicated.
c Based on test review and guidance on evaluating concentration-response relationships (USEPA, 2000a), this test result was
determined to be inconclusive.
D.7.2   Effluent Sample Type

The effluent sample type used for the Champia chronic test method was composed of a municipal
wastewater treatment plant effluent.  This effluent source was selected based on historical testing by the
referee laboratory that demonstrated relatively consistent levels of toxicity.  No spiking of this effluent
was necessary; the unspiked effluent sample produced IC25 values in the range of 0.064% to 0.852%
sample. All tests were performed on unspiked effluent adjusted to a salinity of 30 ppt. Water chemistry
of the effluent on each sample collection date is shown in Table D17.
                                               D-23

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Table D17.  Water chemistry of the effluent sample source for the Champia chronic test method.
Parameters
Salinity (ppt)
Conductivity CuS/cm)
pH
Total residual chlorine
(mg/L)
Total ammonia (mg/L)
Sampling date3
07/28/99
3
2250
7.56
0.15
22.6
08/03/99
1
2740
6.93
0.22
26.0
08/04/99"
1
2300
7.67
0.05
-
08/10/99
2
2550
7.24
0.17
15.8
09/14/99
5
2980
6.99
0.38
14.4
05/09/00
1
2156
6.94
0.84
-
a"-" indicates that the parameter was not tested on the given sampling date.
b Sample collected 07/28/99 and held for 7 days at 4C prior to testing.
Part 1 preliminary testing confirmed that the effluent was relatively consistent in toxicity to Champia
parvula. Results of three separate effluent samples collected on three separate days ranged from 0.119%
sample to 0.240% sample with a mean of 0.177% sample and a CV of 34.3%. The effluent sample
collected on 7/28/99 was held for 7 days at 4C and tested on 8/4/99 for Part 3 preliminary testing. This
test resulted in an IC25 of 0.162% sample, which represents a 5.8% change from the initial test conducted
on that sample. The effluent was tested again on 9/14/99 for Part 4 testing, and resulted in an IC25 of
0.064% sample.  In the spring of 2000, the referee laboratory conducted duplicate testing of the effluent.
The resulting IC25s were 0.407% and 0.852% effluent sample, yielding a CV of 50.0% for the duplicate
samples.

D.7.3   Receiving Water Sample Type

The receiving water sample type used for the Champia chronic test method was composed of a natural
seawater spiked with CuSO4. The receiving water was collected from Rye Harbor, Rye, New Hampshire.
The harbor provides anchorage for small pleasure craft and a limited number of small commercial fishing
vessels. The harbor receives no direct discharges of treated or untreated wastewater.  The water in the
harbor is classified as SA-1 and has  been used by the referee laboratory since 1991 to maintain Champia
parvula cultures. Receiving water was collected from a boat offshore and away from other boat traffic or
potential contamination.  The physical and chemical characteristics of the receiving water are listed in
Table D18 for each sample collection date.  The same water source was used for the reference toxicant
sample matrix and for dilution water in all tests; however, water was filtered and sterilized for these uses.
The receiving water sample type was tested without filtration or sterilization.
                                             D-24

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Table D18. Water chemistry of the receiving water sample source for the Champia chronic test
method.
Parameters
Salinity (ppt)
Conductivity
CuS/cm)
pH
Total residual
chlorine (mg/L)
Total ammonia
(mg/L)
Sampling date3
07/28/99
33
48100
7.92
0.05
0.10
08/03/99
33
48600
8.29
0.05
0.10
08/04/99
33
49100
8.54
0.05
0.10
08/10/99
33
49300
8.04
0.05
0.10
09/14/99
32
45400
7.70
0.05
0.22
05/16/00
32
42500
7.89
0.05
-
05/23/00
33
42700
8.00
0.05
-
05/23/00
33
43000
8.07
0.05
-
05/31/00
33
43010
8.01
0.05
-
1"-" indicates that the parameter was not tested on the given sampling date.
Part 1 preliminary testing initially was conducted using a single concentration (100%) test. Results from
this test showed no toxicity in the receiving water.  In the spring of 2000, the referee laboratory conducted
duplicate testing of the unspiked receiving water. IC25 results from a split sample collected on 5/23/00
were 7.53% and 90.4% sample. This represents high variability between duplicate samples; however,
data review revealed that results from the first test may not be reliable. This test produced an interrupted
concentration-response curve, with statistically significant effects at the 12.5% and 100% test
concentrations but not at the 25% and 50% test concentrations. Based on EPA guidance for evaluating
concentration-response relationships (USEPA, 2000a), this test should be considered inconclusive if the
PMSD is above recommended bounds (USEPA, 2000d). Unfortunately, upper PMSD bounds have not
yet been recommended for the Champia chronic method. In this test, the PMSD was 47%, which is
higher than the recommended PMSD upper bound for other chronic methods (23  - 37%) and higher than
the average PMSD (31%) for other Champia chronic tests conducted during preliminary testing.  Also,
the average control response in this test (10.8 cystocarps per plant) was barely above the test acceptability
criteria (10 cystocarps per plant) for the method and was well below the average control response (17.5
cystocarps per plant) in other Champia chronic tests conducted during preliminary testing.  Based on test
review and guidance on evaluating concentration-response relationships (USEPA, 2000a), this test was
determined to be inconclusive.

