EPA/600/R-96/054
September"! 996
Marine Toxicity Identification Evaluation (TIE)
Phase I Guidance Document
Edited by
Robert M. Burgess
Kay T. Ho
George E. Morrison
National Health and Environmental Effects Research Laboratory
Narragansett, Rhode Island 02882
i
Gary Chapman
National Health and Environmental Effects Research Laboratory
Newport, Oregon 97365
Debra L. Denton
Region IX: U.S. Environmental Protection Agency
San Francisco, Calfornia 94105
National Health and Environmental Effects Research Laboratory
Atlantic Ecology Division
U.S. Environmental Protection Agency
Narragansett, Rhode Island 02828
Printed on Recycled Paper
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Notices
This document has been reviewed in accordance with U. S. Environmental Protection Agency Policy
and approved for publication. Mention of trade names or commercial products does not constitute endorsement
or recommendation for use by the U.S. Environmental Protection Agency.
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Abstract
During the last ten years Toxicity Identification Evaluation (TIE) methods have been used extensively with
freshwater effluents, receiving waters, and sediments. TIEs may be required by state or federal agencies as a
result of enforcement actions, as a condition of the discharger's National Pollutant Discharge Elimination System
(NPDES) permit, or may be conducted voluntarily by permittees. This guidance document, using the freshwater
TEE approach as a model, has been developed to aid in conducting acute and chronic marine TIEs. It focuses on
Phase I of the TIE: Toxicity Characterization. Phase I of a TIE characterizes the classes of toxicants causing
adverse biological effects. These classes may include metals, organics, pH dependent toxicants, volatile toxicants,
filterable toxicants, and oxidants. In this document, information is provided for: (1) salinity adjustment of
freshwater effluents with brine, (2) general guidance for the performance of small volume marine toxicity tests
with Atlantic, Gulf, and Pacific Coast species used in NPDES permit or as a NPDES permit testing requirement,
(3) tolerances to the chemicals added during a TIE, and (4) the conduct of TIE manipulations. These
acute/chronic TIE procedures have been developed for a number of specific macroalgas, echinoids, mysicls,
bivalves, an amphipod, gastropods, and fishes. Recommended manipulations described in this document include
filtration, aeration, EDTA chelation, oxidant reduction, graduated pH, C18 solid phase extraction (SPE), cation
exchange SPE, and sea lettuce Ulva lactuca addition.
m
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Foreword
The Marine Toxicity Identification Evaluation (TIE): Phase I Guidance Document focuses on methods for
characterizing toxicity associated with discharges to marine waters including effluents and receiving waters. Its
purpose is to provide guidance to dischargers, testing laboratory staff, and local, state, and regional personnel
in conducting Phase I of a marine TIE. Methods for conducting freshwater toxicity tests and TJEs have been
produced (EPA 1991a, 1991b, 1993a, 1993b, 1993c); however, these methods were not directly applicable to
marine samples. As stated in EPA 1993c:
These methods are not mandatory but are intended to aid those who need to characterize, identify or confirm
the cause of toxicity in effluents or other aqueous samples such as ambient waters, sediments, and leachates.
Where we lack experience, we have indicated this and have suggested avenues to follow. All tests need not
be done on every sample; the tests are, in general, independent. However, experience has taught us that
skipping tests may result in wasted time, especially in the early stages of Phase I. An exception to this is
when one wants to know only if a specific substance, for example ammonia, is causing the toxicity or if
toxicants other than ammonia are involved. Otherwise, we urge the whole battery of tests.
We assume the reader is familiar with the following documents describing (1) TIE methods: Toxicity
Identification Evaluation: Characterization of Chronically Toxic Effluents, Phase I (EPA 1991a), Methods for
Aquatic Toxicity Identification Evaluations: Phase I Toxicity Characterization Procedures, Second Edition (EPA
1991b), Methods for Aquatic Toxicity Identification Evaluations: Phase II Toxicity Identification Procedures
for Samples Exhibiting Acute and Chronic Toxicity (EPA 1993b), Methods for Aquatic Toxicity Identification
Evaluations: Phase III Toxicity Confirmation Procedures for Samples Exhibiting Acute and Chronic Toxicity
(EPA 1993c); 2) toxicity testing methods: Short-Term Methods for Estimating the Chronic Toxicity of Effluents
and Receiving Waters to Marine and Estuarine Organisms (EPA 1994), Methods for Measuring the Acute
Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms (EPA 1993a), Short-Term
Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to West Coast Marine and
Estuarine Organisms (EPA 1995); and 3) Toxicity Reduction Evaluations (TREs): Toxicity Reduction
Evaluation Protocol for Municipal Wastewater Treatment Plants (EPA 1989a), and Generalized Methodology
for Conducting Industrial Toxicity Reduction Evaluations (TREs) (EPA 1989b). Methodologies for both acute
and sublethal (chronic) toxicity testing have been included in this manual. We invite comments on this document
in order to improve future editions.
IV
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Contents
Notices • • u
Abstract • "i
Forward • iv
Figures • • • • viii
Tables • • «
Acknowledgements • • • xi
Abbreviations xii
1. Introduction 1
1.1. Background ; • 1
1.2. Related Documents 1
1.3. Development of Marine TIE Methods 1
2. Health and Safety 3
3. Quality Assurance • 4
3.1. Tffi Quality Control Plans .4
3.2. Cost Considerations/Concessions 4
3.3. Variability - -.•• 5
3.4. Intra-Laboratory Communication 5
3.5. Record Keeping • 5
3.6. Phase I Considerations 5
3.7. Phase II Considerations 6
3.8. Phase III Considerations 6
4. Equipment, Supplies, and Facilities 7
5. Sample Collection, Handling, Salinity Adjustment, and Dilution 8
5.1. General Collection 8
5.2. Composite versus Grab Samples 8
5.3. Pre- or Post- Chlorinated Samples 8
5.4. Salinity Adjustments and Dilution Water 10
6. Toxicity Testing 11
6.1. Test Species 11
6.2. TestMethods 12
6.2.1. Macroalga Sexual Reproduction or Germination/Growth Tests 12
6.2.2. Echinoid Sperm Cell Tests 12
6.2.3. Echinoid, Bivalve, and Gastropod Development Tests 12
6.2.4. Acute Mysid and Fish Tests 12
6.2.5. Other Marine Species -. 12
7. Statistical Methods • 13
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Contents (continued)
8. Ion Imbalance 14
9. Toxicity Identification Evaluation Procedures 15
9.1. Initial Toxicity Test 15
9.1.1. General Approach 15
9.1.2. Materials 15
9.1.3. Procedural Overview 15
9.2. Baseline Toxicity Test 18
9.2.1. General Approach 18
9.2.2. Materials 18
9.2.3. Procedural Overview 18
9.3. Filtration Procedure 18
9.3.1. General Approach 18
9.3.2. Materials 19
9.3.3. Procedural Overview 19
9.4. Aeration Procedure 19
9.4.1. General Approach 19
9.4.2. Materials 19
9.4.3. Procedural Overview 19
9.5. EDTA Procedure 19
9.5.1. General Approach 19
9.5.2. Materials 19
9.5.3. Procedural Overview 19
9.6. Sodium Thiosulfate Procedure 23
9.6.1. General Approach 23
9.6.2. Materials 23
9.6.3. Procedural Overview 23
9.7. C,8 SPE Procedure 23
9.7.1. General Approach 23
9.7.2. Materials 23
9.7.3. Procedural Overview 25
9.8. Methanol ElutionTest 25
9.8.1. General Approach and Materials 25
9.8.2. Procedural Overview 25
9.9. Graduated pH Procedure 25
9.9.1. General Approach 25
9.9.2. Materials 28
9.9.3. Sample Preparation 28
9.9.4. Procedural Overview 28
9.10. Cation Exchange SPE Procedure 31
9.10.1. General Approach 31
9.10.2. Materials 31
9.10.3. Procedural Overview 31
9.11. Cation Exchange SPE Acid ElutionTest 31
9.11.1. General Approach and Materials 31
9.11.2. Procedural Overview 31
9.12. UlvalactucaProcedure ..33
9.12.1. General Approach 33
9.12.2. Materials 33
9.12.3. Procedural Overview 33
VI
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Contents (continued)
10. TEE Interpretation
10.1 Sodium Dodecyl Sulfate (SDS)
10.2 Copper
10.3 Summary of Results
11. References
Appendix
34
34
34
35
36
41
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Figures
Figure5-l. Example Data Sheet for Logging in Samples 9
Figure 9-1. Overview Flowchart of a Typical Complete Phase I Marine TIE Characterization 16
Figure9-2. Schematic of Marine TIE Experimental Design 17
Figure 9-3. Overview Flowchart of Filtration Procedure 20
Figure 9-4. Overview Flowchart of Aeration Procedure 20
Figure 9-5. Overview Flowchart of EDTA Procedure 22
Figure 9-6. Overview Flowchart of Sodium Thiosulfate Procedure 22
Figure9-7. Overview Flowchart of Ci8 SPE Procedure and Methanol Elution Test 26
Figure 9-8. Apparatus Schematic for Graduated pH Procedure 29
Figure 9-9. Overview Flowchart of Graduated pH Procedure 30
Figure 9-10. Overview Flowchart of Cation Exchange SPE Procdeure and Acid Elution Test 32
Figure 9-11. Overview Flowchart of Ulva lactuca Procedure 33
Vlll
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Tables
Table 1-1. Marine Species Discussed in This Document 2
Table 5-1. Estimated Volumes for Phase I Marine TEE Tests 10
Table 6-1. Marine Species Recommended for Use in Marine TIEs 11
Table 9-1. Guidance on Conduct of Baseline Toxicity Test and TIE Procedures 18
Table 9-2. Volumes of EDTA Stock Solution for Additions 19
Table 9-3. Atlantic and Gulf Coast Species Tolerance to EDTA 21
Table 9-4. Pacific Coast Species Tolerance to EDTA 21
Table 9-5. Volumes of Na^^ Stock Solution for Additions 23
Table 9-6. Atlantic and Gulf Coast Species Tolerance to Na^^ 24
Table 9-7. Pacific Coast Species Tolerance to Na^Oj 24
Table 9-8. Atlantic and Gulf Coast Species Tolerance to Methanol 27
Table 9-9. Pacific Coast Species Tolerance to Methanol ' .27
Table 9-10. Operational Species Tolerance Ranges to pH 28
Table 10-1. Results of Toxicity Test with Sodium Dodecyl Sulfate-Spiked Brine and DI
Using 48hr. Mysid, Mysidopsis bahia. Conditions: 30%o, 21 °C 34
Table 10-2. Results of Toxicity Test with Copper-Spiked Brine and DI Using Sea Urchin,
Arbacia punctulata, Mysid, Mysidopsis bahia, and Fish, Menidia beryllina. Conditions: 30%o, 21 °C 34
Table A.I. Summary of TIE Conditions and Test Acceptability Criteria for Amphipod,
Ampelisca abdita, Acute Toxicity Tests 42
Table A.2. Summary of TIE Conditions and Test Acceptability Criteria for Sea Urchin,
Arbacia punctulata, Fertilization Test 43
Table A.3. Summary of Standard Test Conditions and Test Acceptability Criteria for the Topsmelt,
Atherinops affinis, Larval Survival and Growth Test 44
Table A.4. Summary of TIE Conditions and Test Acceptability Criteria for the Red Macroalga,
Champia parvula, Sexual Reproduction Test 45
Table A.5. Summary of Standard Test Conditions and Test Acceptability Criteria for Oyster,
Crassostrea gigas, and Mussels, Mytilus californianus and Mytilus galloprovincialis,
Embryo-Larval Development Tests 46
Table A.6. Summary of TIE Test Conditions and Test Acceptability Criteria for Fish,
Cyprinodon variegatus, Acute Toxicity Tests 47
Table A.7. Summary of Standard Test Conditions and Test Acceptablity Criteria for Albalone,
Haliotis rufescens, Larval Development Test 48
Table A.8. Summary of Standard Test Conditions and Acceptability Criteria for Giant Kelp,
Macrocystis pyrifera, Germination and Germ-Tube Length Test 49
Table A.9. Summary of TIE Test Conditions and Test Acceptability Criteria for Fish,
Menidia beryllina, Acute Toxicity Tests 50
IX
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Tables (continued)
Table A.10. Summary of TEE Conditions and Test Acceptability Criteria for Bivalve,
Mulinia lateralis, Embroyo-Larval Development Test 51
Table A.11. Summary of TIM Test Conditions and Test Acceptability Criteria for Mysid,
Mysidopsis bahia, Acute Toxicity Test 52
Table A.12. Summary of Standard Test Conditions and Acceptability Criteria for the Purple Urchin,
Strongylocentrotus purpuratus, and Sand Dollar, Dendraster excentricus, Fertilization Tests 53
Table A.I 3. Summary of Standard Test Conditions and Acceptability Criteria for the Purple Urchin,
Strongylocentrotus purpurtatus, and Sand Dollar, Dendraster excentricus,
Embryo Development Tests 54
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Acknowledgments
This document is the result of the hard work and dedication of several individuals. In the area of adapting
existing toxicity test methods for TIE application the following persons are acknowledged: Pamela Comeleo
(formerly of Science Application International Corporation (S AIC), Narragansett, RI), Randy Comeleo (Signal
Corporation), Anne Kuhn, (EPA-AED), Glen Modica (formerly SAIC, Narragansett, RI), Mark Tagliabue
(EPA-AED), Diane Griffin and Paul Krause (MEC Analytical Systems, Inc., Tiburon, CA), Bryn Phillips, John
Hunt, and Brian Anderson (University of California-Santa Cruz). The insightful internal technical reviews of
Anne Kuhn (EPA-AED), Rick McKinney (EPA-AED), Peg Pelletier (EPA-AED), Sherry Poucher (SAIC,
Narragansett, RI), Richard Steele (EPA-AED), and Richard Voyer (EPA-AED) are appreciated. The data
furnished by Bob Berger of the East Bay Municipal Utility District (Oakland, CA) on Pacific coast species is
much appreciated. Further, the financial support of EPA Regions IX and X for developing other Pacific coast
species tolerance data is noted. The input of Anne Dailey (EPA, Region X, Seattle, WA) is also acknowledged.
We also thank the personnel at the EPA Mid-Continent Ecology Division (MED) in Duluth, MN for their
hospitality during our visits, willingness to answer our various questions, and sections of their TIE documents;
specifically we thank: Teresa Norberg-King, Gary Ankley, Marta Lukasewycz, and Elizabeth Durhan of the
EPA-MED, Mary Schubauer-Berigan (University of South Carolina, Georgetown, SC), Tim Dawson (Integrated
Laboratory Systems, Duluth, MN), Don Mount and Joe Amato (Asci, Duluth, MN), and Doug Jensen (Sea Grant,
Duluth, MN). We also wish to thank the reviewers of the draft and current editions of this document for their
comments: Teresa Norberg-King (EPA-MED), David Hutton (D.G. Hutton, Inc. Newark, DE), Stephen Bainter,
Allen Chang, Phillip Jennings, Michael Morton, and Wren Stenger (EPA, Region VI, Dallas, TX), and Patti
Tenbrook (East Bay Municipal Utility District, Oakland, CA). Further, we thank the multitude of individuals we
have discussed TEE related issues with at scientific meetings and during phone conversations. Finally, the
excellent services of Edward Shear (Department of English, University of Rhode Island), our technical writer,
are also acknowledged.
