U" ^i States Office of Research and EPA/600/6-91/005F
Environmental Protection Development May 1992
Agency Washington, DC 20460
Toxicity Identification
Evaluation:
Characterization of
Chronically Toxic Effluents,
Phase
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EPA/600/6-91/005F
May, 1992
Toxicity Identification Evaluation:
Characterization of Chronically Toxic Effluents, Phase I
T.J. Norberg-King
Environmental Research Laboratory
Duluth, MN 55804
D.I. Mount,
J.R. Amato,
D.A. Jensen,
and
J.A. Thompson
AScI Corporation
Duluth, MN 55804
National Effluent Toxicity
Assessment Center
Technical Report 02-92
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Duluth, MN 55804
Printed on Recycled Paper
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Disclaimer
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.
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Foreword
This guidance document has been prepared to assist dischargers and/or their
consultant laboratories in conducting chronic aquatic toxicity identification evaluations
(TIEs). TIEs may be required by the state or federal agencies as a result of enforcement
actions or as a condition of the discharger's National Pollutant Discharge Elimination
System (NPDES) permit or may be conducted voluntarily by permittees. This document
will assist the state and federal agencies and permittees in overseeing and determining
the adequacy of the TIE in toxicity reduction evaluations (TREs).
This document discusses methods to characterize the chemical/physical nature of
the constituents in effluents which cause their chronic toxicity. The general approach for
toxicity identification evaluations is described in the document Methods for Aquatic
Toxicity Identification Evaluations: Phase I Toxicity Characterization Procedures (EPA,
1988A; EPA, 1991 A), hereafter referred to as the "acute Phase I manual." The acute
Phase I manual provides much of the basis for the statements and guidance provided in
this chronic Phase I characterization document. This chronic TIE manual and the acute
Phase I manual should be used as companion documents, because all the guidance of
the acute Phase I manual is not repeated here.
The general approach for the chronic characterization is divided into Tier 1 and Tier
2. Tier 1 consists of the EDTA and sodium thiosulfate additions, the graduated pH test,
aeration and filtration manipulations, and the use of the Clg solid phase extraction (SPE)
resin. For Tier 1, the tests are all done using the effluent sample without any pH
adjustments (i.e., at the initial pH (pH i) of the effluent). Tier 2 manipulations are added
when Tier 1 tests are not definitive in characterizing the toxicity. Tier 2 includes the
aeration, filtration, and C1( SPE steps of Tier 1 performed at pH 3 and pH 10 and returned
to pH i prior to testing.
The chronic Phase I procedures should provide information on whether the toxicants
are volatile, chelatable, filterable, reducible, non-polar, or pH sensitive. These character-
istics are indicated by comparing the results of toxicity tests conducted using unaltered
and manipulated effluent samples. As with the acute TIE, the characterization results
from the chronic TIE can be used for the treatability approach in a TRE (EPA, 1991 A).
These chronic TIE methods are not written as rigid, required protocols, but rather as
general guidance for conducting TIEs with effluents. These acute and chronic methods
should also be applicable to samples from ambient waters, sediment pore and elutriate
waters, and leachates. The methods to identify (Phase II; EPA, 1989A) and confirm
(Phase III; EPA, 1989B) the cause of toxicity in effluent samples evaluated with the acute
Phase I procedure are also applicable to effluent samples evaluated with this chronic
Phase I procedure. The identification and confirmation documents are being revised
(EPA, 1992A; EPA, 1992B) to reflect additional information from this manual and the
revised acute Phase I manual (EPA, 1991 A) to discuss the aspects of TIEs for both acute
and chronic toxicity.
In September of 1991, we solicited peer-review comments until January 31, 1992
from all persons who obtained the document from any of the following locations: EPA's
Office of Water, Washington, D.C., each EPA Regional Water Division Office, EPA's
Environmental Research Laboratory-Duluth, MN, or EPA's Center for Environmental
Research Information (CERI), Cincinnati, Ohio. Appropriate technical comments were
incorporated into this manual.
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Abstract
This manual is intended to provide guidance to aid dischargers in characterizing the
type of toxicants that are causing chronic toxicity in industrial and municipal effluents. In
a regulatory context, a toxicity identification evaluation (TIE) may be required as part of
the National Pollutant Discharge Elimination System (NPDES) permit or as an enforce-
ment action. TIEs may also be conducted by permittees on a volunteer basis to
characterize their discharge toxicity.
The Phase I chronic toxicity methods are modified from those described in the acute
Phase I TIE manual (EPA, 1988A; EPA, 1991 A) and additional techniques are incorpo-
rated. This chronic Phase I manual describes procedures for characterizing the physical/
chemical nature of toxicants in effluents that exhibit chronic toxicity to freshwater species,
although many of the principles and procedures are similar for TIEs on marine species.
Aliquots of effluent samples are manipulated and the resulting effect on toxicity mea-
sured. The objective is to characterize the toxicants so that appropriate analytical
methods can be chosen to identify the toxicants.
The general approach to the chronic toxicity characterization is a two tiered ap-
proach, where usually Tier 1 is applied before proceeding to Tier 2. Tier 1 consists of
filtration, aeration, use of additives to chelate or reduce the toxicants, minor pH adjust-
ments, and use of a separation technique with the C1t solid phase extraction (SPE) resin.
Each effluent is characterized in Tier 1 by performing the manipulations at the initial pH
(pH j) of the effluent. Tier 2 consists of the Tier 1 manipulations combined with pH
adjustments of additional aliquots of the effluent sample, and the Tier 2 characterization
steps include aeration, filtration, and the C1g solid phase extraction of effluent samples
adjusted to pH 3 and pH 10.
The Phase I characterization methods were developed for the short-term "chronic"
test methods using two species, Ceriodaphnia dubia and the fathead minnow (Pimephales
promelas) (EPA, 1989C). Chronic threshold levels for the various additives (sodium
thiosulfate, EDTA, methanol) used in some of the characterization tests are provided for
these species. Although developed for these species, the characterization techniques
should be applicable to other species as well, provided threshold levels are established.
The guidance provided in this manual is intended to be supplemental to that given in
the acute Phase I manual (EPA, 1991 A). Sections of this chronic Phase I TIE manual
discuss quality assurance, effluent handling, facilities and equipment, health and safety,
dilution water, principles of the chronic TIE testing, and the Phase I characterization tests
as a two tiered approach. The use of the whole effluent test as a baseline test (in
manner similar to the acute Phase I characterization procedure), the appropriate treat-
ment of dilution water for blanks and the toxic levels of the additives for two species are
described. Use of short-cuts, reduced test volumes, reduced test duration, and a small
number of replicates are discussed. The importance of sample type, frequency of
sample collection and renewal, and descriptions of all manipulations are discussed, along
with a section on the application of combining several of the characterization tests.
IV
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Contents
Page
Foreword iii
Abstract iv
Contents v
Figures vii
Tables viii
Acknowledgments ix
1. Introduction 1-1
2. Quality Assurance, Health and Safety, and Facilities and Equipment 2-1
2.1 Quality Assurance 2-1
2.2 QA/QC Cost Considerations and Testing Requirements 2-1
2.3 QA/QC and Chronic Testing Considerations 2-2
2.4 QA/QC Blanks and Artifactual Toxicity 2-3
2.5 Health and Safety Issues 2-3
2.6 Facilities and Equipment 2-3
3. Dilution Water 3-1
4. Effluent Samples 4-1
5. Toxicity Testing 5-1
5.1 Principles 5-1
5.2 Test Species 5-1
5.3 Toxicity Test Procedures 5-1
5.4 Concentrations to Test 5-2
5.5 Renewals 5-3
5.6 Toxicity Blanks 5-3
5.7 Renewal of Manipulated Samples 5-4
5.8 Test Endpoints and Data Analysis 5-4
6. Characterization Tests 6-1
6.1 Baseline Test 6-3
6.2 EDTA Addition Test 6-4
6.3 Sodium Thiosulfate Addition Test 6-6
6.4 Aeration Test 6-8
6.5 Filtration Test 6-9
6.6 Post C1( Solid Phase Extraction Column Test 6-11
6.7 Methanol Eluate Test 6-15
6.8 Graduated pH Test 6-17
6.9 Tier 2 Characterization Tests 6-20
6.10 pH Adjustment Test 6-21
6.11 Aeration and pH Adjustment Test 6-22
6.12 Filtration and pH Adjustment Test 6-23
6.13 Post Cia Solid Phase Extraction Column and pH Adjustment Test ....6-23
6.14 Methanol Eluate Test for pH Adjusted Samples 6-24
6.15 Toxicity Characterization Summary 6-24
6.16 Use of Multiple Characterization Tests 6-25
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Contents (continued)
Page
7. Interpreting Phase I Results 7-1
8. References 8-1
VI
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Figures
Number Page
4-1. Example data sheet for logging in samples 4-2
6-1. Overview of characterization tests 6-2
6-2. Tier 1 sample preparation and testing overview 6-2
6-3. Tier 2 sample preparation and testing overview 6-20
VII
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Tables
Number Page
6-1. Outline of Phase I effluent manipulations Tier 1 and Tier 2 6-3
6-2. Chronic toxicity of EDTA (mg/l) to C. dubia and P. promelas
in various hardness waters using the 7-d tests 6-5
6-3. Concentrations of EDTA to add for chronic TIEs. Values given
are the final water concentration in mg/l 6-5
6-4. The chronic toxicity of zinc (jig/l) to C. dubia in very hard
reconstituted water and the toxicity of zinc when EDTA is added 6-6
6-5. Chronic toxicity of sodium thiosulfate (mg/l) to C. dubia and
P. promelas in various hardness waters using the 7-d tests 6-7
6-6. Concentrations of sodium thiosulfate to add for chronic TIEs.