In Part 2 preliminary testing, the receiving water sample was spiked with CuSO4 to provide a consistently
toxic sample. Part 2 testing for the Champia chronic method produced an IC25 of 0.699 /u,g CuSO4/L.
This sample was held for 7 days at 4C and then retested for Part 3 preliminary testing. After holding,
the sample produced an IC25 of 0.438 /j-g CuSO/L, which represents  a 37% change  from the original Part
2 test result. Part 4 testing resulted in an IC25 of 0.866 /u,g CuSO4/L.  An additional test of the spiked
receiving water in the Spring of 2000 produced an IC25  of 1.45 /u,g CuSO4/L.  For the four tests
                                             D-25

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conducted on the spiked receiving water sample type, a mean IC25 of 0.863
was calculated.
                                                                         Cu/L and a CV of 49.7%
D.8     Preliminary Testing for the Holmesimysis Acute Test Method

The  referee laboratory supporting the Holmesimysis acute test method (MEC Analytical, Inc.) was
originally instructed to conduct preliminary testing according to Section 4 (in the main body of this
report). Due to difficulties encountered in obtaining test organisms, a limited number of preliminary tests
were conducted by the referee laboratory from July to September 1999.  On January 28, 2000,
interlaboratory testing for the Holmesimysis acute test method was canceled due to a lack of participant
laboratory support (see Section 2.1 in the main body of this report).  With this cancellation, the objectives
of any uncompleted preliminary tests were adjusted to better direct the use of preliminary test data toward
single-laboratory testing rather than preparation for interlaboratory testing. The referee laboratory
attempted to complete preliminary testing from April through June 2000; however, the laboratory was
unable to obtain test organisms during this period.  On June 29, 2000, any further preliminary testing at
the referee laboratory was canceled due to the unavailability of test organisms.

In all, only five preliminary Holmesimysis acute tests were performed (Table D19); one test was
conducted on receiving water, and two tests were conducted on each of two seawater effluent sources. Of
the five tests conducted, only two met test acceptability criteria for survival, and these two tests were not
conducted according to the WET method manual test requirement for test organism age. Neonates for
these tests were collected directly from the field rather than hatched in the laboratory from field-collected
adults.  For this reason, exact ages of neonates used for testing could not be determined (see Section D.9).
Table D19. Results from Holmesimysis acute preliminary testing.
Sample type
Effluent 1
Effluent 1
Effluent 2
Effluent 2
Receiving water
Test date
7/21/99
7/27/99
8/31/99b
9/7/99b
7/21/99
Control survival
(%)
80a
80a
92.5
90
80a
NOAEC
(% sample)
12.5
12.5
25
12.5
100
LC50
(% sample)
25.3
15.3
35.1
16.6
>100
a Failed to meet test acceptability criteria of >90% control survival.
b Tests were conducted on field collected neonates.
                                              D-26