NHEERL, AED, Narragansett Contribution No. 1788
XI
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Abbreviations
AED Atlantic Ecology Division, EPA, Narragansett, Rhode Island
Cu Octadecyl
CWA Clean Water Act
DI Deionized Water
DO Dissolved Oxygen
EC50 Median Effect Concentration
EDTA Ethylenediaminetetraacetic Acid
EPA U.S. Environmental Protection Agency
GP2 General Purpose Medium Number 2
LCJO Median Lethal Concentration
MEOH HPLC Grade Methanol
MED Mid-Continent Ecology Division, EPA, Duluth, Minnesota
MSDS Materials Safety Data Sheets
Na^S2O3 Sodium Thiosulfate
NPDES National Pollutant Discharge Elimination System
QAP Quality Assurance Plan
SDS Sodium Dodecyl Sulfate
SLP Standard Laboratory Procedure
SOP Standard Operating Procedure
SPE Solid Phase Extraction
TIE Toxicity Identification Evaluation
TRC Total Residual Chlorine
TRE Toxicity Reduction Evaluation
WQC Water Quality Criteria
xn
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Section 1
Introduction
1.1 Background
The Clean Water Act (CWA 1972), in its original and all
subsequent versions, established a "national policy that the
discharge of toxic pollutants in toxic amounts be prohibited."
The goal of the CWA is to eliminate the discharge of pollutants
into waters in the U.S.; however, this goal is not immediately
attainable. Consequently, the CWA allows for National Pollutant
Discharge Elimination System (NPDES) permits for wastewater
discharges. In order to insure that the CWA's prohibition on toxic
discharges are met, an integrated system of testing procedures has
been developed. This document presents additional methods for
the conduct of Toxicity Identification Evaluation (TIE) which are
part of this testing system.
During the last several years, TIE methods were developed and
applied to freshwater effluents and receiving waters (Parkhurst et
al. 1979; Walsh and Garnas 1983; Gasith et al. 1988; EPA 1991a,
1991b, 1993b, 1993c; Burkhard and Ankley 1989; Norberg-King
et al. 1991). Methods for freshwater sediment TIEs have also
been drafted (Ankley et al. 1992a). Implementation of these
methods has demonstrated the regulatory and scientific utility of
the TIE approach.. For example, TIEs have identified specific
'problem toxicants' in effluents (Schimmel et al. 1988;
Goodfellow et al. 1989; Ankley et al. 1990a; Jop et al. 1991a;
Norberg-King et al. 1991; Amato et al. 1992; McCulloch et al.
1993; Ankley and Burkhard 1992; Burkhard and Jenson 1993;
Schubauer-Berigan et al. 1993) receiving waters (Galassi et al.
1988; Schimmel et al. 1988; Norberg-King et al. 1991; Kszos et
al. 1992), and freshwater sediments (Ankley et al. 1990b;
Schubauer-Berigan and Ankley 1991; Ankley et al. 1992b; Hoke
etal. 1992; Krantzberg and Boyd 1992; Schubauer-Berigan et al.
1993; Wenholz and Crunkilton 1995; Gupta and Karuppiah
.1996). Furthermore, improvements have been incorporated as
methods were applied (Doi and Grothe 1989; Ankley et al.
1990b; Durban et al. 1990; Burkhard et al. 1991; Jop et al.
1991b; Mount and Mount 1992; Wong et al. 1996; Bailey et al.
1996; Hewitt et al. 1996).
1.2 Related Documents
As stated in the forward, this report assumes that the reader is
familiar with several related documents. The report, Methods for
Aquatic TIEs: Phase I Toxicity Characterization Procedures,
Second Edition (EPA 1991b), contains essential background
information on Phase I TIE procedures that is not duplicated in
this report; and in addition, that report describes the related
freshwater TIB procedures. Also, this report assumes that the
reader is familiar with the following related documents: Methods
for Aquatic Toxicity Identification Evaluations: Phase II Toxicity
Identification Procedures for Samples Exhibiting Acute and
Chronic Toxicity (EPA 1993b), Methods for Aquatic Toxicity
Identification Evaluations: Phase III Toxicity Confirmation
Procedures for Samples Exhibiting Acute and Chronic Toxicity
(EPA 1993c), Short-Term Methods for Estimating the Chronic
Toxicity of Effluents and Receiving Waters to Marine and
Estuarine Organisms (EPA 1994), Methods for Measuring the
Acute Toxicity of Effluents and Receiving Waters to Freshwater
and Marine Organisms (EPA 1993a), Short-Term Methods for
Estimating the Chronic Toxicity of Effluents and Receiving Waters
to West Coast Marine and Estuarine Organisms (EPA 1995),
Toxicity Reduction Evaluation Protocol for Municipal
Wastewater Treatment Plants (EPA 1989a), and Generalized
Methodology for Conducting Industrial Toxicity Reduction
Evaluations (TREs) (EPA 1989b), and that this report will be
used in conjunction with these related documents. Methodologies
for both acute and sublethal toxicity testing have been included in
this manual. ,
1.3 Development of Marine TIE Methods
Research conducted at the U.S. Environmental Protection
Agency's (EPA) Atlantic Ecology Division (AED) in
Narragansett, RI has focused on the development of marine TIEs
for saline samples using freshwater TIE methods as models. In
addition, two new TIE manipulations are described: a cation
exchange manipulation and macroalga Ulva lactuca addition
(Burgess et al. submitted; Ho et al. in prep.). Marine TIEs are
performed using marine species on waters discharging into or
from marine environments. The marine TIE methods described in
this document are designed specifically for use with the marine
species listed in Table 1-1. Other TIE or toxicity testing directed
fractionation studies performed in marine waters and sediments
used mutagenic (Grifoll etal.1988; Grifoll et al.1990; Grifoll et
al. 1992; Samiloff et al. 1983; Ho and Quinn 1993a; Ho and
Quinn 1993b) and whole organism assays (Walsh and Garnas
1983; Quilliam and Wright 1989; Higashi et al. 1992; Svenson et
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al. 1992; Weis etal. 1992; Burgess et al. 1993; Bailey et al., 1995;
Burgess et al., 1995; Ho et al., 1995).
Tablo 1-1. Marine Species Discussed in This Document.
Region
Organism
Type
Species
Atlantic and
Gulf Coast
Macroaiga
Echinold
Bivalve
Mysid
Amphipod
Rshes
Pacific Coast Macroaiga
Echlnoids
Bivalves
Gastropod
Rsh
Champia parvula
Arbacia punctulata
Mullnia lateralis
Mysidopsis bahia
Ampelisca abdita
Menldla berylllna
Cyprlnodon variegatus
Macrocystis pyrifera
Strongylocentmtus purpuratus
Dendraster excentricus
Crassostrea glgas
Mytilus califomianus
Mytitus galloprovincialis
Halioiis rufescens
Atherinops affinls
Two fundamental questions addressed during the development of
this manual were: (1) can marine species tolerate the chemicals
used in TEE manipulations and (2) are freshwater TIE chemical
manipulations directly applicable to saline effluent samples? The
tolerance of marine species was addressed with most of the
species in Table 1-1 using TIE additives (e.g.,
ethylenediaminetetraacetic acid (EDTA), sodium thiosulfate
(NajSjC^), and methanol). A series of Phase I TIEs, conducted
with several marine species on four industrial (electrical
equipment) and municipal effluents and several mock effluents
and single chemicals, were used to address whether the freshwater
manipulations were compatible with saline samples (Burgess et
al. 1995; Ho et al. 1995; Ho etal. in prep.). It should be noted that
the Atlantic and Gulf coast species in Table 1-1 have undergone
fairly extensive TIE research with "real" effluents for the
preparation of this document. The Pacific coast species have not
undergone similar research; however, they have been used in the
private sector for the past few years.
Results of tolerance tests for EDTA and Na^C^ readily
demonstrated that these marine species can tolerate TIE
manipulations at concentrations sufficient to alter toxicant effects.
Generally, the effect concentrations for various additives by these
marine species were similar to those for freshwater species (EPA
1991b).
The feasibility of using TIE chemicals and manipulations, such as
EDTA, cation exchange solid phase extraction (SPE), and Cw, to
characterize toxicity in a seawater matrix has been illustrated
through several studies. For example, experiments with the
chelator EDTA investigated the toxicity of metals in seawater
(Sunda and Guillard 1976; Anderson and Morel 1978). Cation
exchange has been used extensively for isolating divalent metals
from seawater (e.g., McLaren et al 1985; Pai and Fang 1990).
Similarly, C,8 reverse-phase chromatography has been applied to
measure the marine partitioning behavior of chemicals between
dissolved organic carbon and aqueous phases (Mills et al. 1982;
Hanson et al. 1988).
As the procedures in this manual illustrate, the majority of the
freshwater methods (EPA 1991a, 1991b) functioned acceptably
when used with marine samples. Two primary exceptions were
the graduated pH procedures designed to characterize pH
dependent toxicants and the conduct of each manipulation at pHs
9 and 11 (EPA 1991b). Seawater has a strong carbonate buffering
system that makes any long-term pH adjustments difficult to
maintain. Alteration of seawater pH with acids, bases, or organic
buffers, while often initially successful, does not permanently
repress the natural carbonate buffering and prevent the return to
initial seawater pH. We found the most effective way to
successfully adjust and maintain the pH of seawater samples (for
the durations required for toxicity testing) was to conduct
exposures in controlled atmospheric chambers. Unlike the variety
of procedures used in the chronic and acute freshwater TIE
methods(EPA 1991a, 1991b, 1993b, 1993c), we found that
controlling pH in atmospheric chambers was the least intrusive,
and only efficient, method of those we tested.
The use of 'closed chambers' was also investigated. In this
approach, exposure chambers were completely filled with the
sample, adjusted to the desired pH with acid or base, and the test
organisms added. Tight-fitting lids sealed the chambers from the
atmosphere. Closed chambers, while useful in some applications
(i.e., where dissolved oxygen was not low) were not as
universally applicable as the controlled atmospheric chambers.
Unlike the freshwater graduated pH procedure which is
conducted at three distinctly different pHs (e.g., 6.0,7.0 and 8.0
(EPA 1991b)), exposures on saline waters are performed at pHs
7, ambient seawater (8.2-8.4), and 9. These pH values were
adopted because: (1) some marine test species demonstrated
unacceptable control survival at pHs less than 7 and (2)
maintaining sample pHs at levels two pH units above or below
ambient pH levels was difficult and often ineffective.
Additionally, shifting sample pHs to 11, resulted in the
precipitation of some seawater hydroxides (Stumm and Morgan
1981) and severely altered seawater composition.
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Section 2
Health and Safety
The following section has been reprinted, with minor
modifications from Methods for Aquatic Toxicity Identification
Evaluations: Phase I Toxicity Characterization Procedures,
Second Edition (EPA 1991b).
Since ITEs involve, by definition, working with effluents of
unknown composition, the accompanying safety measures must
be adequate for a wide spectrum of chemical and biological
agents. Often, one may be able to judge probable concerns from
the type of treatment used. For example, extended aeration is
likely to minimize the presence of volatile chemicals and
chlorinated effluents are less likely to contain viable pathogens.
Exposure to water samples during collection and its use in the
laboratory should be kept at a minimum. Inhalation and dermal
absorption can be reduced by using laboratory hoods and wearing
rubber gloves, laboratory aprons or coats, safety glasses, and
respirators. Further guidance on health and safety for toxicity
testing is described in Walters and Jameson (1984).
In addition to taking precautions with effluent samples, a number
of the reagents that might be used during the tests described in
this manual are known or ,suspected to be toxic to humans.
Analysts should familiarize themselves with safe handling
procedures for these chemicals (DHEW, 1977; OSHA 1976), as
well as the manufacturer's Materials Safety Data Sheets (MSDS).
Use of the compounds may also necessitate specific waste
disposal practices.
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Section 3
Quality Assurance
The following section has been reprinted, with minor
modifications from Methods for Aquatic Toxicity Identification
Evaluations: Phase I Toxicity Characterization Procedures,
Second Edition (EPA 1991b).
Quality assurance is composed of two aspects, quality verification
and quality control. Quality verification entails a demonstration
that the proposed study plan was followed as detailed and that
work carried out was properly documented. Some of the aspects
of quality verification include chain of custody procedures,
statements on the objective of the study and what is known about
the problem at its outset, instrumental log books, and work
assignments. This aspect of quality assurance ensures that a
"paper trail" is created to prove that the work plan has been
covered completely. The quality control aspect of quality
assurance involves the procedures which take place such as the
number of samples to be taken and the mode of collection,
standard operating procedures for analyses, and spiking
protocols.
No set quality assurance program can be dictated for a TEE; the
formula to a successful study will be unique to each situation.
However, adherence to some general guidelines in formulating a
Quality Assurance Plan (QAP) may increase the probability of
success.
In preparing a QAP, enough detail should be included so that any
investigator with an appropriate background could take over the
study at any time. Cross checking of results and procedures
should be built into the program to the extent possible. Records
should be of a quality that can be offered as evidence in court.
Generally, the QAP should be provided in a narrative form that
encourages the user to think about quality assurance. To be
effective, the QAP must be more than a paper exercise simply
restating standard operating procedures (SOPs). It must increase
communication between clients, program planners, field and
laboratory personnel and data analysts. The QAP must make clear
the specific responsibilities of each individual. The larger the
staff, the more important this becomes. While QAPs may seem to
be an inconvenience, the amount of effort they require is
commensurate with the benefits derived.
3.1 TIE Quality Control Plans
A successful TIE is dependent upon a strong quality control
program. Obtaining quality TIE data is difficult because the
constituents are unknown in contrast to quality control procedures
for a standard analytical method for a specific chemical. In such
an analysis, one knows the characteristics of the analyte and the
implications of the analytical procedure being used. Without
knowledge of the physical/chemical characteristics of the analyte,
however, the impact of various analytical procedures on the
compound in question is not known. Further, quality control
procedures are specific to each compound; quality control
procedures appropriate to one analyte may be completely
inappropriate to another.
The problem of quality control is further aggravated because
quality control procedures for aquatic toxicity test may be
radically different from those required for individual chemical
analyses. This additional dimension to quality control requires a
unique framework of checks and controls to be successful. The
impacts of chemical analytical procedures on sample toxicity
must be included. Likewise, procedures used to insure quality
toxicity test results should not impact chemical analyses. For
example, in performing a standard aquatic toxicity test, samples
with low dissolved oxygen (DO) are usually aerated. This practice
may, however, result in a loss of toxicity if the toxicant is volatile
or subject to oxidation.
3.2 Cost Considerations/Concessions
The quality control practices required in any given experiment
must be weighed against the importance of the data and decisions
to be based upon that data. The crucial nature of certain data will
demand stringent controls, while quality control can be lessened
in other experiments having less impact on the overall outcome.
Effluent toxicant identification evaluations require a large number
of aquatic toxicity tests. The decision to use the standard toxicity
test methods described in EPA 1993a, 1994,1995 (involving a
relatively high degree of quality control), must be weighed
against the degree of complexity involved, the time required and
number of tests performed; all of these affect the cost of testing.