Values given are the final exposure concentration in mg/l 6-8
6-7. Factors to consider for the size of available pre-packed C14 SPE
columns. Appropriate volumes of sample to apply to each column
with respect to maximum volumes of sample and minimum elution
volumes, and elution volumes frequently used in the TIE process 6-12
6-8. Test volume of eluate needed for methanol eluate test with C. dubia
or P. promelas. Volumes described are based on minimum elution
volumes recommended and the highest test concentration possible
with the methanol level at an acceptable concentration 6-13
6-9. Chronic toxicity of methanol (%) to C. dubia and P. promelas
using the 7-d tests 6-13
6-10. Chronic toxicity of sodium chloride (g/l) to C. dubia
and P. promelas in various hardness waters using the 7-d tests 6-22
VIII
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Acknowledgments
Many people at the National Effluent Toxicrty Assessment Center (NETAC) at the
Environmental Research Laboratory-Duluth (ERL-Duluth) have provided assistance to
produce this manual by performing the toxicity tests, chemical analyses, and data
analyses as well as providing advice based on experience. This document is the result of
the input by the NETAC group which has consisted of both federal and contract staff
members, and includes Gary Ankley, Larry Burkhard, Liz Durhan, Don Mount, Shaneen
Murphy, and Teresa Norberg-King (federal staff), and Joe Amato, Lara Andersen, Steve
Baker, Tim Dawson, Nola Englehorn, Doug Jensen, Correne Jenson, Jim Jenson, Marta
Lukasewycz, Liz Makynen, Greg Peterson, Mary Schubauer-Berigan, and Jo Thompson
(contract staff). The toxicity test data generated for this document and the biological data
upon which this report is based was produced by Doug Jensen, Jo Thompson, Tim
Dawson, Greg Peterson, Nola Englehorn, Shaneen Murphy, Mary Schubauer-Berigan,
Joe Amato, and Jim Jenson. The skillful assistance and dedication of Debra Williams
and Jane Norlander (NETAC) in producing this document are gratefully acknowledged.
Comments were received from the following people and organizations: Charles
Carry and LeAnne Hamilton for the County Sanitation Districts of Los Angeles County,
Whittier, CA.; David Mount and J. Russell Hockett for ENSR Consulting and Engineering,
Fort Collins, CO; Norman LeBlanc for Hampton Roads Sanitation District, Virginia Beach,
VA; Charlie Webster, State of Ohio Environmental Protection Agency (EPA), Columbus,
OH.; Michael Gallaway, State of Ohio EPA, Columbus, OH; Robert Berger for the
Association of Metropolitan Sewerage Agencies (AMSA), Washington, D.C.; and Kerrie
Schurr for EPA Region 10, Seattle, WA. We want to thank those individuals or
organizations for reviewing the report, and in turn improving the document with their
comments.
In the review comments, a suggestion was made to summarize all the effort on TIEs
by government, state, academia, contract laboratories, and industries to date. While the
TIEs at Duluth can be summarized, data from all the possible sources are difficult if not
impossible to obtain. Contract laboratories and industrial data are protected for confiden-
tiality and proprietary reasons, and information about the kinds of toxicants, the types of
discharges, the time-frame for the TIE, and the costs are difficult to obtain. Numerous
toxicity problems have been resolved as TIEs are initiated because of better plant
operation. In fact, during a workshop (Aquatic Habitat Institute, 1992) held March 17 and
18, 1992 in Richmond, CA, these issues were discussed, and presenters of chronic TIE
data indicated chronic TIEs have been much more successful than expected.
This work was supported in part by the Office of Water, Permit Division, Washington,
D.C., through the backing of Rick Brandes and Jim Pendergast, who have provided
strong support for the whole effluent water quality-based approach.
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Section 1
Introduction
The United States Federal Water Pollution Control
Act Amendments (commonly referred to as the Clean
Water Act (CWA); (Public Law 92-500 of 1972) states
that the discharge of toxic pollutants in toxic amounts is
prohibited. In the CWA, the National Pollutant Dis-
charge Elimination System (NPDES) was established;
this system provides a mechanism whereby point source
wastewater discharges are permitted. NPDES permits
contain effluent limits that require baseline use of treat-
ment technologies (best available technology). The
technology-based limits are independent of receiving
water impact, and additional water quality-based limits
may be needed in order to meet the goal of the CWA of
"no toxics in toxic amounts." State narrative and state
numerical water quality standards are used in conjunc-
tion with EPA's water quality criteria and other toxicity
databases to determine the adequacy of the technol-
ogy-based permit limits and the need for any additional
water quality-based controls.
When limits were first written into the permits, they
were based primarily on physical factors such as bio-
logical oxygen demand (BOD), suspended solids (SS),
and color. Additional components were added in sub-
sequent amendments to the CWA; for example, the list
of 126 "priority pollutants" of which many or most were
required to be monitored by the permittees. Water
quality criteria were used to develop the water quality-
based limits for these pollutants. However, water qual-
ity criteria or discharge limits exist for only a few of the
thousands of chemicals in use.
An important objective of the NPDES program is
the control of toxicity of discharges and to accomplish
this objective, EPA uses an integrated water quality-
based approach. Published water quality criteria are
converted to standards that consist of both chemical-
specific numeric criteria for individual toxics and narra-
tive criteria. The states' narrative water quality criterion
generally requires that the waters be free from oil,
scum, floating debris, materials that will cause odors,
materials that are unsightly or deleterious, materials
that will cause a nuisance, or substances in concentra-
tions that are toxic to aquatic life, wildlife or human
health. Use of toxicity testing and whole effluent toxic-
ity limitations is based on a state's narrative water
quality criterion and in some cases, a state numeric
criterion for toxicity.
EPA, in 1984, issued a policy statement (Federal
Register, 1984) that recommends an "integrated ap-
proach' for controlling toxic pollutants. This integrated
approach is referred to as the water quality-based ap-
proach and is described in detail in the Technical Sup-
port Document (hereafter referred to as the TSD; EPA,
1985A; EPA, 1991B). The control regulations for EPA
(Federal Register 23868, 1989) establish specific re-
quirements that the integrated approach be used for
water quality-based toxics control. This integrated ap-
proach results in NPDES permit limits to control toxic
pollutants through the use of both chemical-specific
and whole effluent toxicity limitations as a means to
protect both aquatic life and human health. This com-
bination of chemical specific and whole effluent toxicity
limitations is essential to the control of toxic pollutants.
Once the permit limits are set, compliance is estab-
lished through routine monitoring of effluent quality. In
this manner, water quality-based limits (when following
EPA, 1991B) will protect water quality and prevent the
state water quality standards from being violated.