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D.8.1  Effluent Sample Type

Two seawater effluent sources were tested and considered for use as the effluent sample source. Both
effluent sources were from municipal wastewater treatment facilities, and water chemistry for both
sources is listed in Table D20. Two Holmesimysis acute tests were conducted on effluent 1; these tests
produced LCSOs of 23.9% and 12.4% sample (Table D19). Control survival in each of these tests was
only 80%, which fails to meet the test acceptability criteria for the method.  Synthetic seawater (prepared
using bioassay grade Forty Fathoms artificial sea salts added to deionized water) was used for dilution
in these tests rather than natural seawater as stated in the SOP. The dilution water may have been a factor
in the poor control survival. Due to difficulties in obtaining test organisms, tests conducted on effluent 2
used neonates directly collected from the field, rather than neonates hatched in the laboratory from field
collected adults.  These two tests resulted in LCSOs of 28.2% and 12.6% sample.
Table D20.  Water chemistry of the effluent sample sources for the Holmesimysis acute test method.
Parameters
Alkalinity (mg/L as CaCO3)
Hardness (mg/L as CaCO3)
pH
Temperature (C)
Total residual chlorine (mg/L)
Dissolved oxygen (mg/L)
Salinity (ppt)
Copper (Mg/L)
Total ammonia (mg/L)
Total dissolved solids (mg/L)
Total suspended solids (mg/L)
Biological oxygen demand (mg/L)
Chemical oxygen demand (mg/L)
Effluent 1
Sampling date3
07/21/99
-
-
7.28
19.2
0.07
3.3
0.6
-
22.3
-
-
-
-
07/26/99
250
180
7.26
18.0
0.07
6.0
0.5
10
18.9
562
13.5
32.2
98
Effluent 2
Sampling date3
08/30/99
-
-
7.09
23.3
-
6.2
0
-
18.7
-
-
-
-
09/07/99
-
-
7.88
24.0
0.09
6.9
0
-
14.7
-
-
-
-
1"-" indicates that the parameter was not tested on the given sampling date.
                                             D-27

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D.8.2   Receiving Water Sample Type

The receiving water sample type used for the Holmesimysis acute test method consisted of natural
seawater collected from San Francisco Bay off of Point Chauncey on the Tiburon Peninsula. Samples
were collected away from direct discharges of treated or untreated wastewater. Water from this source
has been used by the referee laboratory as dilution water for several years without exhibiting toxicity.
The physical and chemical characteristics of the receiving water collected from the bay are listed in Table
D21.  A single Part 1 preliminary test was conducted on the receiving water resulting in an LC50 of
>100% sample.  This test failed to meet test acceptability criteria due to control survival of 80%.  As
mentioned above, use of synthetic seawater for dilution may have contributed to low control survival.

Table D21.  Water chemistry of the receiving water sample source for the Holmesimysis acute test
method.
Parameters
Alkalinity (mg/L as CaCO3)
Hardness (mg/L as CaCO3)
pH
Temperature (C)
Total residual chlorine (mg/L)
Dissolved oxygen (mg/L)
Salinity (ppt)
Copper (Mg/L)
Total ammonia (mg/L)
Total dissolved solids (mg/L)
Total suspended solids (mg/L)
Biological oxygen demand (mg/L)
Chemical oxygen demand (mg/L)
Sampling date
07/20/99
122a
4558a
7.85
17.9
0.03
7.6
24
NDa
0.10
27,200a
1.25a
NDa
1930a
a Analyses were conducted on the sample collected 7/27/99, but this sample was not used for Holmesimysis testing due to
insufficient test organisms. ND = not detected.
                                              D-28

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D.9    Problems Encountered in Preliminary Testing

For the Selenastntm chronic test method, spiking levels were originally targeted to produce an IC25 of
50% for the reference toxicant sample type and 25% for the effluent and receiving water sample type. In
Part 2 preliminary testing, IC25  values in repeated tests were variable and the referee laboratory had
difficulty isolating the targeted spiking level. The referee laboratory observed that IC50 values were less
variable than IC25 values in repeated tests. For this reason, the target spiking levels were based on IC50
values rather than IC25 values.  Target spiking levels were set to produce an IC50 of 38% for reference
toxicant, effluent, and receiving  water sample types.  The 38% level was selected in an attempt to allow
IC25 and IC50 results from interlaboratory testing to fall within the test concentration range.