For this reason, toxicity tests used in the early phases of the
evaluation generally do not follow these protocols, nor do they
require exacting quality controls because the data are only
-------
preliminary. Phase I, and to a lesser extent, Phase II results are
more tentative in nature as compared to tests performed for
confirmation of effluent toxicant(s) in Phase III.
The progressions towards increasing definitive results is also
reflected in the use of only a few species in the initial evaluation
studies and multiple species in the later stages. The use of several
species of aquatic organisms to assure that the effluent loxicity
has been reduced to acceptable levels is necessary because species
may have different sensitivities to the same pollutant. Quality
control must relate to the ultimate goal of attaining and
maintaining the designated uses of the receiving water. For this
reason, final effluent test results must be of sufficient quality to
ensure ecosystem protection. The use of dilution water for the
toxicity tests that mimics receiving water characteristics (i.e.,
salinity) will help to ensure that the effluent will remain non-toxic
after being discharged into the environment. In the instances
where the effluent dominates the receiving water, the dilution
water should mimic the characteristics of the effluent. In addition,
it is essential that variability in the cause of effluent toxicity be
defined during the course of the TIE so that appropriate control
actions provide a final effluent safe for discharge.
3.3 Variability
The opportunities to retest any effluent to confirm the quality of
initial TEE results will be limited at best. In addition to the shifting
chemical and lexicological nature of the discharge over time,
individual effluent samples stored in the laboratory change.
Effluent constituents degrade at unknown rates, as each toxicant
has its own rate of change. The change in a sample's toxicity over
time represents the cumulative change in all of the constituents,
plus that variation resulting from experimental error. Some
guidelines for assessing and minimizing changes in sample
chemistry and toxicity are discussed in later sections. Regardless
of the precautions taken to minimize sample changes, a sample
cannot be retested with certainty that it has not changed.
3.4 Intra-Laboratory Communication
Quality control procedures in chemistry and biology can be quite
different. For example, phthalates are a frequent analytical
contaminant requiring special ^precautions that are not of
lexicological concern. The lexicological problem presented by
zinc levels typically associated with new glassware are of no
concern to those performing organic analyses. The difference in
glassware cleanup procedures is an example of one of many
differences thai musl be resolved. Cleaning procedures musl be
eslablished to cover the requiremenls of both. Time schedules for
analyses must be detailed in advance. One cannot assume toxicanl
slabilily; therefore, time delays between the biological and
chemical analysis of a sample cannot be tolerated.
3.5 Record Keeping
Throughout the TEE, record keeping is an important aspect of
quality verification. All observations, including organism
symptoms, should be documented. Details that may seem
unimportant during testing may be crucial in later stages of the
evaluation. Investigators must record test results in a manner such
that preconceived notions about the effluent toxicants are not
unintentially reflected in the data. TEEs required by stale or
federal pollution control agencies may require thai some or all
records be reviewed.
3.6 Phase I Considerations
Effluenl toxicity is "tracked" through Phases I, n, and III using
aquatic organisms. Such tracking is the only way lo deled where
the loxicanls are until their identity in known. The organism's
response musl be considered as the foundation and therefore, the
toxicity tesl resulls musl be dependable. System blanks (blank
sampled carried through procedures and analyses identical lo
Ihose performed on effiuenl sample) are used extensively
tiiroughoul Ihe TIE lo detect toxic artifacts added during the
effluent characterization manipulations. With the exception of
tesls intended lo make Ihe effiuenl more toxic, or silualions in
which a known amounl of toxicity has been intentionally added,
sample manipulation should nol cause Ihe effiuenl toxicity to
change.
There are many sources of toxicity artifacls in Phase I. These
include: excessive ionic slrength resulting from the addition of
acid and base during pH adjustment formation of toxic products
by acids and bases, contaminated air or carbon dioxide sources,
inadequate mixing of test solutions, contaminanls leached from
filters, pH probes, solid phase extraction (SPE) columns, and Ihe
reagents added and their contaminants. The appropriate toxicity
data for the reagent chemicals used in Phase I and common
aquatic tesl organisms are provided as needed in subsequenl
sections of this document
Frequently, toxic artifacls are unknowingly introduced. For
example, some pH meters wilh refillable electrodes can act as a
source of silver which can reach toxic levels in the solutions being
measured for pH. This is especially a problem where there is a
need lo carefully mainlain or track solution pH. Using pH
electrodes wilhoul membranes avoids the silver problem (which
can only be detected by the profuse use of blanks).
Oil in air lines or from compressors is a source of contamination.
Simple aeration devices, such as those sold for use wilh aquaria
are better as long as caution is laken lo prevenl contamination of
die laboratory air which is laken in by Ihe pump.
Worsl case blanks should be used lo belter ensure thai loxicily
artifacls will be recognized. Tesl chambers should be covered to
prevent contamination by dust and to minimize evaporation.
Since small volumes are often used, evaporation must be
controlled. For some manipulations, plastic disposable tesl
chambers are recommended lo avoid problems related lo the reuse
of tesl chambers. Cups from Ihe same lol should be spol-checked
for toxicity.
-------
Glassware used in various tests and analyses must be cleaned not
only for the chemical analyses but so that toxicity is not
introduced either by other contaminants or by residues of
cleaning agents. Since the organisms are sensitive to all chemicals
at some concentrations, all toxic concentrations must be removed
and not just those for which analyses are being made.
Randomization techniques, careful observance of organism
exposure times and the use of organisms of approximately the
same age ensure quality data. Standard reference toxicant tests
should be performed with the aquatic test species on a regular
basis and control charts should be developed (EPA 1993a, 1994,
1995). During Phase I it will not be known how much the toxicity
of the reference toxicants varies over time compared to the
toxicant(s). When the toxicants are known, they should be used
as the reference toxicant. Reference toxicant tests should be
performed to coincide with the TIE testing schedule.
3.7 Phase II Considerations
In Phase II, a more detailed quality control program is required.
Interferences in toxicant analysis are for the most part unknown
initially but as toxicant identifications are made, interferences can
be determined. Likewise instrumental response, degree of toxicant
separation, and detector sensitivity can be determined as
identifications proceed.
3.8 Phase in Considerations
In Phase III of a TEE, the detail paid to quality control and
verification is at the maximum. This phase of the study responds
to the compromises made to data quality in Phases I and II. For
this reason, confidence intervals for toxicity and chemical
measurements must be calculated. These measurements allow the
correlation between the concentration of the toxicants and effluent
toxicity to be checked for significance based on test variability.
Effluent manipulations prior to chemical analyses and toxicity
testing are minimized in this phase in an effort to decrease the
chance for production of artifacts. Field replicates to validate the
precision of the sampling techniques and laboratory replicates to
validate the precision of analyses must be included in the Phase
III quality control program. System blanks must be provided.
Calibration standards and spiked samples must also be included
in the laboratory quality control program. Because an attempt will
be made to correlate effluent toxicity to toxicant concentration,
spiking experiments are important in determining recovery for the
toxicant(s). These procedures are feasible because the identities
of the substances being measured are known.
The toxicants being analyzed can be tested for using pure
compounds, thereby alleviating the need for a general reference
toxicant. Because the test organism also acts as an analytical
detector in the correlation of effluent toxicity with toxicant(s)
concentration, changes in the sensitivity of the test organism must
be known. This is best achieved by using the same chemicals
identified for the reference toxicants.
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Section 4
Equipment, Supplies, and Facilities
Equipment necessary to perform each of the Phase I procedures
is listed in Section 9 under each manipulation. In addition, basic
analytical laboratory equipment such as pH meters, pumps
(vacuum and fluid), pipettors, and the capacity for maintaining
compressed gas cylinders and regulators are required.
A reliable source for large numbers (hundreds) of test organisms
is essential for TIE work. It is recommended that on-site culturing
facilities be used to prevent TIE activity from being subject to
seasonal availability of field collected organisms or delays in
shipping from suppliers.
A supply of "clean" saline water is necessary as a diluent, a
natural seawater control, a performance control for reference
toxicant testing (EPA 1994), and as a source of hypersaline brine.
Large supplies of brine solutions (100%o) can be prepared, stored,
diluted with deionized water (DI) to desired salinities, and used
in batches to insure seawater consistency and to avoid seasonal
fluctuations in water quality. At AED, saline water has been
prepared from both natural seawater and GP2 synthetic seawater
(e.g., EPA 1994). In addition, water used for test organism
culturing should come from the same source (EPA 1994). For a
discussion of acceptable source waters and their quality control,
one should consult the reports: Short-Term Methods for
Estimating the Chronic Toxicity of Effluents and Receiving Water
to Marine andEstuarine Organisms, Second Edition (EPA 1994)
and Short-Term Methods for Estimating the Chronic Toxicity of
Effluents and Receiving Water to West Coast Marine and
Estuarine Organisms (EPA 1995). Further discussion will be
found in Section 5.4: Salinity Adjustments and Dilution Water.
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Section 5
Sample Collection, Handling, Salinity Adjustment, and Dilution
5.1 General Collection
Effluents should be collected in clean plastic or glass containers.
Generally, the collection site should be the same as the monitoring
site specified in the NPDES permit unless a specific concern
suggests otherwise (cf. EPA 1994). Examples of when it would
be appropriate to use alternate or additional collection sites
include: (1) better access to a sampling point between the final
discharge and the discharge outfall; (2) if the processed waste is
chlorinated prior to discharge and it is desired to obtain a sample
prior to chlorination; or (3) there is a desire to evaluate the
toxicity of the influent to municipal waste treatment plants prior
to their being combined with other wastewater streams or non-
contact cooling water. It may be possible to collect enough
additional sample at the time of compliance sampling if a TIE is
to be done. EPA (1991b) provides further guidance on sample
handling and includes a discussion of the choice between plastic
and glass containers that is useful, since certain types of toxicants
may absorb to certain surfaces. Additionally, the documents (EPA
1994,1995) should be consulted for collection requirements.
The time, date, location, duration and procedures used should be
recorded for effluent sample collection. During collection,
aeration and transfer of effluents should be minimized to reduce
the loss of volatile chemicals. Any additional observations such
as color, turbidity, chlorine odor, or unusual sampling conditions
(i.e, heavy rain) should be noted. If an industrial effluent is to be
tested, it may be useful to record any available information on the
current production levels and types of operating processes. The
condition of the facilities treatment system should also be
determined by the individual collecting the sample. In addition,
it is recommended that total ammonia, total residual chlorine
(TRC), pH, dissolved oxygen (DO), salinity/conductivity, and
temperature be recorded upon arrival of the sample. At AED,
salinity is usually measured using a refractometer for marine
samples. Figure 5-1 provides a sample log book page for
recording of sampling data.
Stored or shipped samples should be kept at 4°C and tested for
toxicity within 36 hours. Limited observations on a single
industrial effluent suggest that the timing of salinity adjustment
(i.e., at time of collection or immediately before testing) was not
critical. Parallel tests showed no toxicity differences over a 16 day
period (Ho et al. 1995). However, this observation is not
universal and it is suggested that an initial toxicity test be
conducted on the day that the sample arrives.
The volume requirements for performing Phase I of a TIE will
vary according to the toxicity of the sample. The more toxic the
sample, the less effluent sample will be needed. To a certain
extent, the choice of tests to be performed may also affect the
desired sample volume. Table 5-1 provides estimates of the
volumes of sample needed for the Phase I marine TIE tests.
5.2 Composite versus Grab Samples
There are several factors to consider when designing a sample
collection scheme (EPA 1994). A 24-hour composite sample is
more representative of total effluent toxicity and is more likely to ,
collect the toxic fraction if it is intermittent (i.e., timed with an
industrial process). However, a composite sample may make the
toxic fraction more difficult to detect because of dilution. In
addition, compositing is expensive and time consuming. The
simpler and less expensive grab sample is a "snap shot" of
effluent toxicity at the time of collection. A grab sample,
however, has the disadvantage that it may miss intermittent
toxicity altogether, or conversely, collections synchronized to a
suspected manufacturing process or seasonal discharge can result
in a very toxic sample. The choice of sampling method
consequently will depend on the goals of a given TIE and the
nature of the plant from which it is being collected. For example,
if the sample is being taken from a wastewater treatment plant
with a two-day detention time, there is little need for the use of
composite samples. Please consult EPA 1991b,1993a for further
discussion of this issue.
5.3 Pre- or Post- Chlorinated Samples
The decision to sample a municipal effluent before or after the
addition of chlorine will depend on the objectives of the study.
While addition of sodium thiosulf ate helps determine how much
of the toxicity is due to chlorine, it may also remove other
oxidants and some metals, thus complicating the interpretation of
results. Further, the presence of chlorine will often mask the
effects of other less abundant toxicants. It is recommended to test
both pre- and post- chlorinated samples to determine what portion
of toxicity is attributable to chlorine.
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Sample Log No.:_
Date of Arrival:
Date and Time
of Sample Collection:_
Facility:.
Location:
NPDES No.:_
Contact:
Phone Number:.
Sampler:
Sample Type:
D Grab D Composite
D Glass O Plastic
D Prechlorinated
D Chlorinated
D Dechlorinated
Specific Sampling Information:
Sample Conditions Upon Arrival:
Temperature:
PH:
Total Alkalinity:_
Total Hardness:.
D Conductivity:..
or
D Salinity:
Total Residual Chlorine:.
Total Ammonia:
Dissolved Oxygen:
Conditions of treatment system at time of sampling:
Status of process operations/production (if applicable):
Comments:
Figure 5-1. Example Data Sheet for Logging in Samples.
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Tabla 5-t. Estimated Volumes for Phase I Marine TIE Tests.*
Characterization Step
Volume
Needed (ml)t
Total (ml)
Chemistry -500*
Initial ~ 100
Baseline -120
Filtration ~ 100
Aeration ~ too
EDTA Addition -100
NajSjOj Addition -100
U/vafecfuca Addition -200
C,,SoHd Phase Extraction -100
Cation Solid Phase Extraction -100
Graduated pH ~ 100
pH 7 -100
AmbtentpH -100
pH 9 -100
-2000
Values are for three replicates for initial and baseline tests and two
replicates in the manipulations. Test volumes are assumed to be 20
mi'replfcato. Values are directly applicable to Atlantic and Gulf Coast
species, Pacific Coast species may require greater volumes.
Assumed sample tested at 100% and diluted by 50% splits. Initial and
baseline Include five treatments, and manipulations include three
treatments.
Includes physical measures (e.g., temperature, salinity), pH,
ammonia, chlorine, and dissolved oxygen.
5.4 Salinity Adjustments and Dilution Water
Dilution water for marine TIEs is hypersaline brine (100%o)
adjusted to the desired salinity with DI water. Brine is made by
slowly evaporating filtered natural seawater until the salinity
reaches 100%o (do notexceed this level), filtering it through a one
micron filter, and storing it in. 20 liter cubitaihers® or
polycarbonate water cooler jugs (EPA 1994).. The seawater
should be of high quality and collected on an incoming tide to
minimize the possibility of contamination. The brine and DI
mixture is a very consistent dilution water as any given "batch"
of brine can be used for a year or more.