The whole effluent toxicity limitation aspect involves
using acute and chronic toxicity tests to measure the
toxicity of wastewaters. Acute toxicity refers to toxicity
that occurs in a short period of time, operationally
defined as 96 h or less. Chronic toxicity occurs as the
result of long exposures in which sublethal effects (fer-
tilization, growth, reproduction) are measured in addi-
tion to lethality. The chronic test is used to measure
the effects of long-term exposure to chemicals, waste-
waters, and leachates to aquatic organisms. True chronic
toxicity tests include the life-cycle of the organism. For
fish, the life-cycle test is infrequently conducted (Norberg-
King, 1989A), and abbreviated test methods have been
used to estimate chronic toxicity. These tests are the
7-d growth and survival test (EPA, 1989C), or the 32-d
embryo-larval early life stage test (Norberg-King, 1989A).
These tests rely on the most sensitive life-cycle stages
(i.e., embryos and larval fish) to estimate chronic toxic-
ity (McKim, 1977; Weltering 1983; Norberg-King, 1989A).
Hereafter, chronic tests refer to the short-term tests
that are described in the EPA manuals (EPA, 1992C;
EPA, 1992D; EPA, 1989C; EPA, 1985C).
Toxicity is a useful parameter to protect receiving
waters from potential impacts on water quality and
designated uses caused by the mixture of toxic pollut-
ants in wastewaters. EPA has published manuals
which provide test methods for use of freshwater and
marine organisms to determine acute and chronic tox-
icity of effluents. These manuals have been available
since 1978 and 1985, respectively (EPA, 1978; EPA,
1-1
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1985B; EPA, 1985C; EPA, 1988B; EPA, 1989C) and
have been recently revised (EPA, 1991C; EPA, 1992C;
EPA, 1992D). These methods are used by federal,
state and local governments to assess toxicity and
determine compliance of permitted point source dis-
charges. Since the late 1970's, toxicity has been mea-
sured in wastewaters; permit writers began using toxic-
ity limits in the early 1980's. With the increased use of
toxicity testing, substantial numbers of unacceptably
toxic effluents nave been identified. Now, some permit-
tees are required to perform toxicity reduction evalua-
tions (TREs) as a condition of the NPDES permit. The
TSD defines a TRE as "a she specific study conducted
in a stepwise process designed to identify the caus-
ative agents of effluent toxicity, isolate the sources of
toxicity, evaluate the effectiveness of toxicity control
options, and then confirm the reduction in effluent toxic-
ity." Toxicity identification evaluations (TIEs), which
are a part of the TRE, consist of methods to character-
ize (Phase I; EPA, 1988A; EPA, 1991A; EPA, 1991D),
identify (Phase II; EPA, 1989A; EPA, 1992A), and con-
firm (Phase III; EPA, 1989B; EPA, 1992B) the cause of
acute and chronic toxicity in effluents.
The TIE approach (EPA, 1988A; EPA, 1991 A) re-
lies on the use of organisms to detect the presence of
toxicants in the effluent. Information about the physi-
cal/chemical characteristics of the effluent's toxicity is
gained (by the various manipulations) and if possible
the number of constituents in the effluent is reduced
before any analyses begin. Using this approach, ana-
lytical problems can be simplified and the costs re-
duced. Toxicity throughout the TIE must be tracked to
determine if the toxicity is consistently being caused by
the same substance. Once the physical/chemical char-
acteristics of toxicants are known, a better choice of
analytical methods can be made. Knowledge of physi-
cal/chemical characteristics of any effluent is used for
the treatabilrty approach to TRE's (EPA, 1989D; EPA,
1989E).
As with the acute Phase I TIE approach, the chronic
Phase I TIE is based on manipulations designed to
alter a group of toxicants (such as oxidants, cationic
metals, volatiles, or non-polar organics) so that toxicity
is changed. Chronic toxicity tests are conducted after
each manipulation to indicate the effect on the toxicity
of the effluent. Based upon the manipulations that
change toxicity, inferences about the chemical/physical
characteristics of the toxicants can be made. Using
several samples of the effluent for these characteriza-
tion steps provides information on whether the nature
of compounds causing the chronic toxicity remains con-
sistent. The tests do not provide information on the
variability of toxicants within a characterization group.
From these data the toxicant characteristics can be
identified as pH sensitive, filterable, volatile, soluble,
degradable, reducible, or EOTA chelatable. Such infor-
mation indicates how samples must be handled for
analyses and which analytical methods should be used.
The recommended procedure is to concentrate on
the characterization steps that are most clean-cut and
have the major effect of reducing the toxicity in the
effluent. If toxicity in every effluent sample is not
caused by the same toxicant(s), the characterization
tests should indicate if the type of toxicant(s) is the
same or different. Once identification is initiated, and
suspects identified, the varying causes of toxicity can
be evaluated because the concentration of toxicants
should be tracking with the toxicity. In the earlier
version of this document (EPA, 1991D) we suggested
that samples be subjected to Phase I techniques until
no additional responses are found (which was sug-
gested to be at least three samples). After conducting
several Phase I evaluations for chronic toxicity, we
have determined that if the effluents' toxicity is readily
characterized after Phase I even with one sample it
may be prudent to proceed with Phase II (EPA, 1992C)
to measure the toxicant(s). Use of toxicity patterns as
the TIE progresses can be helpful if patterns are tracked,
beginning with the first samples. Following character-
ization, a decision is made to proceed with identifica-
tion (Phase II; EPA, 1989A; EPA, 1992A) and confir-
mation (Phase III, EPA, 1989B; EPA, 1992B) or to
conduct treatability studies where the identification of
the specific toxicants (cf., acute treatability procedures
(EPA, 1989D; EPA, 1989E)) is not made.
Chronic toxicity must be present frequently enough
so that an adequate number of toxic samples can be
obtained. Enough routine toxicity testing should be
done on each effluent before a TIE is initiated (EPA,
1991B), to ensure that toxicity is consistently present.
It is not important that the same amount of toxicity is
present in each sample; in fact, variable levels of toxic-
ity can assist in determining the cause of toxicity. If
toxicity is not consistently present, when it occurs the
toxicity can be pursued and if a toxicant(s) is sus-
pected, the non-toxic samples may be used to elimi-
nate suspects. One cannot assume that if the effluent
showed acute toxicity and a TIE was completed, identi-
fying the cause(s) of acute toxicity and action taken to
remove the acute toxicant from the effluent, that the
sublethal toxicity exhibited is due to the same com-
pound.
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Section 2
Quality Assurance, Health, and Safety, and Facilities and Equipment
2.1 Quality Assurance
The quality assurance plan (QAP), as described in
Standard Methods for the Examination of Water and
Wastewater (APHA, 1989) (describes standards to con-
duct performance evaluations) is primarily for analytical
analyses. A QAP for toxicity testing can be developed,
but determining the recovery of known additions for
toxicity testing is not possible. For TIEs the combina-
tion of chemistry and biology requires a level of checks
and balances not typically used under other situations.
A step-by-step QAP for all steps of a TIE is not always
possible due to the unknown toxicant(s) requiring vari-
ous follow-up testing and analytical procedures; how-
ever as a TIE progresses, additional or different tests
may be needed and many aspects of the TIE QAP can
be addressed as the TIE proceeds. Adhering to the
general guidelines of a strong QAP is important how-
ever, and should increase the probability of the TIE
succeeding. As additional steps are recognized, the
details should be added to the QAP.
Specific quality control (QC) procedures for aquatic
toxicity tests are different than the specific QC proce-
dures for chemical analytical methods. Both proce-
dures have common goals that are to know that reliable
data are generated, to recognize and eliminate unreli-
able data, and to have methods which assist investiga-
tions in resolving problems for future work. The quality
assurance (QA/QC) guidance given by EPA (1989C)
for the short-term tests lists numerous items of concern
for toxicity testing. These are: (a) effluent sampling/
handling, (b) test organisms, (c) facilities, equipment
and test chambers, (d) analytical methods, (e) calibra-
tion and standardization, (f) dilution water, (g) test con-
ditions, (h) test acceptability, (i) test precision, (j) repli-
cation and test sensitivity, (k) quality of organisms, (I)
quality of food, (m) control charts, and (n) record keep-
ing and data evaluation. Many of these should be
closely followed, and the reader is encouraged to re-
view the guidance in relation to QA/QC in both the
short-term effluent test manual (EPA, 1989C; EPA,
1992C) and the acute Phase I manual (EPA, 1991 A).