For the Mysidopsis chronic and sheepshead acute and chronic test methods, CuSO4 was originally
selected as the spiking agent.  During preliminary testing for these methods, results on spiked samples
were highly variable.  Upon further investigation, the referee laboratory determined that this variability
was due to precipitation of copper in the spiked seawater samples.  A combination of factors including
spiking concentrations, salinity of the sample, pH, other dissolved  ions in the sample matrix, and storage
of the spiked sample at <4C contributed to the precipitation of copper in spiked  samples.  Due to this
precipitation, the spiking agent for these marine methods was changed to KC1. The referee laboratory had
experience in the use of KC1 as the spiking agent for freshwater methods in the WET Variability Study
and had experience in the use  of KC1 as a reference toxicant for these marine methods.  The same
problem was encountered for the silverside acute reference toxicant sample (see Section 5.3 in the main
body of this report), but the problem was not identified in time to change the spiking agent. Precipitation
of copper did not appear to affect the other sample types for the  silverside acute and chronic test methods,
possibly due to the lower  spiking concentrations used for these methods.

For the Holmesimysis acute test  method, problems were encountered in obtaining test organisms.
Organisms for this test method are generally field collected from kelp beds off the coast of California, but
they are not present in sufficient numbers during the winter months. From April  through June 2000, the
referee laboratory attempted to collect organisms to complete preliminary testing, but Holmesimysis
costata populations were still  not at sufficient densities at potential sites in San Diego or Santa Cruz, CA.
Even when field-collected adult Holmesimysis costata were available (July through September 1999),
obtaining sufficient neonates within the required age range was difficult.  Field-collected gravid females
were held in the laboratory and culled daily to obtain neonates within a 24-hour age  range. This required
maintaining a large number of gravid females to produce the necessary neonates. In addition, survival of
newly hatched neonates was poor, which added to difficulties in obtaining a sufficient number of test
organisms. To avoid these difficulties, the  referee laboratory collected neonates directly from the field for
use in two preliminary tests.  The smallest of collected organisms  were used in these tests. This
technique  of obtaining test organisms did not allow an exact determination of the age of test organisms, so
the age of test organisms could have been outside of the required range.
                                              D-29

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A second problem encountered in Holmesimysis acute preliminary testing was poor survival of neonates
in test controls. Three of the five preliminary tests conducted failed to meet the test acceptability criteria
of 90% survival. It is believed that poor control survival in these tests was due to the use of a synthetic
seawater rather than a natural seawater for organism holding and test dilution.
                                              D-30

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                  Appendix E:
Analysis of Percent Minimum Significant Differences

-------
The percent minimum significant difference (PMSD) is a measure of within-test variability and test
sensitivity. The PMSD for a given WET test can be defined as the smallest percentage difference
between the control and a treatment (an effluent dilution) that could be declared as statistically significant.
As test variability increases, the ability of a test to detect small toxic effects diminishes and the test
becomes a less sensitive measure of toxicity. Appendix C of the WET method manuals (USEPA, 1994a;
USEPA, 1994b) describes the calculation of the minimum significant difference (MSB) as:
                      MSD =dxSw ^(l/n,) + (]/)
where,         d = critical value for the Dunnett's procedure
               Sw = the square root of the within mean square
               n = the number of replicates at each concentration, assuming an equal number of
               replicates at all treatment concentrations
               nl = number of replicates in the control

The PMSD is the MSD expressed as a percentage of the control response (i.e., PMSD = MSD/control
mean * 100).

In June 2000, EPA published guidance on WET test variability that recommended placing upper and
lower bounds on the PMSD to control variability and ensure a specified range of test sensitivity (the WET
Variability Guidance Document; USEPA, 2000d).  Based on this guidance, tests for which the PMSD
exceeds an upper bound would not be acceptable, if the test leads to a decision that there is no significant
toxicity at the concentration identified in the permit as a limit ("Instream Waste Concentration" (IWC) or
"Receiving Water Concentration"). This guidance also applies lower PMSD bounds for the purpose of
determining the no observed effect concentration (NOEC). The purpose of the lower PMSD bound is to
avoid declaring as "significant" toxic effects that are smaller than can generally and routinely be detected
by the method as currently conducted by qualified laboratories.

To derive recommended PMSD bounds for the WET Variability Guidance Document, EPA compiled and
analyzed a database of more than 1800 reference toxicant tests conducted for 23 different methods
between 1988 and 1999 in 75 laboratories. EPA derived the lower and upper bounds as the 10th and 90th
percentiles, respectively, of PMSDs from this reference toxicant test database.