Directions for the use of hypersaline brine for salinity adjustment
is also described in EPA 1993a. Basically, for freshwater salinity
adjustment (0%o), the volume of brine (V^^) added is described
by the relationship: V^^S^ x V^/S^, where SM is the
desired test salinity, V^is the test sample volume, and S^^ is the
brine salinity.
Using hypersaline brine for effluent salinity adjustment causes a
degree of sample dilution that is dependent upon the initial sample
salinity and the desired test salinity. For example, the greatest
concentration of a freshwater effluent (i.e., 0%o) adjusted to 30%o
with 100%o hypersaline brine is 70%. For purposes of continuity
and simplicity, all further discussion of effluent concentration in
this document refers to salinity adjusted samples. Therefore,
100% salinity adjusted sample means the effluent concentration
is between 70% and 100%.
An alternative approach to adjust effluent salinity is the addition
of artificial seawater salts like GP2. Although this method has not
been tested at AED with Phase I Marine TIEs, this method has the
advantage that it does not dilute the effluent sample, and
consequently may be useful in certain circumstances. It is not
recommended that the artificial seawater be substituted for brine
as dilution water, as brine contains the necessary trace metals,
biogenic colloids, and some of the microbial components
necessary for the adequate growth, survival, and/or reproduction
of marine and estuarine organisms (EPA 1994). Consequently,
the use of artificial seawater salts may be problematic in some
cases. Conversely, for a very weakly toxic samples, where brine
dilution would be problematic, the addition of sea salts may be
required. Finally, if a sample is hypersaline (i.e., >34%o), dilution
with DI water may be needed. In general, a TIE should be
performed using dilution waters similar to that used in the toxicity
test(s) which triggered the TIE.
Concentrations selected for testing should be bracketed around
known or estimated LC50 and EC50 values. Determining test
concentrations for initial testing requires some estimations, unless
the effluent has been previously tested. Starting at the highest
possible concentration and using logarithmic splits results in a
wide distribution of concentrations. Concentrations for the
baseline and the manipulations testing should be established by
bracketing theLC50 or EC50 values generated in the initial test.
10
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Section 6
Toxicity Testing
6.1 Test Species
The toxicity testing species described in this document are listed
in Table 6-1. The table indicates species recommended for use in
Pacific, Atlantic, and Gulf Coast testing. The reader may note
small changes to these methods compared to methods reported
elsewhere (EPA 1993a, 1994, 1995). Changes were made to
adapt methods for TIE use.
Both acute and chronic (i.e., sublethal) endpoints are presented.
In the table, endpoints are labeled as "mortality" for acute toxicity
tests while short-term chronic tests specify an endpoint other than
Table 6-1. Marine Species Recommended for Use in Marine TIEs
mortality. The chronic tests include the macroalga sexual
reproduction and germination and growth test using Champia
parvula and Macrocystis pyrifera, and the echinoid sperm cell test
using sea urchins Strongylocentrotus purpuratus and Arbacia
punctulata, and the echinoid fertilization test with the sand dollar
Dendraster exeentricus. Bivalve and gastropod development tests
with Mulinia later alls, Crassostreagigas, Mytilus californianus,
Mytilus galloprovincialis, and Haliotis rufescens are used. The
acute tests include those for fishes: Menidia beryllina,
Cyprinodon variegatus, and Atherinops cffinis, the mysid
Mysidopsis bahia, and the amphipod Ampelisca abdita.
Organism
Species
Region
Endpoint*
Exposure (hr.)
Macroalga
Echinoid
Bivalve
Gastropod
Mysid
Amphipod
Fish
Champia parvula
Macrocystis pyrifera
Arbacia punctulata
Strongylocentrotus purpuratus
Dendraster excentricus
Mulinia lateralis
Crassostrea gigas
Mytilus californianus
Mytilus galloprovincialis
Haliotis rufescens
Mysidopsis bahia
Ampelisca abdita
Menidia beryllina
Cyprinodon variegatus
Atherinops affinis
Atlantic and Gulf Coasts
Pacific Coast
Atlantic and Gulf Coasts
Pacific Coast
Pacific Coast
Atlantic and Gulf Coasts
Pacific Coast
Pacific Coast
Pacific Coast
Pacific Coast
Atlantic and Gulf Coasts
Atlantic and Gulf Coasts
Atlantic and Gulf Coasts
Atlantic and Gulf Coasts
Pacific Coast
sexual reproduction
germination/growth
fertilization
fertilization
or development
fertilization
or development
mortality/development
development
development
V
development
development
mortality
mortality
mortality
mortality
mortality/growth
48
48
1
"*1
72
~1
72
24
48
48
48
48
48
48
48
48
48-168
' Acute tests are indicated by an endpoint of mortality, chronic tests by an endpoint other than mortality.
11
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6.2 Test Methods
This section provides brief descriptions of the marine Phase I TIE
toxicily tests. The TIE toxicity testing methods are very similar to
conventional methods described in EPA 1993a, 1994, and 1995
except for minor changes to account for exposure volume
reductions and feeding protocols. The Appendix provides test
parameters of the methods.
In addition to the noted tests, we have conducted sediment
interstitial water TiEs with the marine amphipod Ampelisca
abdita and bivalve Mulinia lateralis. Further, we have used
conventional NPDES toxicity tests, using the mysid Mysidopsis
bahia and sea urchin Arbacia punctulata, in sediment interstitial
water TIEs.
6.2.1 Macroalga Sexual Reproduction or
Germination/Growth Tests
These methods use sexual reproduction of the macroalga
Champia parvula and the germination and growth of the kelp
Macrocystis pyrifera to measure toxicity. The Champia parvula
procedure involves measuring the development of cystocarps on
female plants. The Macrocystis pyrifera procedure quantifies the
germination of settled zoospores and length of the germination
tube.
Changes to the Champia parvula method (EPA 1994) for TIE
purposes include a reduction in test solution volume from 100 mL
to 20 mL and use of 50 mL petri dishes as the exposure chambers.
Further, when conducting the Graduated pH Procedure,
photosynthesis will increase pH by approximately 0.1 - 0.4 units.
This is to be expected but should not exceed 0.5 pH units. Test
parameters of these methods are presented in the Appendix.
6.2.2 Echinoid Sperm Cell Tests
The echinoid sperm cell tests have reduced fertilization of
exposed gametes as an indication of toxicity. Dilute sperm
solutions are exposed to test samples for 20 to 60 minutes.
Following this exposure eggs are added to the samples and
fertilization is allowed to occur. Twenty minutes after egg
addition the test is terminated by the addition of a fixative.
Fertilization is determined by microscopic examination of an
aliquot from each treatment, and is shown by the presence of a
membrane surrounding the egg.
Little has been changed in the sperm cell test methods to
accommodate TIE applications. The existing method (EPA 1994,
1995) is extremely useful for TIE applications due to its use of
very small exposure volumes (i.e., 5 mL), demonstrated
sensitivity, and relatively rapid exposure. For conducting the
Graduated pH Procedure, we have found it useful to keep the test
scintillation vials in the atmosphere controlled chambers during
the 20-60 minute sperm exposure to maintain desired pH values
(cf. Section 95). Test parameters are presented in the Appendix.
6.2.3 Echinoid, Bivalve, and Gastropod
Development Tests
The development tests involve several marine species and
developmental endpoints (EPA 1994,1995). Echinoid procedures
assess the formation of the larval test. Bivalve and gastropod tests
evaluate the growth of the larval shell. Microscopic analysis is
used to determine test and shell condition. All tests are performed
in small volumes (5-10 mL) and are amenable for TIEs. Test
parameters of the methods are found in the Appendix.
6.2.4 Acute Mysid and Fish Tests
For TIEs, three Atlantic and Gulf Coast test methods are
conducted similarly and use a mortality endpoint. Experimental
designs consist of static 48-hour exposures with five organisms
in 10 to 20 mL of test solution (i.e., 30 mL exposure cups).
Mysid (Mysidopsis bahia) toxicity tests use 1-5 day animals. For
fish testing, 9 to 14 day old Menidia beryllina, and 1-14 day old
Cyprinodon variegatus are used. A TIE method for using 9-15
day old fish Atherinops affirds with small test volumes has not
been fully developed. Test parameters are given in the Appendix.
Noteworthy changes to the standard marine acute methods (EPA
1993a) are the reduction in sample volume from approximately
100 mL to 10 or 20 mL and reduction in exposure period from 96
hours to 48 hours. When conducting the Graduated pH Procedure
the organisms will add CO2 to the exposure chambers resulting in
decreases in sample pHs. Also, feeding test organisms Anemia
will further reduce chamber pHs. To avoid drastic reductions in
sample pH, especially in the pH 9.0 treatment, feed test organisms
small rations. The Appendix details these and other changes to the
standard methods.
6.2.5 Other Marine Species
Included in various sections of this document are references to
other marine species, besides some of the common marine
NPDES toxicity testing species, which can be incorporated into
the marine TIE. Currently, these species are the amphipod
Ampelisca abdita and the bivalve Mulinia lateralis. At AED, they
have proven valuable in developing marine sediment TIE
methods, but they can also be used to assess effluent toxicity. At
the time this document was prepared, insufficient information was
available to include the West Coast survival and growth method
using the mysid Holmesimysis costata.
As with the other marine toxicity tests that use "whole
organisms," major changes to the current methods with Ampelisca
abdita (Scott and Redmond 1989) include reducing exposure
volumes to approximately 10 mL and exposure duration to 48
hours. An evaluation of a 24-hour embryo-larval development
test using the bivalve Mulinia lateralis is continuing.
12
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Section 7
Statistical Methods
Test results are used to calculate point estimates (e.g., LC50s and
EC50s). EPA recommends probit, Spearman-Karber, trimmed
Spearman-Karber, and Inhibition Concentration (ICp; p is the
percent'effect, e.g., mortality, reduced growth, etc.) as means to
calculate point estimates (EPA 1993a, 1994,1995).
Conversion of point estimates to toxic units (e.g., Toxic Units =
100/LQoOr 100/ICp) eliminates the inverse relationship between
toxicity and LC50 or EC50 values making TIE interpretation easier.
Furthermore, if the concentration of toxicants are known for a
given sample, the toxic units for the individual toxicants can be
compared to the total sample toxic units. The sum of the toxic
units of the individual toxicants should be similar to the total toxic
units of the sample, assuming they are all measured, bioavailable,
and that their toxicities are additive.
13
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Section 8
Ion Imbalance
The methods in this document do not directly address toxicity
caused by ion imbalance as recorded in some types of effluents
(e-g., McCulloch et al. 1993). If an ion imbalance is suspected in
& sample, several studies are available that discuss how to
characterize and identify such toxicity (McCulloch et al. 1993;
Mount et al. in press; Douglas and Home in press; Douglas et al.
in press; Tietge et al. in press). It should be noted that although an
ion imbalance may impart an apparent 'salinity' to a sample, in
most cases the sample is not truly marine. Marine salinity has a
specific composition of ions at relatively consistent proportions
to one another. Effluents with ion imbalances seldom will have
truly marine composition.
An approach for determining if an ion imbalance may be present
in a given sample is to perform an anion and cation analysis for
major elements (e.g., sodium, calcium, potassium, magnesium,
chloride, sulfate, and bromide). Measured values can be
compared to toxicity information (Douglas et al. in press), marine
Water Quality Criteria (WQC), and known marine background
levels (Millero and Sohn 1992) to assess if an imbalance may
occur.
14
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Section 9
Toxicity Identification Evaluation Procedures
A Phase I marine TIE characterization consists of the following
recommended components (see also Figure 9-1):
• Initial Toxicity Test (§9.1, §6, Appendix)
• Baseline Toxicity Test (§9.2, §6, Appendix)
• Filtration Procedure (§9.3)
• Aeration Procedure (§9.4)
• EDTA Procedure (§9.5)
• Na^Oj Procedure (§9.6)
• C18 Solid Phase Extraction (SPE) Procedure (§9.7)
• GIS SPE Methanol Elution Test (§9.8)
• Graduated pH Procedure (§9.9)
• Cation Exchange SPE Procedure (§9.10)
• Cation Exchange SPE Acid Elution Test (§9.11)
• Ulva lactuca Procedure (§9.12)
Figures 9-1 and 9-2 give an overview of the design of a typical
marine Phase I TIE. One should note, however, that because of
the varying durations of the toxicity tests used in a marine Phase
I TIE that the indications of 'DAY 1' and 'DAY 2' may not
always be appropriate.
While the Initial and Baseline Toxicity Tests are based on routine
toxicity testing exposures, the other procedures (e.g., EDTA and
Na^Oj) are specialized and require some knowledge of the
sensitivity of the testing organisms to specific chemicals. The
following sections describe the objectives and general procedures
for conducting the TIE manipulations. Familiarity with the
freshwater TIE procedures (EPA 1991a, 1991b) is recommended.
Specific information concerning numbers of treatments, types of
species to test, volumes of effluent to prepare, and duration of
exposures are only recommendations and may require
modification depending upon each application. Blanks are
described for each procedure and involve using the control
seawater (often brine and DI) in the manipulations before the
sample.
9.1 Initial Toxicity Test
9.1.1 General Approach
The objective of an Initial Toxicity Test for a TIE is to determine
the toxicity of a given sample. The Initial Toxicity Test is
performed on DAY 1 of the marine TIE process, while the
Baseline Toxicity Test and procedures are generally conducted on
DAY 2 (Figure 9-1 and Figure 9-2).
9.1.2 Materials
• Materials, organisms and apparatus necessary to conduct
toxicity test (See Section 6 and Appendix).
9.1.3 Procedural Overview
Design of Initial Toxicity Test
Initial Toxicity Tests • have a serial dilution design. We
recommend five concentrations (post-salinity adjusted): 100%,
50%, 25%, 12.5%, 6.25% and a control (i.e., 0%) with one to
three replicates (three preferred) per concentration (Figure 9-2).
However, if a sample is very toxic, this range of concentrations
will be too high and a set of lower concentrations will be needed.
Therefore, if data from compliance testing suggests high toxicity,
one should adopt a different set of concentration ranges including,
the necessary lower non-toxic concentrations.
Results of Initial Toxicity Test
Initial Toxicity Test results are used to judge how toxic the
sample is toxic and if a TIE on the given sample is warranted. If
so, Initial Toxicity Test results will be used to establish effluent
test concentrations for subsequent TIE manipulations.
From our experience, it may be difficult, but not impossible, to
conduct a TIE when the toxic units of a sample from the Initial
Toxicity Test using the most sensitive species are <2 (i.e., LC50 >
50%). It is critical, however, to insure that the toxic units are <2
by repeating toxicity tests and using smaller concentration
intervals (i.e., bracketing the effect concentrations more closely).
Table 9-1 provides some other criteria as to when decisions can
be made about proceeding with the Baseline Toxicity Test and
TIE procedures.