2.2 QA/QC Cost Considerations and Testing
Requirements
For the chronic TIE, cost considerations are impor-
tant and concessions in the requirements of the QC
may have to be made. In some instances, the data will
demand stringent control while in others, the QC can
be lessened without impact to the overall endpoint of
the TIE.
TIEs can require a great number of toxicity tests.
The use of all aspects of the standard test protocols
(EPA, 1989C; EPA, 1991C) is not necessary in Phase
I. The factors of time requirements, number of tests
and the test design (i.e., five replicates versus ten, four
dilutions versus five) must be considered and weighed
against the type of questions that are posed. For
example, the need for water chemistry data are specific
for each Phase I test. The testing requirement (EPA,
1989C) according to the permit requirement most likely
included pH, daily measurements of DO, temperature,
conductivity, alkalinity, and hardness measurements in
the low, middle, and high concentrations for the five
test dilutions of the effluent. However, hardness mea-
surements are not pertinent for the methanol eluate
collected from a solid phase extraction column. The
post C18 SPE column effluent samples are more similar
to the effluent and a concern for low dissolved oxygen
(DO) exists, while the test solutions of the methanol
eluate are more similar to the dilution water and the
possibility of low DOs is not as great a concern. In
contrast, frequent pH measurements on all test con-
centrations are needed to determine the impact of pH
sensitive compounds.
As TIEs are reliant on a strong QAP, there are
several aspects of a QA/QC program for chronic TIEs
that should be delineated. In regard to test organism
quality, there are steps for culturing organisms that
should help provide the necessary QC verification that
is needed to ensure the animals are representative in
their sensitivity. These steps are simply routine items
such as monitoring and recording the young production
(for cladocerans) of the culture brood animals once a
month, conducting monthly reference toxicant tests (in-
cluding maintaining control charts), monitoring the prepa-
ration dates for the reconstituted waters used, and
monitoring the types and age of the foods fed (Norberg-
King, 1989B). For fathead minnows, it is useful to
monitor the survival of the breeding stock, and the
percent hatchability of the embryos, to verify that new
genetic stock is introduced on a regular basis, and to
conduct monthly reference toxicant tests (Norberg-King
and Denny, 1989; Denny, 1988). Similar parameters
for other species that are used are also desirable.
Since toxicity tests in the early part of the chronic
Phase I do not generally follow all the effluent testing
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requirements (EPA, 1989C), the QC measures are not
as strict because the data are primarily informative
rather than definitive. When Phases II (identification)
and III (confirmation) are initiated, then QC aspects
should be reconsidered and the tests modified. Phase
I procedures frequently use one species and later stages
of the TIE (Phase III) use more than one species to
determine whether the cause of toxicity is the same for
other species of the aquatic community.
Reference toxicant tests are not conducted with
each set of Phase I manipulations because of the
amount of labor and large numbers of animals required
for testing. In general, the utility of the reference
toxicant test is to know that the organisms are respond-
ing as expected. Since only relative differences are
needed at this stage (Phase I), reference toxicant data
are much less useful for the characterization interpreta-
tion but are important for the knowledge of the quality
of the test organisms and general test procedures. For
various manipulations of the TIE, organism responses
are compared to either the baseline test (see Section
6) or the response of organisms in the dilution water
treatments. Monthly reference toxicant tests should
provide the necessary information about the quality of
the organisms for the laboratory conducting the TIE.
When a toxicant has been identified (Phase II) and
tests for Phase III confirmation indicate it is the
toxicant(s), that chemical should become the reference
toxicant with the species used in the TIE.
Using receiving water as the dilution water in Phase
III confirmation will help ensure that receiving water
effects are properly considered (see Section 3, Dilution
Water). The variability of the effluent, by nature of the
TIE, is defined during the TIE, and this information will
aid in determining the appropriate control option in
order that the final effluent is safe upon discharge.
2.3 QA/QC and Chronic Testing
Considerations
An inherent problem with effluents is that no efflu-
ent test can be repeated to assure that the toxicity is
the same and that the toxicants are the same. How-
ever, repeated baseline tests (Section 6) can be done
with the same effluent sample to determine how long
that effluent sample can be used. The chemical and
lexicological nature of the effluent shifts as an effluent
is discharged or as an effluent sample is stored. Efflu-
ent constituents degrade (at unknown rates) and each
constituent has its own rate of change. Analysis of
each sample should be initiated as soon as the sample
is received in the testing laboratory (generally <24 h).
Until an effluent sample has been tested several times,
there is no way to predict how long a sample can be
stored before the toxicity changes. Testing of each
sample can be done provided the toxicity remains and/
or stabilizes; however this cannot be determined at the
beginning of the Phase I battery of tests and will be
known only through testing several samples a few times.
Even though the toxicity remains, it is possible that the
toxicant may change with time. The number of samples
to evaluate and the number of tests to conduct must be
weighed against the cost of the effort and how repre-
sentative each effluent sample is of the effluent. Efflu-
ents that have low and non-persistent toxicity may
need to be approached with the Tier 1 and Tier 2
characterization steps applied simultaneously (see Sec-
tion 6).
In a chronic TIE, information obtained from a test
should be maximized. This may mean paying particu-
larly close attention to details such as small differences
in the number of neonates the cladocerans are produc-
ing or the lack of food in the stomach of the larval fish.
These parameters and any other observed characteris-
tics during a test may be subtle indicators and quite
informative about small changes in toxicity. For ex-
ample, if all the animals exposed to the whole effluent
die on day 4, and in some characterization test the
animals don't reproduce or grow but are alive at day 7
of the exposure, that characterization manipulation re-
duced the toxicity, but did not remove it completely.
Observations such as these may be just as useful as
reductions in young production or growth.
While some abbreviations in the test design are
made, the general principles for toxicity testing still
apply. For example, all animals must be added to test
solutions randomly. Animals must be placed in a test
chamber one at a time. For the fathead minnows, use
of an intermediate vessel to hold all 10 animals is
preferable to ensure that animals are assigned ran-
domly and that the volume of water added with the fish
is minimized (1-2 ml). Also, transferring animals may
require separate pipettes for each concentration or clean-
ing of the pipettes between concentrations to prevent
cross contamination. However, we have observed that
C. dubia do not have to be placed under the water;
they can be added or transferred by dropping the water
droplet containing the animal into the test solution. The
problem frequently observed with D. pulex where ani-
mals are caught at the surface of the test solution
(called "floaters") does not occur with C. dubia. Ran-
domization, careful exposure time readings, use of ani-
mals of uniform narrow-age groups (i.e., Ceriodaphnia
neonates 0-6 h old rather than 0-12 h old) should assist
in quality data generation.
Standard operating procedures (SOPs) should be
developed for each Phase I test, for preparing the
reconstituted waters, preparing the foods for the test
organisms, calibration and standardization for all mea-
surements (temperature, DO, pH, conductivity, alkalin-
ity, hardness, ammonia, chlorine), and other general
routine practices.
An important aspect of TIEs is accurate and thor-
ough data recording. All observations should be docu-
mented. Items that were not thought to be important at
first may be useful in later stages of analysis and
actually assist in the confirmation of the toxicant(s).
These observations can be as simple as large bubbles
produced during the aeration and filtration manipula-
tions, large particles present in whole effluent, and low
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pH upon arrival. It is best to record data so that any
preconceived ideas of the toxicants are avoided. Data
records should include records of test organisms (spe-
cies, source, age, date of receipt, history and health),
calibration records, test conditions, results of tests, and
summaries of data. Once a control chart is developed
using point estimates for reference toxicant tests, 1 out
of 20 reference toxicity test results will be predicted to
fall outside the acceptable limits if the 95% confidence
intervals are used to develop the control chart (EPA,
1991C). If TIEs are conducted during such a period,
the TIE data generated must be used with caution, and
the investigator must carefully examine the TIE data to
determine if the results are usable. The decision may
be based on consistency of the concentration response
data, control blank performance, and the consistency of
the TIE results with those obtained with the same
effluent sample.