This appendix reports PMSD values calculated for short-term chronic tests in the WET Variability Study,
and compares the distribution of those values to the PMSD distributions  and bounds derived in the WET
Variability Guidance Document (USEPA, 2000d).  While the WET Variability Study results contain
fewer tests than the database analyzed for the guidance document, the number of laboratories contained in
each database is comparable.  The WET Variability Study database contained only 25% to 67%
(depending on the method) of the number of tests contained in the guidance document database but 63%
to 142% (depending on the method)  of the number of laboratories included in the guidance document
database.  For four of the six chronic methods, the WET Variability Study  database contained a larger
                                             E-l

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number of laboratories. Also, while the guidance document database contains only data from reference
toxicant tests, the WET Variability Study database contains results from the analysis of blank, reference
toxicant, effluent, and receiving water samples.

The percentiles of PMSD values calculated in the WET Variability Study for chronic test methods are
displayed in Table El. This data represents the PMSDs calculated for valid tests (i.e., those that met the
test acceptability criteria). This data is from 28 to 100 tests per method and 7 to 32 laboratories per
method. In the WET Variability Study, median PMSD values ranged from 12% to 23% for the various
methods. The median PMSD values from the WET Variability Study were also very similar to the
median PMSD values in the WET Variability Guidance Document. Median PMSD values from both
databases were within 2% of each other for the Ceriodaphnia chronic (23% in both databases),
Mysldopsls chronic (18% in the WET Variability Study database and 20% in EPA's reference toxicant
database), sheepshead chronic (12% in the WET Variability Study database and 13% in EPA's reference
toxicant database), and silverside chronic (19% in the WET Variability Study database and  18% in EPA's
reference toxicant database) test methods. The median PMSD for the fathead chronic test was 4% lower
in the WET Variability Study (16%) than the reference toxicant database (20%); and the median PMSD
for the Selenastrum chronic test was 3% higher in the WET Variability Study (17%) than the reference
toxicant database (14%). The PMSDs for the fathead chronic test may have been lower in the WET
Variability Study because all tests used four replicates, while some tests in the reference toxicant database
used only three replicates. The PMSDs for the Selenastrum chronic test may have  been higher in the
WET Variability Study due to the inclusion of tests conducted both with and without EDTA. PMSD
values for this method are more similar when only tests conducted with EDTA in the WET Variability
Study (median of 15%) are compared to the reference toxicant database (median of 14%).

While median PMSD values were very similar between the two databases, more variability was exhibited
in the tails of the distributions. This is not unexpected, given the differences in the size of each database.
Table E2 compares the 10th and 90th percentile PMSDs between the WET Variability Study and the WET
Variability Guidance Document. PMSD 10th percentiles from the two databases differed by less than 3%
for all test methods, and PMSD 90th percentiles differed by 5 to 10%. PMSD 90th percentiles were higher
in the WET Variability Study than the guidance document for the Ceriodaphnia chronic, Selenastrum
chronic, and Mysidopsis chronic test methods; but were lower for the fathead chronic, sheepshead
chronic, and silverside chronic test methods.

Table E3 shows the number of tests in the WET Variability Study that had PMSD values outside of the
lower or upper PMSD bounds recommended in the WET Variability Guidance Document (USEPA,
2000d). From 2 to  15% of tests had PMSDs below the recommended lower bound, and from 0 to 31% of
tests had PMSDs above the upper bound. These upper and lower PMSD bounds were not used to exclude
or modify test results in the WET Variability Study. Based on the guidance (USEPA, 2000d), decisions
regarding the validity of test results exceeding the upper PMSD bounds are dependent upon the permit
                                             E-2

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IWC concentration. Because IWC concentrations were not established or applicable to an interlaboratory
variability study, determinations of test validity were not made based on PMSD bounds.
                                               E-3