15
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Table 9-1. Guidance on Conduct of Baseline Toxicity Test and TIE
Procedures
Toxldty Test Species
Guidelines to Make Decision to Proceed
Cbampta parvirfa
Aibada punctulata
Mu!ini3 lateralis
Mysktopsls bahla
AmpeHsca abdita
Menldla beryttlna
Cyprinodon variegatus
Macrocystis pyrifera
Strongylocontrotus
purpwatus
Dsndraster excontrlcus
Cmssostreaglgas
Mytilus californianus
Mytilus galtoprovlnclalls
Halhtis rvfonscens
Athorinops afffnls
Due to duration of exposure, one may
have to use results of other tests or
delay Initiation of TIE
Results of initial toxidty test (Day 1)
48 hr. results
24 hr. results; if no toxicity, use 48 hr.
results
24 hr. results; if no toxicity, use 48 hr.
results
24 hr. results; if no toxicity, use 48 hr.
results
24 hr. results; if no toxicity, use 48 hr.
results
48 hr. results
fertilization: Day 1 results;
development: 72 hr. results
fertilization: Day 1 results;
development: 72 hr. results
48 hr, results
48 hr. results
48 hr. results
48 hr. results
24 hr. results; if no mortality, use 48 hr.
results, up to 168 hr.
Because of the long duration of the algal Champia parvula
reproduction test, it is difficult to follow the standard TIE format.
Therefore, it is necessary to use test results from other species to
predict Champia parvula's response or perform the initial test five
to seven days earlier than the other species (assuming no
alterations in toxicity due to storage). Champia parvula is often
the most sensitive NPDES toxicity testing species when tested
with municipal and industrial effluents (Schimmel et al. 1989)
and therefore, a prediction of high toxicity is warranted.
Conversely, because of the short duration of the sea urchin
Arbacia punctulata sperm cell test, an entire TIE can often be
conducted in two days, or even one day, if prior information
about the toxicily of the sample is available and appropriate
dilutions can be prepared. The fertilization endpoints of toxicity
test using Strongylocentrotus purpuratus and Dendraster
-n be used similarly.
9.2 Baseline Toxicity Test
92.1 General Approach
Results of the Baseline Toxicity Test are used for comparison
with the Initial Toxicity Test and TIE manipulations. Objectives
are to: (1) determine if sample toxicity has changed relative to
Initial Toxicity Test and (2) provide a baseline for comparison
with results of TIE procedures. A Baseline Toxicity Test is
performed following the Initial Toxicity Test, in conjunction with
the TIE Manipulations (Figures 9-1 and 9-2). In Figure 9-2, we
indictate the use of one replicate per test concentration and three
concentrations per procedure. These values for the study design
are not recommendations but must be determined according to
study objectives, logistics and economic constraints.
92.2 Materials
• Materials, Organisms and Apparatus necessary to conduct
toxicity test (See Section 6 and Appendix).
92,3 Procedural Overview
Design of Baseline Test
Baseline Toxicity Tests have a serial dilution design. Usually five
concentrations: 100%, 50%, 25%, 12.5%, 6.25% and a control
(0%) with three replicates/concentration are used. However, if the
Initial Toxicity Test demonstrates greater toxicity, lower dilutions
may be justified.
Results of Baseline Toxicity Test
Because of the variety of species potentially being tested, Baseline
Toxicity Test results will be dependent on the toxicity test being
used. However, regardless of species, the questions being
answered are the same for each toxicity test, "Did sample toxicity
change relative to the Initial Toxicity Test and did the TIE
procedures decrease or increase toxicity compared to the Baseline
Toxicity Test?" Quantitatively, these questions are answered by
comparing toxic units between the various procedures. Sources
of toxicity are implied from the magnitude of difference between
the baseline and TIE procedures results. However, statistical
evaluations of significance may be precluded, for most TIE tests,
because of insufficient replication within TIE experimental
designs. See Section 10 for further discussion of the interpretation
of TIE results.
9.3 Filtration Procedure
9.3.1 General Approach
Filtration is used to determine whether toxicants pass through a
filter or are associated with particles. Note for effluents, samples
can be filtered before being passed through the C18 column (See
Section 9.7). However, filtration may create artifacts (e.g.,
toxicant sorption to filter) that may need to be addressed in
evaluating results. Filtrates are the substances that pass through
the filter.
18
-------
9.3.2 Materials
• Oil-free air pump and tubing—to force sample through
filtration apparatus.
• 0.45 um (or similar size) glass fiber filters and filtration
apparatus—4o separate particles from sample. For samples
that are suspected to contain toxic metals, organic
membrane filters may be used instead of glass filters.
However, a comparison of filter types may be necessary.
9.3.3 Procedural Overview
(1) Filter brine and DI blank; remove brine and DI blank filtrate
for testing (Figure 9-3).
(2) Without changing filters, filter the effluent. Change filters as
often as necessary to prevent clogging, repeating step 1 as needed.
Save all filters for possible later analysis (i.e., wrap in aluminum
foil or Parafilm® and store at 4°C). Remove filtrate for testing.
(3) Use filtered brine and DI blank as diluent.
9.4 Aeration Procedure
9.4.1 General Approach
Samples are aerated to determine if toxicity is due to volatile
toxicants (e.g., ILjS or volatile hydrocarbons).
9.4.2 Materials
• Oil-free air pump and tubing—to aerate sample.
• Graduated cylinders—to hold sample while aerating.
• 1-10 mL pipettes—attached to tubing and placed in sample
during aeration. Fritted end on pipettor tubing will
improve aeration.
9.4.3 Procedural Overview
(1) Samples should be aerated in a hood.
(2) Separately pour sample, and brine and DI blank into
graduated cylinders (Figure 9-4).
(3) Connect 1-10 mL pipettes to air pump tubing and place
pipettes into graduated cylinders.
(4) Turn pump on, adjust air flow to establish many small
bubbles, and let sample aerate for 1 hour.
(5) Test aerated sample using aerated brine and DI as diluent.
9.5 EDTA Procedure
9.5.1 General Approach
EDTA (ethylenediaminetetraacetic acid) is an organic chelating
agent that preferentially binds with divalent cationic metals, such
as copper, nickel, lead, zinc, cadmium, mercury, and other
transition metals (Garvan 1964). Studies have demonstrated that
when a metal is bound to the EDTA molecule, the toxicity of the
metal is greatly reduced (e.g., Sunda and Guilliard 1976). In this
procedure, EDTA is added to samples to evaluate metal toxicity.
Table 9-2 provides recommended exposure concentrations and
Tables 9-3 and 9-4 report results of tolerance testing with
Atlantic, Gulf, and Pacific Coast species.
9.5.2 Materials
• EDTA stock solution (25 g EDTA/L DI (74.4 mmols
EDTA/L) refrigerated)
• Glass Erlenmeyer flask (100-250 mL), microbalance,
weighing pan, and Teflon®-coated stirbar—-for preparing
EDTA stock solution.
• Adjustable microvolumepipetter (10-1000 uL range) and
tips—for dispensing EDTA stock solution to exposure
chambers.
Table 9-2. Volumes of EDTA Stock Solution for Additions (25g EDTA/L
stock solution)
Replicate
Volume (mL)
5
10
20
40
100
200
Volume (uL) EDTA
Solution/Replicate
12
24
48
96
240
480
Volume (uL) EDTA
Solution/Replicate
for M. pyrifera
10
20
40
80
200
400
9.5.3 Procedural Overview
(1) Prepare EDTA stock solution: weigh-out 2.78 g of
EDTA°2ILp reagent (sodium salt) and add to 100 mL of DI. Mix
with a Teflon*-coated stirbar until EDTA is completely in
solution. This stock solution is stable and can be stored
refrigerated (Figure 9-5).
(2) Set-up dilution series with sample. Generally, a TIE dilution
series consists of three effluent concentrations and a blank (brine
and DI), however, the statistical design of the TIE should be
based on the objectives of the study, logistics, and economic
constraints. The concentrations tested should bracket observed
toxicity, based on the Initial Toxicity Test. Do not add the
organisms yet!
(3) Tolerance testing of several Atlantic, Gulf, and Pacific Coast
species indicates that most organisms can tolerate 60 mg EDTA/L
(0.22 mmols EDTA/L) (Table 9-3,9-4). Given the ECSO of 100
mg/L for M. pyrifera, it is advisable to use 50 mg/L (0.14
mmol/L) for the EDTA Procedure with that species. This
concentration of EDTA is sufficient to chelate about 22 mg Total
M^/L (equal molarity of metals). Use Table 9-2 to determine the
volume of EDTA stock (25 g EDTA/L) to add to test containers:
(4) Add specified volume, mix thoroughly and allow EDTA and
sample to interact for about 3 hours. Do not add the organisms
yet!
(5) After 3 hours, add test organisms to dilution series.
19
-------
Prepare
Equipment
V
Filter Brine/DI
for Blank
Fitter Sample
Jsing Same Filter
Save Filter
Save Brine/DI
for Blank/Diluent
Save Filtered
Sample
Test Blank
1,
Test Filtered
Sample Using
Filter Blank as
Diluent
Figure 9-3. Overview Flowchart of Filtration Procedure.
Pour Sample into
Graduated Cyl.
Pour Blank into
Graduated Cyl.
V V
Connect Pipette
to Pump
V
Place Pipette
into Grad. Cyl.
Connect Pipette
to Pump
V
Place Pipette
into Grad. Cyl.
ii i. « "•— —
"-i»
Turn on Pump
V V
Adjust Air Flow
V V
Aerate for
1 hr
Test Blank and
Sample (Using
Blank as Diluent)
Flflur* 9-4. Overview Flowchart of Aeration Procedure.
20
-------
Table 9-3. Atlantic and Gulf Coast Species Tolerance to EDTA (mg/L) (see Appendix for specific salinity and temperature).
Duration
(hr)
Species
(± 95% Confidence Intervals)
1.2
24
48
72
96
Champia
parvula
(EC*)
-
-
165
(94.2-240)
-
-
Arbacia
punctulata
(ECSO)
300
(300-300)
-
-
-
'
Mulinia Mysidopsis
lateralls bahia
(EC*,) (LCM)
-
318
(309-323)
288 313
(283-295) (300-326)
318
(303-327)
315
(298-325)
Ampelisca
abdita
(LCM)
-
240
(150-350)
175
(65.6-205)
164
(50-200)
150
(28.2-188)
Menidia
beryllina
(LCso)
-
362
(354-369)
353
(347-359)
353
(344-359)
350
(344-359)
Cyprinodon
variegatus
(LCM)
-
>600
(-)
542
(534-547)
348
(345-349)
346
(344-349)
-Not Available
Table 9-4. Pacific Coast Species Tolerance to EDTA (mg/L) (see Appendix for specific salinity and temperature).
Duration
(hr)
Species
, of EC50 (± 95% Confidence Intervals)
Macrocystis
pyrifera*
(EC*)
Strongylocentrotus
purpuratusf
(EC60)
Dendraster
excentricusf
Crassostrea
gigas
(EC50)
Mytilus
califomianus
Mytilus
galloprovincialis
(EC*,)
Haliotis
rufescens
Atherinops
(EC60)
-Not Available
* Germination Endpoint
t Fertilization Endpoint
i 7 Day Growth Endpoint
>750
>750
24
48 100 -
72
96 - -
-
>750 >750 >750 300
. - . . .
- - 300
21
-------
Prepare EDTA
Stock Solution
V
Set up Dilution
Series and Blank
V
Determine Volume of
EDTA Stock Solution to
Add to Test Containers
Add EDTA
Solution and Mix
V Y
Allow Sample and
EDTA to Interact for 3 hr
Add Organisms
Rgure 9-5. Overview Flowchart of EDTA Procedure.
Prepare Sodium
Thiosulfate Stock Solution
*
Set Up Dilution
Series and Blank
i
! }
Determine Volum
Stock Solution
Add to Test Conta
1
eof
to
iners
- — -
Add Sodium
Thiosulfate Solution
V *
Allow Sample and
SodiumThiosulfate
to Interact for 1 hr
1 V
Add Organisms
Rgure 9-6. Overview Flowchart of Sodium Thiosulfate Procedure.
22
-------
9.6 Sodium Thiosulfate Procedure
9.6.1 General Approach
Addition of sodium thiosulfate (Na^C^), a reducing agent, to a
sample containing oxidants (e.g., chlorine or bromine), results in
a reduction reaction (White 1972) that may decrease sample
toxicity. For example, chlorine (Clj) added to sewage effluent
prior to release would undergo the following reaction:
Table 9-5. Volumes of Na,S2O3 Stock Solution for Additions (15g
jCVL stock solution)
C/+2e-
2CI-
where the 2 electrons (e") provided by the thiosulfate (S2O3)
reduce the toxic diatomic chlorine (CLj) to nontoxic chlorine ions
(CT). In this test, Na£2Q3 is added to effluent samples to evaluate
whether toxic oxidants are present. Table 9-5 provides
recommended exposure concentrations and Tables 9-6 and 9-7
report the results of tolerance testing with Atlantic, Gulf, and
Pacific coast species.
9.6.2 Materials
Stock Solution (15 g NaAOs/L DI (94.9 mmols
). This solution cannot be stored. Make up
prior to use.
• Glass Erlenmeyer flask (100-250 mL), microbalance,
weighing pan, spatula and TefW-coated stirbar—for
preparing Na^C^ stock solution.
• Adjustable microvolume pipetter (10-1000 uL range) and
tips—for dispensing Na^C^ stock solution to exposure
chambers.
9.6.3 Procedural Overview
(1) Make-up Na&Oj Stock Solution
• Weigh-out 2.35 g of Na2S2O3°5H2O reagent, add to 100
mL of DI in a flask with a TefW-coated stirbar, and
allow, to mix until all the Na^Oj is completely in solution
(Figure 9-6).
(2) Use of NajSjOs in TIE
• (a) Set up dilution series with sample. Generally, a TIE
dilution series will consist of three effluent concentrations
and blank (brine and DI). Concentrations should bracket
observed toxicity, based on the Initial Toxicity Test. Do
not add organisms yet!
• (b) Use Table 9-5 to determine the volume of Na^O,
stock to add to test chambers. Tolerance testing of several
Atlantic, Gulf, and Pacific coast toxicity testing species
indicates that all organisms can tolerate 50 mg Na^Oj/L
(0.32 mmol Na^Os/L) (Table 9-5).
(3) Add NajSjC^and allow to interact for about one hour. Do not
add organisms yet!
(4) After one hour, add test organisms to exposure chambers.
Replicate Volume (mL)
5
10
20
40
100
200
Volume (uL) Na.,S.,O3
Solution/Replicate
17
34
68
136
340
680
9.7 C18SPE Procedure
9.7.1 General Approach
The C,8 solid phase extraction (SPE) column manipulation is used
to determine if toxic components are nonionic organic
compounds. In the manipulation, reverse phase liquid
chromatography is applied to extract nonionic organic toxicants
from the aqueous sample. Operationally, filtered test solutions
(i.e., samples and controls) are passed through a disposable C18
column and the post-column effluent tested for toxicity (Figure 9-
5). Absence of toxicity in the post-column effluent suggests that
organic toxicants were active in the original sample. Elution of the
column with methanol can return toxicants to aqueous solution to
confirm toxicity (see Section 9.8).
Tables 9-8 and 9-9 provide information on the tolerance of
Atlantic, Gulf, and Pacific coast species to methanol.
9.7.2 Materials
• Disposable C18 column(s)—for performing C18
manipulation (e.g., Waters (Sep-Pak Environmental Plus
1000 mg / 2.0 mL column))
• HPLC Grade Methanol (MEOH)—for activating C18
column(s).