2.4 QA/QC Blanks and Artifactual Toxicity
Throughout the TIE, dilution water samples are
subjected to most of the procedures and analyses per-
formed on the effluent sample (see Section 5.6). This
is done to detect toxic artifacts (i.e., toxicity due to
anything other than the effluent constituents causing
toxicity) that are created during the effluent character-
ization manipulations (see Section 6). These manipu-
lations can make QC/QA verifications difficult, as the
use of such blanks for interpreting toxicity results is not
standard toxicology. For example, typically organism
responses from any toxicity test in standard aquatic
toxicology are compared to the performance of control
organisms which were in dilution water only. In the
TIE, controls are used to judge organism performance
(Section 5), and toxicity controls and blanks are used to
evaluate whether a manipulation affected the toxicant(s),
therefore the results of all characterization tests are not
necessarily compared to the baseline test. For in-
stance, post-column effluent samples that are collected
and tested following concentration on a resin column
have been filtered first. Therefore it is only logical to
compare the post-column effluent toxicity (post C1f SPE
column test, Section 6.6) to the toxicity observed in the
filtered effluent sample (filtration test. Section 6.4) rather
than to the unfiltered whole effluent (baseline test, Sec-
tion 6.1) (see Section 5).
Artifactual toxicity can occur in several of the ma-
nipulations, particularly from the major pH adjustment
manipulation (Tier 2). Toxicity results from tests relying
on the addition of the reagents (EDTA, sodium thiosul-
fale, acids/bases) must be interpretable. Addition of
both the acid (HCI) and the base (NaOH) can form a
toxic product (e.g., NaCI). The addition of the acid and
base may interfere with the growth and reproduction of
the test organisms for the short-term chronic test, at
lower levels than cause mortality in the acute test.
Whether additives act in an additive, synergistic, or
independent manner with the compounds in the efflu-
ent must be determined during the TIE but this is not
likely to be clear during Phase I. Artifactual toxicity
can occur in the aeration process, where contaminated
air can be introduced. Also, contaminants can be
leached from solid phase extraction (SPE) columns,
and methanol leaching off the column can cause bacte-
rial growth that will confound the results in the post-
column blank and post C1t SPE column tests. Original-
ity and judgement are needed to devise tests that will
reveal artifactual toxicity (see Section 6) and some of
these methods to deal with artifactual toxicity will be
effluent specific.
2.5 Health and Safety Issues
For the toxicity identification work, hazards present
in any effluent may not be known until Phase II identifi-
cation steps have been started. Therefore, safety re-
quirements for working with effluents (or other samples)
of unknown composition must follow safety procedures
for a wide spectrum of chemical and biological agents.
Because all of the hazards in an effluent sample may
not be known when a toxicant is identified, effluent
samples should be treated as hazards of unknown
composition throughout the TIE. Knowledge of the
types of wastewater treatment applied to each effluent
can provide some insight for the possible hazards. For
example, unchlorinated primary treatment plant efflu-
ents containing domestic waste may contain patho-
gens. Chlorinated secondary effluents are less likely to
contain such agents. Effluents from activated sludge
treatment plants are less likely to contain volatile toxi-
cants.
Because effluent characteristics are unknown, per-
sonnel should follow the guidelines for hazardous ma-
terials (EPA, 1991 A; 1991C). Also, if any sample
contains human waste, personnel should be immu-
nized for diseases such as hepatitis B, tetanus, polio,
and typhoid fever.
Each laboratory should provide a safe and healthy
work place. All laboratories should develop and main-
tain effective health and safety programs (APHA, 1989;
EPA, 1991C). Each program should consist of: (a)
designated health and safety officers, (b) formal written
health and safety plans, (c) on-going training programs,
and (d) periodic inspections of emergency equipment
and safety violations. Further guidance on safety prac-
tices is provided in other documents (APHA, 1989;
EPA, 1991A; 1991C).
2.6 Facilities and Equipment
The laboratory facilities and equipment needed to
conduct TIEs are discussed in the acute Phase I manual
(EPA, 1988A; EPA, 1991 A). Most of the equipment for
conducting the short-term tests are delineated else-
where (EPA, 1989C; EPA, 1992C). The reagents used
for the chronic Phase I characterization are identical to
those described in the acute Phase I manual (EPA,
1991 A). Compressed air systems with oil-free com-
pressors and air filters to provide high purity air are
very important (EPA, 1991 A). All glassware should be
rigorously cleaned, and the glassware used for filtering
must be rigorously cleaned to remove residual contami-
nants from the glass frit(s). Filtering equipment may
2-3
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need to be made of plastic to avoid leaching of metals and cause toxicrty. Ultra pure acids and bases (e.g.,
or other toxicants from glass when acid washes are Suprapur*, E. Merck, Darmstadt, Germany) should be
used (see Section 6). Use of stainless steel frits can used to prevent impurities in the acids/bases from inter-
be used provided pH adjustments are not made since fering in the toxicity results.
metals will rinse off the stainless steel at extreme pH's
2-4
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Section 3
Dilution Water
Dilution water used for chronic TIE's must meet
several requirements. Obviously it must support ad-
equate performance of the test animals in regard to
growth, survival, and reproduction since these are the
effects measured in the tests. Secondly, it must not
substantially change the animals' response to the sample
toxicants. Because the characteristics of the toxicants
are not known, there is no way to be sure which dilution
water characteristics are important. Hardness and al-
kalinity are most often used to select the dilution water
but these parameters are generally of little importance
for non-polar organics. Rarely is the organic matter
content considered and yet for both non-polar organics
and metals, organic matter has more effect on toxicity
than hardness. Experience in the acute TIE work has
shown pH to be the single most important water quality
characteristic for characterizing the cause of toxicity.
The most important consideration, in addition to
those mentioned above, is that the water be consistent
in quality and not contain contaminants that could pro-
duce artificial toxicity. For example, if there was a
nontoxic concentration of a non-polar organic present
in the dilution water, when samples are concentrated, it
might be toxic and this can confound the identification
of the components causing toxicity in the effluent. The
best policy is to use a high purity reconstituted water or
a well water of known suitability. Receiving water
should not be used until Phase III, when it is the water
of choice to evaluate the toxicant in the receiving water
system (see Section 2.2).
A reconstituted water of similar pH, hardness and
alkalinity to that of the effluent is a first approximation
of an appropriate water; however, organic matter is
hard to duplicate. Experience has shown that for the
Ceriodaphnia test, the addition of food1 to the water
has been helpful to provide some organic material.
With food added, traces of contaminants can be less
toxic. If higher concentrations of effluent are to be
used, the choice of the dilution water is less important
because the characteristics of the effluent dilution mix-
ture will resemble those of the effluent. As information
is gained about the toxicant characteristics, the choice
of dilution water can be improved.
Food added for the C. dubia tests are the yeast-cerophyll-trout food
(YCT) and the algae (Selenastrum capricomutum) at a rate of 0.1 ml/
15 ml (EPA, 1989C). Although at ERL-Duluth the algae has been
added at the rate of 0.05 ml/15ml until May of 1991 when we increased
the level (EPA, 1989C).
The impact of dilution water choice depends on the
IC25 (see Section 5.8) of the effluent. If toxicity changes
substantially from sample to sample, but the dilution
water selected does not match the effluent in water
characteristics yet is kept the same throughout several
samples for Phase I, then the effect of the effluent in
the dilution water can also vary across samples. As
the TIE progresses into Phase II, attributing relative
toxicity to various constituents must be more refined.
For instance, suppose the suspect toxicant is a cationic
metal whose toxicity is hardness dependent. Also,
suppose that the whole effluent has a hardness of
300 mg/l as CaCO3 (very hard water) but the dilution
water has a hardness of 40 mg/l as CaCO3. In this
case, the hardness in each of the test dilutions will be
different from that of either the whole effluent or the
dilution water. Provided the cationic metal concentra-
tions vary over the course of the TIE period, the amount
of toxicity (as toxic units2, TUs) due to a particular metal
concentration will also vary depending upon the effect
concentration in the effluent. If the first whole effluent
sample contains 160 ug/l of zinc (for this example, 160
|ig/l is 1.0 TUc in very hard water) and the test is
conducted using a dilution water of 40 mg/l as CaCO3
(soft water), the no effect concentration would be 100%
where hardness is 300 mg/l and the effluent would
have <1 TUc. The second whole effluent sample con-
tains 480 u.g/1 of zinc. One would expect this sample to
possess 3 TUs (480 u.g/1 + 160 u.g/1). The toxicity due
to the second effluent sample would likely contain more
than 3 TUs because the hardness at the effect level
(<100%) would be much lower than at 100% effluent
(where hardness is 300 mg/l as CaCO ). The effect
TUs is a means of normalizing the concentration term (i.e., LC50,
NOEC, IC25 as percent effluent; see Section 5.8) to a unit of toxicity.