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Table El. Percentiles of PMSD for sublethal endpoints of chronic WET methods evaluated in the WET Variability Study.
Parameter
No. of testsb
No. of labs
Endpoint
PMSD
Percentile
5%
10%
15%
20%
25%
50%
75%
80%
85%
90%
95%
Test Method
Ceriodaphnia
chronic
100
32
reproduction
10
13
14
16
17
23
34
35
39
47
53
Fathead
chronic
99
27
growth
10
12
13
13
14
16
21
23
25
30
34
Selenastrum
chronic3
57
11
growth
8.2
9.5
10
11
12
17
23
27
30
32
41
Selenastrum
chronic
(EDTA)
28
8
growth
8.0
9.1
10
11
11
15
21
22
27
29
31
Selenastrum
chronic
(w/o EDTA)
29
9
growth
9.1
10
10
11
12
18
26
30
33
38
52
Mysidopsis
chronic
43
11
growth
11
11
12
15
15
18
26
29
31
37
61
Sheepshead
chronic
28
7
growth
7.0
7.3
8.1
9.1
9.4
12
15
16
16
17
19
Silverside
chronic
40
10
growth
11
11
12
13
14
19
23
25
26
28
31
a Results of tests conducted with EDTA and without EDTA are combined.
b Number of valid tests. Tests failing to meet test acceptability criteria were excluded from analysis.
                                                                    E-4

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Table E2. Comparison of PMSD percentiles observed in the WET Variability Study and those
reported in the WET Variability Guidance Document (USEPA, 2000d).
Test method
Ceriodaphnia
chronic
Fathead chronic
Selenastmm
chronic
Mysidopsis chronic
Sheepshead chronic
Silverside chronic
WET Variability Guidance Document
PMSD 10th
percentile
11
9.4
9.3
12
6.3
12
PMSD 90th
percentile
37
35
23
32
23
35
WET Variability Study
PMSD 10th
percentile
13
12
9.5
11
7.3
11
PMSD 90th
percentile
47
30
32
37
17
28
Table E3. Percentage of tests in the WET Variability Study with calculated PMSDs outside of
recommended bounds (USEPA, 2000d).
Test Method
Ceriodaphnia chronic
Fathead chronic
Selenastrum chronic
Selenastrum chronic
(EDTA)
Selenastrum chronic
(w/o EDTA)
Mysidopsis chronic
Sheepshead chronic
Silverside chronic
Total
no. of
tests3
100
99
57
28
29
43
28
40
Below lower PMSD bound
No. of tests
6
2
5
3
2
7
1
6
% of tests
6.0
2.0
8.8
11
6.9
16
3.6
15
Above upper PMSD bound
No. of tests
18
4
14
5
9
6
0
2
% of tests
18
4.0
25
18
31
14
0.0
5.0
a Number of valid tests. Tests failing to meet test acceptability criteria were excluded from analysis.
                                             E-5

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        Appendix F:

Method Performance Including
   Referee Laboratory Data

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The WET Variability Study evaluated the successful test completion rate, false positive rate, and
precision of WET test methods. In the analysis of these test performance measures, data from the referee
laboratories were not included. Referee laboratories conducted testing of each sample type
simultaneously with participant laboratories; however, the identity and expected result of test samples was
not blinded to referee laboratories as they were to participant laboratories. For this reason, referee
laboratory results were excluded from the calculation of test performance measures. This appendix
presents summarized results of the study including referee laboratory data.

Table Fl shows the successful test completion rates  achieved in the WET Variability Study when referee
laboratory data is included.  Including referee laboratory data had very little effect on successful test
completion rates.  Successful test completion rates remained unchanged for three test methods, increased
for five test methods, and decreased for two test methods. Successful test completion rates increased by
only 0.1 to 1%, and decreased by only  1.8 to 1.9%.

Table F2 shows the false positive rates reported in the WET Variability Study when referee laboratory
data is included.  Inclusion of referee laboratory data only affected the false positive rates for the
Ceriodaphnia chronic and fathead chronic test methods.  For these two methods, false positive rates
decreased by 0.13% and 0.18%, respectively.