• Low flow metering pump (~10 mL/min) and tubing—for
forcing sample through C18 column.
•. Separatory funnel—to serve as a sample reservoir.
• Erlenmeyer flasks—for collecting post-C18 effluent.
23
-------
Table 9-6. Atlantic and Gulf Coast Species Tolerance to Na2S2O3 (mg/L) (see Appendix for specific temperature and salinity)
Duration
(hr)
Species
L.CK, or ECs,, (± 95% Confidence Intervals)
1,2
24
48
72
96
Champla
parvula
(ECM)
-
-
181
(141-773)
-
-
Artacla Mullnia Mysidopsis
punctulata lateralis bahia
ICf* \ tGf* \ /I /"* \
I'-^-'HV l^^so) t'-'-'so)
>15000
>200
9400 164
(8990-9760) (155-169)
121
(116-126)
119
(113-125)
Ampelisca
abdita
(LCM)
-
>300
>300
223
(122-283)
150
(87.5-214)
Menidia
beryllina
(LCJ
-
12200
(11300-13000)
12000
(11500-12600)
11500
(10700-12400)
9550
(8330-10600)
Cyprinodon
variegatus
(LCM)
-
>15000
>15000
>15000
>15000
-Not Available
Ttblo 9-7. Pacific Coast Species Tolerance to Naj,S2O3 (mg/L) (see Appendix for specific temperature and salinity).
Duration
(lw)
<1.0
24
48
72
96
Species
LCM or ECM (± 95% Confidence Intervals)
Macrocystis Strongylocentrotus Dendraster Crassostrea Mytilus Mytilus Haliotis
pyrifera* purpuratusf excentricust gigas califomtanus gallopmvincialis rufescens
(ECM) (EC^) (ECJ (EC^j) (ECJ (EC^) (EC60)
>1000 >1000 - ...
-
200 - - >500 >500 >500 10000
-
-
Atherinops
affinis$
(ECM)
-
-
-
.
10000
-Not Available
* Germination Endpolnt
t Fertilization Endpoint
i 7 Day Growth Endpolnt
24
-------
9.7.3 Procedural Overview
(1) Preparation of Tubing
• (a) Connect pump, sample reservoir and column with
tubing. Do not attach column. Pump 25 ml of DI water
followed by 25 ml of MEOH through the entire system to
remove any contamination. Throughout this procedure a
flowrate of 10 mL/min is used (Figure 9-7).
(2) Preparation of C18 Column
• (a) Attach C18 column to tubing (check manufacturer's
recommendations for wetting volumes and total capacity
of the column). Pass recommended volume of MEOH
through the column. Do not let the column dry out.
• (b) Pass recommended volume of DI through the column.
Do not let the column dry out; to avoid drying the
column, leave a small volume of DI in the tubing.
(3) Blank Sample
• (a) Pass the brine and DI filtered blank through the wet
prepped column.
• (b) Allow first 10-20 ml of brine and DI to pass into waste
container before collecting sample. Collect enough post-
column brine and DI to conduct toxicity tests (the column
can now go dry).
(4) Re-prepare Column
• From Step 2, the same column may be used. Do not let
the column dry out in between the preparatory steps or
before adding the filtered sample.
(5) Sample
• (a) Pass the filtered sample through the wet prepped
column.
• (b) Collect enough post-column sample to perform toxicity
tests. Column can now go dry.
(6) Toxicity Testing
• (a) Prepare test dilutions using post-column sample and
post-column brine and DI.
• (b) Add organisms.
9.8 Methanol Elution Test
9.8.1 General Approach and Materials
If following the C^ Column SPE Procedure (Section 9.7), and the
post-column effluent shows reduced toxicity, it is recommended
that the column be eluted with methanol to attempt to verify
sample toxicity is due to an organic toxicant. Tables 9-8 and 9-9
provide information on the tolerance of several marine species to
methanol.
Materials are the same as in the Cw Column SPE Procedure
(Section 9.7.2) except the column is now "loaded."
9.8.2 Procedural Overview
(1) Preparation of Tubing
• Same as C18 Column SPE Procedure, Section 9.7.3.(l).(a)
(Figure 9-7).
(2) Elution of Column
• (a) The reader is advised to consult EPA 1993b for
specific details of column elution. The information here is
only cursory.
• (b) Attach loaded column to tubing. Pass at least one
column bed volume of methanol through column twice
using a flowrate of 10 mL/min. Volume reduce eluate if
necessary.
• (c) Collect methanol hi container and return to initial
sample volume with clean brine and DI. Use only enough
methanol to be well below toxicity values in Table 9-8 and
9-9.
(3) Toxicity Testing
• (a) Prepare test dilutions using reconstituted sample and
brine andDI.
• (b) Add organisms.
9.9 Graduated pH Procedure
9.9.1 General Approach
The pH of marine waters is largely controlled by the
concentration of dissolved CO2 present:
CO2
H+ + HCO3" * H+ + CO3
As the concentration of CO2 increases, the carbonic acid
and bicarbonate (HCO3~) dissociate and the reaction goes to the
right, generating an excess of hydrogen ions (EP) which decreases
sample pH. Conversely, if CO2 is absent the hydrogen ions are
found in an associated form and sample pH increases. In this
procedure, sample pH is manipulated to determine if pH
dependent toxicants are responsible for observed toxicity. For
example, if sample toxicity increases with increasing sample pH,
toxicants such as ammonia (NH3) are suspected (Miller et al.
1990). Conversely, if sample toxicity increases with decreasing
sample pH, toxicants such as hydrogen sulfide (HjS) are
suspected. Also, in freshwater, the toxicity of some metals is
known to change as a function of pH (Schubauer-Berigan et al.
1993). For marine samples, exposures are conducted at three
pHs: 7, ambient (7.9-8.4), and 9 using atmosphere-controlled
chambers (Figure 9-8).
25
-------
Connect Pump,
Reservoir, and
Column with Tubing
Pump 25mL Dl
through Entire
System
I
Pump 25 mL MEOH
through Entire
System
M
Prepare Column*
Pass Brine Blank over
Conditioned Column
Recondition Column*
V
Pass Filtered Sample
over Column
* Column Preparation
Attach Column to Tubing
*
Pass Recommended Volume
of MEOH over Column
V
Pass Recommended Volume
of Dl over Column
Allow First 10-20 mL
of Blank or Sample to
Pass into Waste
Container before
Collecting
V V
Prepare Test
Dilution Series
i i
Methanol Elution Test
Prepare Tubing
I
Attach Loaded Column
I
Pass Methanol
through Column
I
Collect Methanol and Return to
Sample Volume If Necessary
Using Clean Brine and Dl
Prepare Test Dilutions
Using Reconstituted Sample
Toxicity Tests
Toxicity Tests
Perform Methanol
Elution Test"
Figure 9-7. Overview Flowchart for C18 SPE Procedure and Methanol Elution Test (** Consult EPA 1993b).
26
-------
Table 9-S. Atlantic and Gulf Coast Species Tolerance to Methanol (%v/v) (see Appendix for specific temperature and salinity)
Duration
(hr)
Champ/a Arbacia
parvula punctulata
(ECM) (EC60)
1.2 - 9.31
(9.30-9.33)
24 -
48 0.13 -
(0.10-0.26)
72 -
96 -
Species •
LC60 or EC50 (± 95% Confidence Intervals)
Mulinia Mysidopsis
lateralis bahia
(EC^) (LCa,)
•- •
2.43
(2.37-2.46)
2.18 2.35
(2.14-2.25) (-)
2.35
(-)
2.30
(2.26-2.33)
Ampelisca
abdita
(LCJ
-
3.75
(3.75-3.75)
3.21
(3.01-3.33)
1.25
(0.98-1.91)
0.75
(0.59-0.86)
Menidia
beryllina
(•Ac)
-
2.56
(2.44-2.63)
2.33
(2.14-2.50)
1.77
(1.50-2.17)
1.55
(1.32-181)
Cyprinodon
variegatus
(LC60)
-
3.89
(3.81-3.95)
3.67
(3.38-3.94)
3.39
(2.90-3.93)
3.33
(2.85-3.75)
-Not Available
Table 9-$. Pacific Coast Species Tolerance to Methanol (%v/v) (see Appendix for specific temperature and salinity).
Duration
(hr)
Macmcystis Strongylocentrotus
pyrifersf purpuratus\
(ECM) (ECM)
<1.0 - 3.78
(3.47-4.11)
24 - -
48 - -
72 -
96 - -
Species
LCM or ECM (± 95% Confidence Intervals)
Dendraster
excentricus\
(ECM) .
3.50
(3.32-3.69)
-
-
-
Crassostrea Mytilus Mytilus Haliotis
gigas californianus galloprovincial/s rufescens
(ECjo) (ECjo) (ECso) (ECSO)
-••••. - - /
-
3.14 2.26 3.55
(2.69-3;59) (2.02-2.57) (3.34-3:74)
-
-
Athefinops
affinisj.
.(ECJ
• -
• -
'--
: -
'-
- Not Available
* Germination Endpoint
f Fertilization Endpoint
i 7 Day Growth Endpoint
27
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9.9.2 Materials
• pH 7.0 and pH 9.0 atmospheric chambers — for
maintaining sample pHs at desired levels. Our atmospheric
chambers were constructed from plexiglass in two sizes:
30 cm wide x 25 cm deep x 16 cm high and 80 cm x 40
cm x 30 cm. These chambers are not completely sealed
from the ambient atmosphere but do maintain a positive
pressure ensuring atmospheric gases do not enter. Locating
the gas ports in the center of the chambers is advised to
improve gas mixing.
• pH meter, stir plate, Teflon*-coated stirbars and calibration
buffers — for monitoring sample pHs.
• Cylinders of CO2, air, low CO2 or low hydrocarbon air
(e.g., Zero-Grade® or CO2-Free®, (M.G. Industries,
Valley Forge, PA)), and regulators for above cylinders
(CGA 320 (COj), CGA 346 (Air) & CGA 590 (low
COj)) — to flow into pH chambers.
• CO2 Scrubber — to remove CO2 contamination from low
CO2 air (e.g., Merck, Damstadt, Germany).
• Precision flow meters (CO2 meter should be capable of 2
mL/min) — for metering gas flow to chambers.
9.9.3 Sample Preparation
Samples are prepared for testing as described in the other TIE
procedures, but with the following special preparations
(Figure 9-9).
pH7
(1) Approximately 24 hours before the manipulations are to be
conducted, initiate CO2 and air flowlnto the pH 7.0 chamber.
Adjust the COjflow to approximately 2% of the air flow (e.g., ~2
mL/min CO2to 98 mL/min of air).
(2) Approximately 18 hours before toxicity testing is to begin,
check gas flow and place separate containers of the sample and
blank (brine and DI) into the chamber. Let equilibrate overnight.
pH 8 (Initial)
Generally, pH 8 is the blank (brine and DI) and sample under
initial atmospheric conditions. Because of the strong carbonate
buffering capacity of seawater, the pH of these samples will
usually range from 7.90 to 8.40. Set up this series at the same time
as the pH 7 and 9.
pH9
(1) Approximately 24 hours before manipulations are to begin,
adjust the low CO2 air flow to the pH 9.0 chamber to 150 - 300
(2) Adjust needed volumes of blank (brine and DI) and sample
with 1 M sodium hydroxide (NaOH) to pH 9.0±0.3. CAUTION!
The amount of NaOH needed varies based on the sample;
overshooting pH 9.0 can result in excessive toxicity due to high
salinity from excess sodium addition. After adjusting the pH,
place the blank and sample volumes into the pH 9 chamber and
close tightly.
(3) Approximately 18 hours before toxicity testing is to begin,
check the pHs of the blank and sample to ensure that pH 9 is
being maintained.
9.9.4 Procedural Overview
(1) Before conducting the toxicity test, check pHs of test
solutions. For tests with marine animals (except for bivalves),
pHs should be 7.0+0.3 for pH 7, ambient pH for pH 8, and
9.0±0.3 forpH 9 (Table 9-10). When testing marine plants, pHs
should be 7.5+0.2 for pH 7, ambient pH for pH 8 and 9.0±0.3 for
pH 9 (Table 9-10). Adjusted pH samples can be maintained
outside of the chambers for short time periods (e.g., 5-10
minutes) to allow for preparing and monitoring the test.
(2) Set up toxicity test with test solutions and place dilution series
in the appropriate chambers for the duration of test. Table 9-10
provides acceptable pH ranges for exposing Atlantic, Gulf, and
Pacific coast marine organisms. Note that bivalve species are
particularly sensitive to low pHs.
(3) Check gas flow and pH at least every 24 hrs. NOTE: Because
of organism respiration or photosynthesis, pHs in the respective
chambers will decrease or increase from nominal values, but
changes should not exceed ± 0.3 pH units. If necessary, adjust
gas flow to maintain desired pHs.
Table 9-10. Operational Species Tolerance Ranges to pH*
Species
pH Range
Atlantic and Gulf Coasts
Champia pan/via
Arbacia punctulata
Mulinia lateralls
Mysidopsis bahia
Ampelisca abdita
Menidia betyllina
Cyprinodon variegatus
Pacific Coast
Macrocystis pyrifera
Strongylocentrotus purpurtatus
Dendraster excentricus
Crassostrea gigas
Mytilus califomianus
Mytilus galloprovincialis
Haliot/s rufescens
Atherinops affinls
7.4-9.2
7.2-9.1
8.0-8.8
6.8-8.8
7.1-9.0
Insufficient Data
6.6-8.8
7-9
-7.8-8.5
Insufficent Data
7.5-8.5
8.0-8.5
7.5-8.5
7-9
7-9
* See Appendix for specific salinity and temperature.
28
-------
CO2 Scrubber
pH9
Chamber
pH7
Chamber
Figure 9-8. Apparatus Schematic for Graduated pH Procedure.
29
-------
24 hr Before Manipulations
Initiate Carbon Dioxide
Flow into Chamber
Adjust Carbon Dioxide
Flow to 2% of Air Flow
18 hr before Testing,
Check Air Flow and Place
Sample and Blank
in Chamber
J.
Set Up Sample
and Blank
/
24 hr Before Manipulations
Initiate Low Carbon Dioxide
Air Flow into Chamber
JL
Adjust Low Carbon Dioxide
Air Flow
Adjust pH of Sample
and Blank with NaOH
Place Sample and
Blank in Chamber
JL
18 hr Before Testing,
Check pH of Sample
and Blank
\
Before Testing, Check pH Values
JL
*
Set Up Toxicity Test with Test Solutions, Place Dilution Series in Appropriate
Chambers for Duration of Test. Check Gas Flow and pH at Least Every 24 hr
Figure 9-9. Overview Flowchart for Graduated pH Procedure.
30
-------
9.10 Cation Exchange SPE Procedure
9.10.1 General Approach
The cation exchange manipulation is used to determine if toxic
components are cationic in nature (e.g., metals). Cation exchange
chromatography is applied to remove cationic toxicants from the
aqueous sample. This manipulation can be used to support the
EDTA manipulation (cf. Section 9.5) and with elution verify
potential metal toxicity. Operationally, filtered test solutions (i.e.,
samples and controls) are passed through a disposable cation
exhange column and the post-column sample tested for toxicity
(Figure 9-10). Reduced toxicity in the post-column sample
suggests that cationic toxicants are active (Burgess et al.
submitted). Not all interferences with the cation exchange SPE
procedure have been identified; therefore, it is important to
perform the acid elution to verify metal toxicity.