The use of the TUs approach allows effluent toxicity to be compared
(provided test species and test duration are the same) to a suspect
toxicants toxicity. The toxicity of an effluent and a chemical are
different and different concentrations of each equal one LC50 (1 TU).
TUs of an effluent can be calculated for either acute or chronic toxicity
endpoints. The number of acute TUs in the effluent is 100% + LC50
= TU and the chronic TUs in the effluent is 100% * NOEC = TU or
100% + IC25 = TUc. (EPA, 1991B). For specific chemicals the TU is
equal to the concentration of the compound present in the effluent
divided by the acute test LC50 for TUa or the chronic test NOEC or
IC25 for the TU.. The assignment of TUc is necessary for the
correlation step (Phase III) when effluent toxicity TUs are compared
to suspect toxicant(s) TUs.
3-1
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level would be near 20-25% effluent where hardness water for the diluent, the hardness might change dra-
would be <100 mg/l as CaCO3 and 1 TU of zinc would matically and confound calculation of TU's in a like
be <160 ng/l. In addition, if one were to use receiving manner if the effect concentration was <100% effluent.
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Section 4
Effluent Samples
To determine whether an effluent sample is typical
of the wastewater discharge may require a number of
samples to be tested. Experience has shown that the
use of several samples spanning two to three months
has been successful in characterizing many effluents.
TIE work on atypical samples may be problematic and
these TIE procedures were not developed for one-time
episodic events. However, the very nature of atypical
samples may provide valuable assistance in the TIE
effort by identifying the type of toxicant(s) that previ-
ously was not suspect. This is probably more likely
when an atypical sample has greater toxicity than the
other samples. In addition, the atypical toxic sample
may aid a discharger in recognizing wastewater treat-
ment plant upsets and assist the discharger in imple-
menting prevention procedures or generally improve
and maintain better wastewater plant housekeeping
efforts, which in turn may eliminate the episodic toxicity
problems.
The acute Phase I manual discusses the quantita-
tive and qualitative changes in effluents (EPA, 1988A;
EPA, 1991 A) that may affect toxicity. Varying concen-
trations of toxicants, different toxicants, water quality
characteristics, and analytical and toxicological error
are all factors in determining the toxicity of an effluent.
Although the toxicity of an effluent over time appears
unchanged, there may be more than one toxicant in-
volved in each sample, and not necessarily the same
ones.
At the same time a sample is collected, information
on the facilities treatment system (normal operation;
aberrant processes) may be useful. When dealing with
industrial discharges, details of the process being used
may be helpful. These details and others should be
recorded and provided to the laboratory conducting the
TIE at the time of sample shipment. When samples
are received, temperature, pH, chronic toxicity, hard-
ness, conductivity, total residual chlorine (TRC), total
ammonia, alkalinity and DO should be measured. Fig-
ure 4-1 provides a typical format to record such infor-
mation.
Since most TIEs are not performed on-site, the
effluent samples must be shipped on ice to the testing
location. The samples should be cooled to 4°C or less
prior to shipment and they should be shipped in sturdy
ice chests to prevent either temperature increases or
container breakage during shipment. Primary require-
ments of the TIE are that toxicity occurs frequently in
the effluent samples and that the toxicity of each sample
(held at 4°C) remains in the effluent sample for a
sufficient period of time. If samples repeatedly lose
their toxicity after shipment, steps should be taken to
preserve toxic fractions (Section 6.7) for later testing
and analysis. For example, if the initial characterization
tests indicate the presence of non-polar organics, one
tool to use is to concentrate large volumes (5-10 L) of
effluent when the sample arrives (see Section 6). Use
of the Phase II (EPA, 1992A) non-polar fractionation
procedure is the preferred way to concentrate the non-
polar toxicants for subsequent analysis and testing.
While efforts must be expended on this procedure, it
can be a crucial step to aid in identifying potential
toxicants (in instances where toxicity is present and lost
in the effluent). The information on when toxicity de-
grades or is lost may become useful as the toxicant(s)
is identified (see Section 9; EPA, 1991 A). Filterable
toxicants which degrade quickly in the effluent may be
recovered from the filters with solvent and stored for
future use (cf., filtration test, Section 6.4).
For one chronic Phase I TIE, a typical volume of
effluent needed to ship is 19 L (5 gal) but of course this
will depend on the options chosen for the TIE (Section
6) and 38 L (10 gal) may be more helpful once identifi-
cation and confirmation begin on any sample. The
second edition of the acute Phase I TIE manual (EPA,
1991 A) recommends that samples be initially collected
and stored in both glass and plastic to determine whether
effluent stored in either container affects the toxicity.
Some compounds (such as surfactants) are less toxic if
water samples containing them are stored in plastic
containers. Prior to initiating the characterization it
may be useful to collect and test several preliminary
samples to determine which containers to use during
the TIE to provide samples that are the most represen-
tative of the effluent (see Phase I, Section 6 (EPA,
1991 A) for more details). Less volume (<2 L) is needed
for these tests.
Composite samples should be used for Phase I.
Later, in Phases II and III, where variability is desired,
grab samples should be used. Samples that are con-
sistent (i.e., composite samples) give results that are
easier to interpret and lead more rapidly to identifica-
tion (Phase II) and confirmation (Phase III) of the cause
of toxicity. Grab samples can provide the maximum
effluent toxicity; however, it is more difficult to catch
4-1
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Rgure 4-1. Example data sheet for logging in samples.
Sample Log No.:
Date of Arrival:
Date and Time
of Sample Collection:
Facility:
Location:
NPDES No.:
Contact:
Phone No.
Sampler
Sample Type: Q] Grab
D Glass
CD Prechlorinated
G Chlorinated
G Dechlorinated
Sample Conditions Upon Arrival:
Temperature
pH
Total Alkalinity
Total Hardness
Composite
Plastic
Conductivity/Salinity.
Total Residual Chlorine
Total Ammonia
Condition of treatment system at time of sampling:
Status of process operations/production (if applicable):
Comments:
intermittent peaks of toxicity (such episodic events may
not be caused by the same toxicant that causes routine
toxicrty).
Multiple effluent samples in each test should not be
used in Phase I as is done for permit testing (EPA,
1989C). We have found that using only one composite
sample for each set of Phase I characterization tests is
adequate. If several effluent samples are used for
renewals during the chronic Phase I TIE and the toxi-
cants are different or change in their ratios one to
another, the interpretation of Phase I will be nearly
impossible. Indeed such variability must be identified
but rt should be done after at least one or preferably
most of the toxicants are known. The use of one
sample is more important in Phase III, (EPA, 1992B)
where toxicrty data are correlated to the measured
concentrations in the effluent. If multiple samples are
used, this correlation can not be readily done because
the same toxicant may not be present in each sample,
or it is present in varying concentrations and other
toxicants may appear.
Existing routine toxicity test data should be exam-
ined. If one notes a sudden response such as death in
the middle to the end of the test period and especially if
it is associated with a new sample, the effect being
measured may actually be acute rather than chronic
and if so the approach may be switched to an acute
TIE approach. The investigative approach should be
adjusted to respond to such situations. When the
permit test is conducted and the test fails, it may be
desirable to try to identify the toxicants in those permit
compliance samples. This can be done by collecting
the appropriate volume needed for a chronic TIE of
either the daily samples or the three samples used for
the short-term toxicity test (EPA, 1992C). Additional
short-term toxicity tests can be conducted on each
4-2
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sample prior to any TIE tests on each sample or prefer- to demonstrate that the effluent is toxic in less than the
ably additional short-term tests would be initiated on full 7-d of the C. dubia or fathead minnow tests. When
each new sample during the 7-d test to evaluate whether the toxicrty that occurs in <48 h (C. dubia) or <96 h
it is the cumulative toxicrty from all samples or whether (fathead minnow) with any one of the samples from the
one or two samples are driving the toxicrty. We have permit compliance samples or any sample collected for
observed in several effluent tests that the toxicrty dur- the TIE, is observed as >50% mortality, acute TIE
ing the short-term chronic test can be caused by one or procedures can be applied to more quickly characterize
two samples and these samples cause the chronic test the toxicant(s).