Table F3 shows the precision of WET methods achieved in the WET Variability Study when referee
laboratory data is included.  For most test methods, the inclusion of referee laboratory data had very little
effect on measured test precision.  Interlaboratory CVs (based on total variance) of LCSOs for acute tests
and IC25s for chronic tests remained unchanged for two methods, increased for two methods, and
decreased for six methods. For the six methods that decreased in variability, interlaboratory CVs
decreased by 0.2 to 2.1%. For the sheepshead chronic method, the CV increased by 0.5%, and for the
Selenastrum chronic method the CV increased by 8.6% (when conducted with EDTA) and 8.4% (when
conducted without EDTA).  Referee laboratory results for the Selenastrum chronic test method were
consistently more sensitive (i.e., lower IC25) than results  from most participant laboratories.
                                              F-l

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Table Fl. Successful test completion rates for test methods evaluated in the WET Variability Study
(including referee laboratory data).
Test method
Ceriodaphnia acute
Ceriodaphnia chronic
Fathead acute
Fathead chronic
Selenastrum chronic (with EDTA)
Selenastrum chronic (without
EDTA)
Mysidopsis chronic
Sheepshead acute
Sheepshead chronic
Silverside acute
Silverside chronic
N
108
126
111
105
48
48
48
32
32
40
44
No. of invalid tests
5
22
2
2
17
16
2
0
0
2
0
Successful test
completion rate
(%)
95.4
82.5
98.2
98.1
64.6
66.7
95.8
100
100
95.0
100
                                            F-2

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Table F2.  False positive rates for test methods evaluated in the WET Variability Study (including
referee laboratory data).
Test method
Ceriodaphnia acute
Ceriodaphnia chronic
Fathead acute
Fathead chronic
Selenastrum chronic
(with EDTA)
Selenastrum chronic
(without EDTA)
Mysidopsis chronic
Sheepshead acute
Sheepshead chronic
Silverside acute
Silverside chronic
N
33
28
28
25
5
6
7
8
8
7
8
False positive rate (%)
Survival endpoint
LC50
0.00
0.00
0.00
0.00
-
-
0.00
0.00
0.00
0.00
0.00
NOEC
-
0.00
-
0.00
-
-
0.00
-
0.00
-
0.00
Growth endpoint
IC25
-
-
-
4.00
0.00
33.3
0.00
-
0.00
-
0.00
NOEC
-
-
-
4.17a
0.00
20.0b
0.00
-
0.00
-
0.00
Reproduction endpoint
IC25
-
3.57
-
-
-
-
o.ooc
-
-
-
-
NOEC
-
3.57
-
-
-
-
o.ooc
-
-
-
-
a N for the growth NOEC endpoint was 24.
b N for the growth NOEC endpoint was 5.
0 N for the fecundity endpoints was 4.
                                                F-3

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Table F 3.  Within-laboratory, between-laboratory, and total variability observed for test methods
evaluated in the WET Variability Study (including referee laboratory data).
Test method
Ceriodaphnia acute
Ceriodaphnia chronic
Fathead acute
Fathead chronic
Selenastrum chronic
(with EDTA)
Selenastrum chronic
(without EDTA)
Mysidopsis chronic6
Sheepshead acutef
Sheepshead chronicf
Silverside acute
Silverside chronic
CV (%)a
Survival endpointb
Within-
laboratory
12.0
7.03
8.96
7.84
-
-
6.44
-
-
10.1
11.0
Between-
laboratory
23.5
21.5
19.4
11.2
-
-
26.6
-
-
49.2
40.3
Total"
28.4
21.2
19.8
13.2
-
-
30.1
24.1
8.17
38.5
41.3
Sublethal endpoint0
Within-
laboratory
-
17.3
-
14.5
25.6
25.5
6.89
-
-
-
14.7
Between-
laboratory
-
26.6
-
15.0
28.0
78.4
36.9
-
-
-
41.7
Total"
-
33.5
-
20.7
42.9
66.9
39.2
-
11.0
-
43.8
3 Within-laboratory, between-laboratory, and total CVs presented are averaged across sample types.
bCVs for the survival endpoint are based on LC50 values.
c CVs for the sublethal endpoint are based on IC25 values.
dCVs based on total variance may not necessarily be greater than CVs based on within and between-laboratory variance because
the CVs presented are averaged across sample types. No within-laboratory replication was provided for the receiving water
sample type, so CVs based on within and between-laboratory variance are averaged across only the reference toxicant and
effluent sample types; CVs based on total variance are averaged across the reference toxicant, effluent, and receiving water
sample types.
e For the Mysidopsis chronic test method, sublethal enpdoint CVs are for the growth endpoint.
f Within and between-laboratory components of variability were not estimated for the sheepshead acute and chronic test methods
because no within-laboratory replication was provided for these methods.
                                                      F-4

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