Resulting post-cationic exchange column effluent is then tested to
determine if the toxicity has been removed. The cation exchange
column is activated with a combination of methanol and DI.
9.10.2 Materials
• Disposable cation exchange column(s)—for performing
cation exchange manipulation (e.g., Supelco LC-WCX
(500 mg/3mL tube))
« IMHClAcid
• IMNaOH
• Low flow metering pump (-0.5-10 mL/min) and
tubing—for forcing sample through cation exchange
column.
• Separately funnel—to serve as effluent sample reservoir.
• Erlenmeyer flasks—for collecting post-column effluent.
9.10.3 Procedural Overview
(1) Preparation of Tubing
• (a) Connect pump, sample reservoir and column to tubing.
Do not attach column. Pump 10 mL of 1 M HC1 followed
by 25 mL of DI through the entire system to remove any
contamination. Throughout column preparation a flow of
7-10 mL/min is used.
(2) Preparation of Cation Exchange Column
• (a) Attach cation exchange column to tubing. For Supelco
LC-WCX (3 mL/500 mg) column, the following
procedure is recommended; for other types, check
manufacturer recommendations. Using a flow rate of 2.5
mL/min, pass 2 mL of methanol through column. Do not
let the column dry out
• (b) Pass 6 mL of DI through the column. Do not let the
column dry out. To avoid drying the column, leave a
small volume of DI in the tubing.
(3) Blanks
• (a) Pass the brine and DI filtered blank through the wet
prepared column.
• (b) Allow first 5 mL of brine and DI to pass into a waste
container before collecting blank. Collect enough post-
column brine and DI to conduct toxicity tests. Check pH
to insure residual acid is not contaminating the sample. Do
not let the column dry out
(4) Effluent Sample
• (a) Pass the filtered sample through the wet prepared
column.
• (b) Collect enough post-column sample to perform toxicity
test. Column can now go dry. Check pH to insure residual
acid is not contaminating sample.
(5) Toxicity Testing
• (a) Prepare test dilutions using post-column sample and
post-column brine and DI. . .
• (b) Add organisms.
9.11 Cation Exchange SPE Acid Elution Test
9.11.1 General Approach and Materials
If following the Cation Exchange SPE procedure (Section 9.10),
the post-column sample is non-toxic, it is recommended that the
column be eluted with 1 M HC1 to verify sample toxicity due to
metal toxicants.
Materials for this test are the same as the Cation Exchange SPE
Procedure (Section 9.10.2).
9.11. 2 Procedural Overview
(1) Preparation of Tubing
• Same as Cation Exchange SPE Procedure, Section
(2) Elution of Column
• (a) Attach loaded column to tubing. Pass 6 mL 1 M HC1
through column using a flowrate of 0.5 mL/min.
• (b) Collect HC1 in container and return sample to original
volume with clean brine and DI and adjust pH with
sodium hydroxide (Figure 9-10).
(3) Toxicity Testing
• (a) Prepare test dilutions using reconsituted sample and DI.
• (b) Add organisms.
31
-------
Connect Pump,
Reservoir, and
Column with Tubing
Pump10mL1 MHCI
through Entire
System
Pump 25mL Dl
through Entire
System
Prepare Column*
V
Pass Brine Blank over
Conditioned Column
Pass Filtered Sample
over Column
Allow First 5mL
of Blank to Pass into
Waste Container
before Collecting
Sample
Check pH
\
1
Prepare Test
Dilution Series
*-^
Check pH
V
Toxicity Tests
* Column Preparation
Attach Column to Tubing
V
Pass 2 mL Methanol
over Column
*
Pass Dl
over Column
Acid Elution Test
Prepare Tubing
*
Attach Loaded Column
*
Pass HCI
over Column
V
Collect HCI, Increase
Volume Using
Clean Brine and Dl
and Check pH
Prepare Test Dilutions
usinq Reconstituted Sample
*
Toxicity Tests
Perform Acid
Elution Test
Rgure 9-10. Overview Flowchart for Cation Exchange SPE Procedure and Acid Elution Test.
32
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9.12 Viva lactuca Procedure
9.12.1 General Approach
The objective of this manipulation is to remove ammonia from
seawater samples by addition of a marine macrophyte Ulva
lactuca, commonly known as sea lettuce. Ulva lactuca is a
macrophyte that has the ability to uptake, store, and utilize large
amounts of ammonia. Ulva lactuca has historically been used to
clean-up effluents in aquaculture (Cohen and Neori 1991; Neori
etal. 1991) and has proven effective in removing environmental
concentration of ammonia from seawater (Ho et al. in prep.).
9.12.2 Materials
• Ulva lactuca 5g/60mL of sample
• Oil-free air pump, tubing, and pipettes
• Containers—to hold 60 mL sample, Ulva lactuca, and
allow for aeration
• Light source (~75 uE/m2/s)
• Temperature 15-20°C. Temperatures over 20°C hasten the
degradation of Ulva lactuca during storage.
9.12.3 Procedural Overview
(1) Ulva lactuca Collection and Storage
• Collect Ulva lactuca from a clean site. Sort through plants
and discard any with white or yellowing tips. Remove any
surficial organisms and hold static in 30%o clean seawater
in aerated jars under 16:8 light:dark condition until use.
Sea lettuce is held in static systems, not flow-through
conditions to minimize the exposure of the plant to
nutrient concentration. Presumably, if the plant is
"starved", it will uptake ammonia more quickly when
placed in the sample. Maximum holding time for Ulva
lactuca is four days but should be used within 24 hr for
optimal results (Figure 9-11).
(2) Ulva lactuca Addition
• Remove Ulva lactuca from holding jars using forceps,
gently pat dry and place in salinity adjusted sample under
lights with gentle aeration for five hours.
• (b) Remove Ulva lactuca from sample.
(3) Ulva lactuca Removal
• (a) Remove Ulva lactuca from sample.
• (b) Prepare toxicity dilutions with Ulva lactuca treated
brine and DI and sample.
Collect
Ulva lactuca
and Store for Use
Remove
Ulva lactuca
from Holding Jars
Pat Plants Dry
and Weigh 5 g
per Replicate
Place Plants in
Salinity Adjusted
Sample and Blank
Hold under Lights
and Gentle Aeration
for Five Hours
Remove Plants
Prepare Test Dilutions
from Blank and Sample
Treatments
Figure 9-11. Overview Flowchart for Ulva lactuca Procedure.
33
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Section 10
TIE Interpretation
To determine the efficacy of these methods in characterizing
unknown toxicants, we performed some marine TIE
manipulations on two spiked brine and DI samples (i.e., mock
effluent). One sample contained 40 mg/L of the reference toxicant
sodium dodecyl sulfate (SDS) and the other copper sulfate (1.0
mg copper/L). Results from these TIEs conducted on very simple
samples provide insight into the complexity of interpreting marine
TIE data.
10.1 Sodium Dodecyl Sulfate (SDS)
In this TIE, tests were conducted with the mysid Mysidopsis
bahia. Results are presented in Table 10-1.
Tablo 10-1. Results of Toxicity Test with Sodium Dodecyl Sulfate-Spiked
Brine and DI Using Mysid, Mysidopsis bahia. Conditions:
30%.,21°C.
Manipulation
Toxic Units
Initial
Baseline
Not Performed'
6.8
EDTA Addition 6.7
Naaspa Addition 7.5
Filtration 6.7
PostC,.
No Toxicity t
* Historic data used to determine baseline exposure concentration.
t 0% Mortality In highest concentration (40 mg SDS/L)
As these data demonstrate, the C,8 column removed all toxicity,
and there was no significant change in toxicity in the other
manipulations except for the possible increase in toxicity caused
by sodium thiosulfate. These results should be interpreted that
organic compounds are responsible for all or most of the toxicity.
Although C18 column elution data for this example analysis is not
available, the reader is reminded that that procedure is highly
recommended (cf. Section 9.8).
10.2 Copper
Copper toxicity tests were conducted with the sea urchin Arbocia
punctulata, mysid Mysidopsis bahia, and fish Menidia beryllina.
Results are presented in Table 10-2
Table 10-2. Results of Toxicity Test with Copper-Spiked Brine and DI
Using Sea Urchin, Arbacia punctulata, Mysid, Mysidopsis
bahia, and Fish, Menidia beryllina. Conditions: 30%», 21 °C.
Manipulation
Toxic Units
Arbacia Mysidopsis
punctulata bahia
Initial 5.0 2.4
Baseline 11.9 1.7
EDTA <2.0* <2.0f
Addition
Na-.S-.O.j 2.2 5.3
Addition
Filtration 5.0 2.1
Aeration 14.5 5.8
PostC,, 3.1 <2.0^
100% Fertilization at 50% effluent.
t 1 00% Survival at 50% effluent.
if 60% Survival at 50% effluent.
§ 100% Survival in 25% effluent.
# 90% Survival in 25% effluent.
** 60% Survival in 25% effluent.
Menidia
beryllina
8.6
5.3
<4.0§
<4.0#
<4.0 **
6.4
<4.0§
Results of this TIE are not as easily evaluated as was SDS;
clearly, EDTA removed the most toxicity in all cases with all
three species, but other manipulations removed toxicity as well.
Toxicity to Arbacia punctulata increased between the Initial
Toxicity Test and the Baseline Toxicity Test by 6.9 toxic units.
This significant variablility in the response of the sea urchin
sperm cell test is not uncommon when measuring copper toxicity.
Morrison et al. (1989) reports a coefficient of variation of 46%
for Arbacia punctulata in reference toxicant tests with copper.
34
-------
All manipulations removed some amount of toxicity to A.
punctulata except aeration, which increased toxicity about 2.5
toxic units. Toxicity to the mysid was fairly low but both the
sodium tniosulfate and aeration manipulations increased toxicity.
Exposures to the fish demonstrated a small reduction in toxicity
between the Initial and Baseline Toxicity Tests and all
manipulations reduced toxicity except for aeration.
Possible reasons for these results are: 1) sodium thiosulfate
reduces the toxicity of some metals (EPA 1991b; MED, Duluth,
personal communication), 2) filtration of metals through a glass
fiber filter may result in adsorption of copper to the filter surface,
and 3) C,8 chelates some metals like copper. Aeration results that
were consistent for all species suggest that the sample volume was
reduced, and consequently, metal concentrations increased.
However, it has been observed that EDTA seldom reduces the
toxicity of any other toxicants except metals (MED, Duluth,
personal communication); therefore, Table 10-2 results strongly
support the presence of metals toxicity. If this sample had been a
complex mixture of toxicants from an industrial; or municipal
plant, evaluation of these initial results would have suggested a
combination of metals and organics as being the sources of
toxicity.
10.3 Summary of Results
Phase I as described in this guidance'document is dedicated to
toxicity characterization. In Phases n and in, the TIB includes
more advanced approaches: for example, the use of analytical
chemistry (EPA 1993b, 1993c). For the exercise with copper
above, analytical chemistry would progress the characterization
from types of toxicants to specific toxicants by demonstrating the
presence of elevated levels of copper. In general, comparison of
these concentration data for various contaminants to the
sensitivities of the test species in the scientific literature, including
EPA WQC, may help to elucidate which types of toxicants to
include or exclude from consideration. Specifically, toxicity
information on toxic metals, organics and ammonia are readily
available from these sources. Use of this information will help
individuals conducting marine Tffis to establish sensitivity
patterns for the various marine species (e.g., Arbacia punctulata
is very sensitive to most divalent transition metals and insensitive
to most organics and ammonia). These sensitivity patterns in turn
become diagnostic TIE tools contributing to the determination of
what toxicants are active. Any complementary data (e.g.,
historical, collection, site) will assist in the characterization.
The investigator needs to keep in mind potential interferences to
the TIE manipulations; although the methods are designed to be
specific to single classes of toxicants, they may not be so in
practice. Documented interferences or 'side effects' include: the
pH manipulations changing the toxicity of both metals and ionic
organic toxicants (Schubauer-Berigan et al. 1993; Spehar et at.
1984); and the C18 SPE can sorb certain metals from seawater;
• filtration may remove metals and nonionic organic toxicants from
solution while Ulva lactuca removes nonionic toxicants (Ho et al.
in prep.). Also, not all possible interferences associated with the
cation exchange SPE have been determined. Despite the problems
interferences can create when interpreting a TIE, advantage may
be taken of interferences to aid in the characterization of
toxicants.
Following the Phase I of a marine TIE are Phases n
(Identification) and HI (Confirmation). The reader is advised to
refer to EPA I991b, 1993b, and 1993c for guidance in
performing these phases.
35
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Section 11
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40
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Appendix
Summary of Test Conditions and Acceptability
The tables in this appendix summarize test conditions and acceptability for the Phase I Marine TIE characterization tests. Because routine
TIE toxicity testing methods are not currently available for all Pacific Coast species, the standard test conditions are provided. Tables
correspond to those in EPA 1993a, 1994,1995. Readers should refer to these references for detailed procedural outlines of the toxicity
tests, and use the tables in this appendix for Marine Phase I TEE-specific variations.
41
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Tabla A.1. Summary o{ TIE Test Conditions and Test Acceptability Criteria for Amphipod, Ampelisca abdite, Acute Toxicity Tests.
1. Test Type
2. Saltnlty
3. Temperature
4. Light quality
5. Light Intensity
6. Photoperlod
7. Test chamber size
8. Test solution volume
9. Size of test organisms
10. No. of organisms per chamber
11. No. replicate chambers per concentration
12. Feeding regime
13. Dilution water
14. Test concentrations
15, Dilution series
16, Tost duration
17.EndpoInts
18. Tost acceptability criteria
Static non-renewal
30±2%»
20±2°C
Ambient laboratory light
10-20 ME/m% (50-100-ft-c) (ambient laboratory levels)
16 h light, 8 h darkness
25 mL chambers
10-20 mL
0.5-0.7 mm
5
1-3 (TIE manipulations)
3 (Initial and Baseline)
none
Natural seawater or hypersaline brine
6 (Initial and Baseline toxicity tests)
4 (TIE procedures)
0.5
24,48, or 96 h
Mortality (LC50)
s90% survival in controls
42
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Table A.2. Summary of TIE Test Conditions and Test Acceptability Criteria for Sea Urchin, Arbacia punctulata, Fertilization Test.