4-3
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Section 5
Toxicity Testing
5.1 Principles
The test organism is used as the detector of chemi-
cals causing chronic toxicity in effluents and other aque-
ous media. The response to toxic levels of chemicals
is a general one; however the organism is the only tool
that can be used specifically to measure toxicity. Only
when the cause of toxicity is characterized can chemi-
cal analytical methods be applied to identify and quan-
tify the toxicants.
Chronic TIE's will usually be triggered by the use of
the toxicity test methods as found in the short-term
chronic toxicity test manuals (EPA, 1989C; EPA, 1992C).
These methods rely on sublethal endpoints as the indi-
cator of chronic toxicity for the Phase I manipulations,
therefore conducting the tests strictly as detailed in
those manuals is not always necessary and sometimes
not possible. Modifications have been developed and
these include: (a) reduced test volumes, (b) shorter
test duration, (c) smaller number of replicates, (d) re-
duced number of test concentrations, and (e) reduction
in the frequency of the test solution renewal. In addi-
tion, the frequency of preparation of manipulated
samples for test solution renewal must be established
and this issue is discussed in the following section.
Any loss of test precision due to these modifications is
not as critical during Phase I characterization as it is in
Phase II and Phase III (EPA, 1992A; EPA, 1992B).
During Phase I the analyst is searching for an obvious
alteration in effluent toxicity, which may be obtained
using modified chronic test methods. Confirmation
testing (Phase III) conducted according to the standard
methodologies will confirm whether the toxicant(s) de-
tected in the characterization and identification steps
(Phases I and II) is the true toxicant.
5.2 Test Species
In most cases, freshwater effluents will be sub-
jected to this evaluation because they have been found
to be chronically toxic to the cladoceran, C. dubia, or to
the fish, fathead minnow (P. promelas), or possibly to
the cladocerans, D. magna or D. pulex. Freshwater
effluents discharged into marine environments are evalu-
ated for toxicity using marine species or may be as-
sessed with freshwater species (EPA, 1991D). TIE
guidance for the marine species will be forthcoming in
the fall of 1992 (George Morrison, personal communi-
cation, ERL-Narragansett, Rl).
The species which detected the toxicity which in
turn triggered the TIE, is the first choice for the TIE
species. When an alternative species is chosen one
must prove that it is being impacted by the same
toxicant(s) as the species which initially detected the
toxicity. The species need not have the same sensitiv-
ity to the toxicant(s), but each species' threshold must
be at or below the toxicant concentration(s) present in
the effluent. One method of proving that the species
are being affected by the same compound(s) is to test
several samples of the effluent over time to both spe-
cies. If the effluent possesses sufficient variability, and
the two species IC25s (see Section 5.8 below for a
description of the IC25) change in proportion to one
another, the analyst may assume that the organisms
are reacting to changing concentrations of the same
compound. Further proof that the two species are
responding to the same toxicant should surface during
Phase III. If the toxicant is the same for both species,
then characterization manipulations which alter toxicity
to one species should also alter toxicity to the second
species. The extent to which toxicity is altered for each
will depend upon the efficiency of the manipulation and
the organism's sensitivity to the toxicant. Steps applied
in Phase III will confirm whether the two species are
indeed sensitive to the same toxicant in the effluent.
Extensive time and resources may be wasted if one
discovers during Phase III that the organism of choice
is not responding to the same toxicant as the species
which triggered the TIE.
For the above mentioned reasons, we recommend
when at all possible to use the organism which prompted
the TIE. Our chronic TIE experience has been based
on tests with C. dubia and/or larval fathead minnows.
Obvious constraints on the use of other species are
availability, size, age, and adaptability to test condi-
tions. Also, the threshold levels for additives and re-
agents must be determined for other species.
5.3 Toxicity Test Procedures
Measures to conserve time and resources required
to conduct a chronic Phase I must be used in order to
make the procedures cost-effective. The application of
all aspects of the standard short-term chronic tests to
Phase I in terms of replicates, routine water chemis-
tries, test duration, and volume is not practical due to
time constraints and expense. Variations of the proce-
dures need to be implemented whenever possible.
As mentioned above, smaller test volumes can be
used in all tests with C. dubia and in most instances
with fathead minnows. For example, 10 ml in a 1 oz
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plastic cup (or 30 ml glass beaker) has been adequate
for C. dubia and 50 ml in a 4 oz plastic cup (10 fish per
cup) has been used successfully to test the fathead
minnows (or 100 ml in a 400 ml glass beaker). There
are two precautions to watch for in the chronic TIE
tests—1) evaporation of test solutions and 2) transfer
of toxicants while moving the animals. If evaporation
reduces test volumes, efforts to reduce the evaporation
must be made or larger volumes must be used. The
volume of water added with each transfer should be
minimized, because the volume used in the test is
small, and the resultant test concentration could be
diluted, thereby reducing toxicity. Using the same size
test chambers and consistent volumes should be main-
tained in Phase I; when Phases II and III are initiated,
tests should be conducted following the test protocol
that was used to trigger the TIE. This may be impor-
tant in Phase I to be as sure that the oxygen require-
ments for the test species are met and that toxicity is
not due to physical restrictions of the test procedure.
If a reduction in the number of replicates per test
concentration is used, one must assume that precision
is sufficient enough to decipher changes in toxicity that
must be measured. For the C. dubia test, five animals
per concentration (one per cup) and for the fathead
minnow test, two replicates per concentration and 10
fish per replicate have been found to be adequate for
interpreting the changes in toxicity. However this smaller
data set is not amenable to all statistical requirements
as described for the short-term tests (EPA, 1989C; see
Section 5.8). Use of more organisms and more repli-
cates may be preferable if Phase I data are likely to be
used in Phase III confirmation (see Sections 2.2 and
2.3).
A shortened version of the 7-d C. dubia test, re-
ferred to as the 4-d test, may be useful in the TIE. The
4-d test does not have to be as sensitive as the 7-d
test, just sensitive enough that the toxicity changes
occurring in Phases I and II of the TIE (using 4-d tests)
would be the same as the 7-d tests. The 4-d day test
was found to produce similar results for single chemi-
cals (Oris et al., 1991), but in tests in our laboratory
with effluents, the 4-d test has not been as sensitive for
all effluents tested as the 7-d test in determining the
effects on young production and survival. Masters et
al. (1991) tested C. dubia to one effluent (three times),
three surfactants, three metals, and three organic com-
pounds with the 4-d and 7-d exposures. They found
that for the most part the effluent toxicity was similar for
the 4-d and 7-d test results but for the surfactants the
7-d test was more sensitive. For the metals (cadmium,
lead, and zinc), ethylene glycol, and pentachlorophe-
nol, the chronic toxicity values for both tests were very
similar while the 4-d test was more sensitive for phenol.
In the 4-d test, when animals are initially exposed
at 72 h they are ready to produce their first brood.
Therefore, toxicity can be underestimated because these
animals are predisposed to produce their first brood,
unlike the animals exposed as neonates (24 h old). The
exposure during a 4-d test may miss their most sensi-
tive life stage. However for the Phase I where the
purpose is to detect differences following various ma-
nipulations, this issue is not as important as the ability
to rapidly conduct the characterization. Use of the
shorter term test will decrease the cost of Phase I
TIE's. In the confirmation of toxicity (Phase III), the 7-d
test is required because the toxicity as measured in the
7-d test (with more replicates, more dilutions, more
volume) was used to detect toxicity for the permit, and
should be used to confirm the cause of toxicity.
To conduct a 4-d test with C. dubia, neonates (0-12
h old) are placed in the dilution water that will be used
to conduct the TIE. At present these animals are held
in groups of three, two or individually in test containers
(with 15 ml of culture water) and fed daily until they are
72 h (±6 h) old in a similar test fashion (Oris et al.,
1991). The animals are then transferred to the baseline
test solutions or the various characterization test solu-
tions. The test is then continued for 4-d using the
endpoint of three broods.
The use of known parentage (EPA, 1989C) for the
C. dubia test is important when the number of repli-
cates is reduced, and helpful for Phase I, II or III tests
and in routine tests as well (EPA, 1992C). For Phase I,
this known parentage approach allows the young of
one female to be used across one replicate of all
dilutions and the control (i.e., 5 animals), the young
from another female for the next replicate set of dilu-
tions and control, and so on until all test cups contain
one young animal. By this technique, animals from a
given female that later appear atypical in appearance
or movement or produce no young when others in the
same test concentration are producing normally can
legitimately be dropped from the data set without statis-
tical bias (Norberg-King et al., 1989). The ability to
discard such data without bias improves precision. Pre-
cision will be better when n > 7 per treatment for
C. dubia or n > 4 for the fathead minnow test.
5.4 Concentrations to Test
The level of toxicity for any given discharger most
likely will have been established with some degree of
certainty from previous tests that were conducted on
the effluent that triggered the TIE.