1. Test Type
2. Salinity
3. Temperature
4. Light quality
5. Light intensity
6. Test chamber size
7. Test solution volume
8. No. of sea urchins
9. No. egg and sperm cells per chamber
10. No. replicate chambers per concentration
11. Dilution water
12. Effluent concentrations
13. Test dilution factor
14. Test duration
15. Endpoints
16. Test acceptability criteria
Static
30±2%o
20±1°C
Ambient laboratory light during test preparation
10-20 uE/rn%, or 50-100 ft-c (ambient laboratory levels)
Disposable (glass) liquid scintillation vials (20 mL capacity), presoaked
in control water
5mL
Pooled sperm from four males and pooled eggs from four females are
used per test
About 2000 eggs and 5,000,000 sperm cells per vial
4 (minimum of 3)
Uncontaminated source of natural seawater; deionized water mixed
with hypersaline brine or artificial sea salts (HW Marinemix®, FORTY
FATHOMS®, GP2, or equivalent)
Effluents: Minimum of 5 and a control
Receiving waters: 100% receiving water and a control
Effluents: aO.5
Receiving waters: None, or 20.5
1 hour and 20 min
Fertilization of sea urchin eggs
70%-90% egg fertilization in controls
43
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Table A.3. Summary of Standard Test Conditions and Test Acceptability Criteria for the Topsmelt, Atherinops affinls, Larval Survival and
Growth Test (NOTE: for Phase I TIE, conditions may need to be altered (e.g., test volume)).
1. Tost Type
2. Salinity
3. Temperature
4. Light quality
S, Light Intensity
6. Photoperiod
7. Test chamber size
8. Test solution volume
9. Renewal of test solutions
10. Age of test organism
11. No. of larvae per test chamber
12. No. replicate chambers per concentration
13. Source of food
14. Feeding regime
15. Cleaning
16. Aeration
17. Dilution water
18. Tost concentrations
19. Dilution factor
20. Test duration
21.Endpolnts
22. Test acceptability criteria
Static-renewal
5 to 34%. (± 2%. of the selected test salinity)
20±1 °C
Ambient laboratory illumination
10-20 uE/m2/s (ambient laboratory levels)
16 h light, 8 h darkness
600 mL
200 mL/replicate
Daily
9-15 days post hatch
5
5
Newly hatched Anemia nauplii
Feed 40 nauplii per larvae twice daily (morning and night)
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.
Uncontaminated 1 um-filtered natural seawater or hypersaline brine
prepared from natural seawater
Effluent: Minimum of 5 and a control
Receiving waters: 100% receiving water and a control
effluents: 2 0.5
Receiving waters: None, or aO.5
7 days
Survival and growth (weight)
a80% survival in controls, 0.85 mg average weight of control larvae (9
day old), LC60 with copper must be s205 Mg/L, <25% MSD* for survival
and 50% MSD for growthf
* MSD Mean Standard Deviation
t Provisional, check with appropriate Region or State for latest guidance.
44
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Table A.4. Summary of TIE Test Conditions and Test Acceptability Criteria for the Red Macroalga, Champia parvula. Sexual Reproduction Test.
1. Test type
2. Salinity
3. Temperature
4. Light source
5. Light intensity
6. Photoperiod
7. Test chamber size
8. Test solution volume
9. No. of organisms per test chamber
10. No. replicate chambers per concentration
11. No. of organisms per concentrations
12. Dilution water
13. Test concentrations
14. Dilution factor
15. Test duration
16. Endpoints
17. Test acceptability criteria
Static, Static non-renewal
30±2%»
23±1 °C
Cool-white flourescent lights
100 uE/m2/s (500 ft-c)
16 h light, 8 h darkness
50 ml polystyrene or borosilicate petri dishes
or 125 mL Erlenmeyer flasks
20 mL (minimum)
5 female branch tips and 1 male plant
4 (minimum of 3)
24 (minimum of 18)
Uncontaminated source of natural seawater; deionized water mixed
with hypersaline brine or artificial sea salts (HW Marinemix®, FORTY
FATHOMS®, GP2, or equivalent)
Effluent: Minimum of 5 and a control
Receiving waters: 100% receiving water and a control
Effluents: aO.5
Receiving waters: None, or sO.5
Two day exposure to effluent, followed by 5 to 7 day recovery period in
control medium for cystocarp development
Reduction in cystocarp production compared to controls
80% or greater survival, and an average of 10 cystocarps per plant in
controls
45
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Tablo A.5. Summary of Standard Test Conditions and Test Acceptability Criteria for Oyster, Crassostrea gigas and Mussels,
Mytilus califomlanus and Mytilus galfoprovincialis, Embryo-Larval Development Test. '
1. Tost type
2. Salinity
3. Temperature
4. Light quality
5. Light intensity
6. Photoperiod
7. Test chamber size
8. Test solution volume
9. No. of larvae per chamber
10. No. replicate chambers per concentration
11. Dilution water
12. Test concentrations
13. Dilution factor
14. Test duration
15. Endpoints
16. Test acceptability criteraia
Static non-renewal
30±2%o
20±1 "C (oysters)
15 or 18 ±1°C (mussels)
Ambient laboratory illumination
10-20 nE/m'Vs (ambient laboratory levels)
16 h light, 8 h darkness
30 mL
10 mL
150-300
4
Uncontaminated 1-urn-filtered natural seawater or hypersaline brine
prepared from natural seawater
Effluent: Minimum of 5 and a control
Receiving waters: 100% receiving water and a control
effluents: aO.5
Receiving waters: None, or a 0.5
48 hours (or until complete development up to 54 hours)
Survival and normal shell development
Control survival must be k70% for oyster embryos or a50% for mussel
embryos in control vials; ;>90% normal shell development in surviving
controls; and must achieve %MSD* of <25%t
* MSD Mean Standard Deviation
t Provisonal, check with appropriate Region or State for latest guidance.
46
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Table A.6. Summary of TIE Test Conditions and Test Acceptability Criteria for Fish, Cyprinodon variegatus, Acute Toxicity Tests.
1. Test type
2. Salinity
3. Temperature
4. Light quality
5. Light intensity
6. Photoperiod
7. Test chamber size
8. Test solution volume
9. Age of test organisms
10. No. replicate chambers per concentration
11. No. organisms per chamber
12. Feeding regime
13. Dilution water
14. Test concentrations
15. Dilution series
16. Test duration
17. Endpoints
18. Test acceptability criteria
Static non-renewal
25±10
20±2°C
Ambient laboratory light
10-20 uE/ms/s (50-100-ft-c) (ambient laboratory levels)
16 h light, 8 h darkness
25 mL chambers
10-20 mL
1-14 days old at start
1 (TIE manipulations)
3 (Initial and Baseline)
Feed one drop of concentrated Artemia nauplii suspension daily
(approximately 100 nauplii per mysid)
Natural seawater or hypersaline brine
6 (Initial and Baseline toxicity tests)
4 (TIE procedures)
0.5
24,48, or 96 h
Mortality (LC50)
:>80% survival in controls
47
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Table A.7. Summary of Standard Test Conditions and Test Acceptability Criteria for Albalone, Haliotis rufescens, Larval Development
Test. (NOTE: for Phase I TIE, conditions may need to be altered (e.g., sample volume)).
1. Test Typo
2. Salinity
3. Temperature
4. Light quality
5. Ught Intensity
6. Photopsrlod
7. Test chamber size
8. Test solution volume
9. Larvae density per chamber
10. No. Replicate chambers per concentration
11. Dilution water
12. Test concentrations
13. Dilution factor
14. Test duration
15. Endpofnt
16. Test acceptability criteria
Static non-renewal
34±2%o
15±1°C
Ambient laboratory illumination
10 uE/m2/s (ambient laboratory levels)
16 h light, 8 h darkness
600 ml*
200 mL/replicate*
5-10 per mL
5
Uncontaminated 1-urn-filtered natural seawater or hypersaline brine
plus reagent water
Effluent: Minimum of 5 and a control
Receiving waters: 100% receiving water and a control
Effluents: aO.5
Receiving waters: None, or aO.5
48 h
Normal shell development
i80% normal shell development in the controls; must have statistical
significant effect at 56 ug/L zinc; must acheive a %MSDf of <20%t
* Successful tests performed at 10 mL volume in 20 mL scintillation vials (Hunt et al. In press).
t MSD Mean Standard Deviation
$ Provisional, check with appropriate Region or State for latest guidance
48
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Table A.8. Summary of Standard Test Conditions and Test Acceptability Criteria for Giant Kelp, Macrocystis pyrifera, Germination and Germ-tube
Length Test. (NOTE: for Phase I TIE, conditions may need to be altered (e.g., sample volume)). .
1. Test Type
2. Salinity
3. Temperature
4. Light quality
5. Light intensity
6. Photoperiod
7. Test chamber size
8. Test solution volume
9. Spore density per test chamber
10. No. Replicate chambers per concentration
11. Dilution water
12. Test concentrations
13. Dilution factor
14. Test duration
15. Endpoints
15. Test acceptability criteria
State non-renewal
34±2%o
15±1°C
Ambient laboratory light during test preparation
50±10 uE/mVs
16 h light, 8 h darkness
600 mL
200 mL/replicate
7500 /mL of test solution
5
Uncontaminated 1-urn-filtered natural seawater or hypersaline brine
prepared from natural seawater
Effluent: Minimum of 5 and a control
Receiving waters: 100% receiving water and a control
Effluents: aO.5
Receiving waters: None or aO.5
48 h
Germination and germ-tube length
a70% germination in the controls; a 10um germ-tube length in the
controls and the NOEC must be below 35 ug/L in the reference toxicant
test; must achieve a %MSD* of <20 for both germiniation and germ-
tube length in the reference toxicant.f
* MSD Mean Standard Deviation
t Provisional, check with appropriate Region or State for latest guidance.
49
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Tabla A.9. Summary of TIE Test Conditions and Test Acceptability Criteria for Fish, Menidia beryllina, Acute Toxicity Test.
1. Test Type
2. Salinity
3. Temperature
4. Light quality
5. Light Intensity
6. Photopertod
7. Test chamber size
8, Test solution volume
9. Age of test organisms
10. No. replicate chambers per concentration
11. Organisms per chamber
12. Feeding regime
13. Dilution water
14. Test concentrations
15. Dilution series
16. Test duration
17. Endpoints
18. Test acceptability criteria
Static non-renewal
25±10%o
20±2°C
Ambient laboratory light
10-20 pE/m2/s (50-100-ft-c) (ambient laboratory levels)
16 h light, 8 h darkness
25 mL chambers
10-20 mL
9-14 days old at start
1 (TIE manipulations)
3 (Initial and Baseline)
Feed one drop of concentrated Artemia nauplii suspension daily
(approximately 100 nauplii per mysid)
Natural seawater or hypersaline brine
6 (Initial and Baseline toxicity tests)
4 (TIE procedures)
0.5
24, 48, or 96 h
Mortality (LC60)
^80% survival in controls
.
50
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Table A.10. Summary of TIE Test Conditions and Test Acceptability Criteria for Bivalve, Mulinia lateralis, Embryo-Larval Development Test.
1. Test type
2. Salinity
3. Temperature
4. Light quality
5. Light intensity
6. Photoperiod
7. Test chamber size
8. Test solution volume
9. No. of larvae per chamber
10. No. Replicate chambers per concentration
11. Dilution water
12. Test concentrations
13. Dilution factor
14. Test duration
15. Endpoints
16. Test acceptability criteria
Static non-renewal
30±2%=
20±2°C
Ambient laboratory illumination
10-20 uE/m% (ambient laboratory levels)
16 h light, 8 h darkness
30 ml
10 mL
-300
3-4
Uncontaminated 1-urn-filtered natural seawater or hypersaline brine
prepared from natural seawater
Effluent: Minimum of 5 and a control
Receiving waters: 100% receiving water and a control
Effluents: a 0.5
Receiving waters: None, or aO.5
48 hours
Survival and normal shell development
> 70% Survival; >90% Development
51
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Tabla A.11. Summary of TIE Test Conditions and Test Acceptability Criteria for Mysid. Mysidopsis bahla, Acute Toxicity Tests.
1. Test type
2. Salinity
3. Temperature
4. Light quality
5. Light intensity
6. Photopertod
7. Test chamber size
8. Test solution volume
9. Age of test organisms
10. Number of organisms per chamber
11. No. Replicate chambers per concentration
12. Feeding regime
13. Dilution water
14. Test concentrations
15. Dilution series
16. Test duration
17. Endpolnts
17. Test acceptability criteria
Static non-renewal
25±10%«
20±2°C
Ambient laboratory light
10-20 uE/m2/s (50-100-ft-c) (ambient laboratory levels)
16 h light, 8 h darkness
30 mL chambers
10-20 mL
48 hold at start
5
1 (TIE manipulations)
3 (Initial and Baseline)
Feed one drop of concentrated Anemia nauplli suspension daily
(approximately 100 nauplii per mysid)
Natural seawater or hypersaline brine
6 (Initial and Baseline toxicity tests)
4 (TIE procedures)
0.5
24, 48, or 96 h
Mortality (LCEO)
280% survival in controls
52
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Table A.12. Summary of Standard Test Conditions and Test Acceptability Criteria for the Purple Urchin,Strongylocentrotuspurpuratus,
and Sand Dollar, Dendraster excentricus, Fertilization Tests. .
1. Test Type
2. Salinity
3. Temperature
4. Light quality
5. Light intensity
6. Test chamber size
7. Test solution volume
8. Number of spawners
9. No. Egg and sperm cells per chamber
10. No. Replicate chambers per concentration
11. Dilution water
12. Test concentrations
12. Dilution factor
13. Test duration
14. Endpoint
15. Test acceptability criteria
Static non-renewal
34±2%o
12±1°C
Ambient laboratory light during test preparation
10-20 uE/m% (ambient laboratory levels)
16x100 or 16x125 mm
5mL
Pooled sperm from up to four males and pooled eggs from up to four
females are used per test.
About 1,120 eggs and not more than 3,360,000 sperm per test tube
4
Uncontaminated 1-urn-filtered natural seawater or hypersaline brine
prepared from natural seawater or artificial sea salts
Effluent: Minimum of 5 and a control
Receiving waters: 100% receiving water and a control
Effluents: kO.5
Receiving waters: None or a 0.5
40 min (20 min plus 20 min)
Fertilization of eggs
a 70% egg fertilization in controls; %MSD* of <25%; and appropriate
sperm countst
* MSD Mean Standard Deviation
t Provisional, check with appropriate Region or State for latest guidance.
53
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Table A.13. Summary of Standard Test Conditions and Test Acceptability Criteria for the Purple Urchin,Strongylocentrotus purpuratus,
and Sand Dollar, Dendraster excentricus, Embryo-Larval Development Test.
1. Test Type
2, Salinity
3, Temperature
4. Light quality
5. Light intensity
6, Photoperiod
7. Test chamber size
8. Test solution volume
9. No. Replicate chambers per concentration
10, Dilution water
11. Test concentrations
12. Dilution factor
13, Test duration
14. Endpoint
15. Test acceptability criteria
Static non-renewal
34±2%o
15±1°C
Ambient laboratory illumination
10-20 uE/m2/s (ambient laboratory levels)
16 h light, 8 h darkness
30 mL
10 mL
4
Uncontaminated 1-urn-filtered natural seawater or hypersaline brine
prepared from natural seawater
Effluent: Minimum of 5 and a control
Receiving waters: 100% receiving water and a control
Effluents: aO.5
Receiving waters: 100% receiving water and a control
72±2h
Normal development; mortality can be included
a80% normal shell development in the controls; must acheive a
%MSD* of <25%f
* MSD Mean Standard Deviation
f Provisional, check with appropriate Region or State for latest guidance.
54
•&U.S. GOVERNMENT PRINTING OFFICE: 1997 - 549-001/60110
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