Therefore during Phase I of the TIE, we have found
that four effluent dilutions and a control are adequate to
define the toxicity of the sample while reducing the cost
of the tests. Now for the TIE, the key to choosing the
concentrations to test is to select those that will assist
in the detection of small changes in toxicity, which is
essential in the chronic TIE. For example, if the NOEC
(from a previous data set) is 12% (or IC25 is 10%),
then a concentration series such as 6.3%, 12.5%, 25%,
and 50% would be logical; or perhaps closer concen-
tration intervals may be desired. Using 20% as the
high concentration and a dilution factor of 0.7, would
mean the concentrations to test would be 7%, 10%,
14%, and 20%. If the NOEC (from historical data) is
40-50% (or above 50%), then the concentrations to test
should be, for example, 25%, 50%, 75%, and 100% or
5-2
-------
40%, 60%, 80%, and 100%. Choice of dilution factor
and test concentration range is a matter of judgement
and depends on needed precision and practicality.
In nearly all examples in this document, the con-
centrations of 12.5%, 25%, 50%, and 100% are used.
We are assuming that if effluents have ICp (or NOEC)
values below 10%, the effluent is likely to show acute
toxicrty and if so, an acute TIE approach should be
used. If chronic work is to be done on a highly toxic
effluent, the same recommendations given in the acute
manual should be used; that is, use concentrations of
4x, 2x, 1x and 0.5x the IC25 or IC50 value (see Section
5.8 for which value to select). For example, if the IC25
is 5% effluent, we would suggest using a range such as
20%, 10%, 5% and 2.5% for the various tests. It is
best to use the same dilution sequence within a series
of tests (Tier 1) when tests are to be compared to each
other for differences in toxicity.
5.5 Renewals
For C. dubia, daily renewals of the test media (as
required in the chronic manual, EPA, 1989C) are not
necessary in Phase I as long as the toxicity of the
effluent can be measured with one or two renewals.
Because available sample volume is limiting in some
manipulations, fewer renewals are desirable. As with
the test duration (4-d vs. 7-d) the acceptability of less
frequent renewals must be established by comparison
with whichever test duration is selected. However in
Phase III, tests must be conducted similarly to the
routine biomonitoring test. For the fathead minnow test
the frequency of sample replacement must be daily to
maintain adequate water quality because the live food
organisms (brine shrimp, Artemia salina) die 2-8 h after
being added to the freshwater test solutions. A baseline
test (see Section 6) is always conducted when the
sample is received. The suitability of reduced renewal
frequency can efficiently be evaluated at this time by
conducting comparative baseline tests simultaneously
with different renewal frequencies.
The number and types of chemical measurements
taken initially and at the renewal intervals (referred to
as finals) should be based on the need for these mea-
surements and their usefulness (see Section 2). Ini-
tially, little judgement about the value of these can be
made, but as toxicant characteristics are identified, the
usefulness of various measurements can be judged.
Initially, the usual measurements (hardness, alkalinity,
conductivity; EPA, 1989C) should be made but some of
these can be dropped as the TIE progresses. For
example, if non-polar toxicity is found, then hardness
and alkalinity need not be closely monitored. However
if a metal is suspected, then these measurements are
important. Low levels of dissolved oxygen in the
fathead minnow test are a greater concern than in the
C. dubia test, and the pH between the two tests will be
dissimilar after 24 h of exposure. The pH measure-
ment is frequently needed and for toxicants such as
ammonia it is extremely important (EPA, 1992A). If an
effluent contains greater than 5.0 mg/l of ammonia, the
pH should be carefully measured at least daily (or more
often) in all test concentrations. Since ammonia is a
highly pH dependent toxicant, one must be aware of
variable pH drift in the Phase I treatments which may
lead to erroneous conclusions. One generalization,
however, can be made. For characteristics that are
unlikely to change, such as conductivity and hardness,
both initial and final measurements need not be made-
once is enough.
5.5 Toxicity Blanks
A risk of the reliance on a toxicity response in the
characterization step of TIEs is the probability that
artifactual toxicity is created during sample manipula-
tions (see Section 2.4). While a particular manipulation
may cause some degree of artifactual toxicity, if the
toxicity is predictable the test may still retain its validity.
Since chronic tests are more sensitive to artifactual
toxicity, lower concentrations of additives or less se-
vere conditions must be used as compared to the acute
test.
The presence of artifactual toxicity caused by con-
taminated acids, bases, air, filters and columns and by
intentional additives are detected by treatment blanks
and toxicity controls. A blank is dilution water manipu-
lated the same as the effluent, and then it is toxicity
tested to determine if the manipulation added any toxic-
ity. The toxicity control is the reference used to judge
the impact of a manipulation. Sometimes the toxicity
control is the baseline test, at other times it will be a
characterization test. For example, the toxicity control
for the EDTA addition test is the baseline test while the
toxicity control for the post C SPE column test is the
filtration test (filtered whole effluent). Treatment blanks
for either the EDTA addition test or the sodium thiosul-
fate addition test are not appropriate as the testing of
these additives in clean dilution water is not represen-
tative of the effluents' characteristics. The toxicity con-
trol must be distinguished from the control treatment
(animals in standard culture or dilution water; also de-
scribed as "performance controls") which is always
used. Controls provide information on the health of
the test organism and the test conditions while the
blanks provide information on the cleanliness of the
acids and bases, the aeration system, the filter appara-
tus, the C1( SPE column, and other apparatus used.
Although artifactual toxicity may appear in the dilu-
tion water blanks, artifactual toxicity in the effluent
matrix may not be observed. One must decide whether
the test results from that manipulated sample are mean-
ingful. For example, if the aeration manipulation caused
toxicity in the dilution water blank but aeration removed
the effluents' toxicity then the conclusion that aeration
was an effective treatment is valid. However, if the
dilution water blank was toxic and it appeared aeration
did not remove the effluent's toxicity then one cannot
conclude that aeration was not effective without further
investigation.
5-3
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5.7 Renewal of Manipulated Samples
One must decide whether a manipulated sample to
be used for renewal during the test should be prepared
(e.g., aerated or passed over a C18 SPE column) as a
batch sample for the entire test or prepared separately
for each renewal. This choice may be dependent on
the persistence of the effluent toxicity, but whether daily
samples are prepared or batch samples are prepared
and used for renewals of the tests should be decided
by the investigator, and the same methods should be
performed consistently throughout the TIE. As a gen-
eral guideline, we have chosen to discuss these Phase
I steps as though one aliquot of effluent samples pre-
pared for the characterization tests is used for all re-
newals. However for either daily or batch samples, the
same techniques should be used for all the manipula-
tions. For example, a sample for the filtration test
(Section 6) may be batch prepared on day 1. Then on
day 2, a batch sample for the aeration test should be
prepared. Yet for the EDTA and sodium thiosulfate
addition tests, these additives should be added to the
effluent dilutions on the day of each renewal as batch
solutions for each dilution (e.g., add EDTA to 50 ml of
100% effluent, let sample sit and dispense to test
cups). This is true for the methanol addition and the
graduated pH manipulations as well. To test the post
C1( SPE column samples for some effluents, daily
samples may need to be prepared because of bacterial
growth problems in samples stored for several days.
Since Phase I TIE work is often concerned with the
qualitative evaluation of toxicity, rather than quantita-
tive, there is no reason why a test could not be termi-
nated sooner than 7 d, if the answer to the particular
question posed has been found. For example, if the
baseline test with a sample indicates a complete inhibi-
tion of C.dubia reproduction by day 5 of a 7-d test, and
one of the manipulated samples (e.g., aeration) shows
normal reproduction, there may be little point in con-
tinuing that test, because toxicity was altered. This
type of judgmental decision is harder to make in a
chronic fathead minnow test based on growth; how-
ever, by careful observation of factors such as survival
or behavior, the trend of the toxicity response may be
discerned earlier than 7 d. Sufficient measurable growth
of the fathead minnows may have been achieved by
5-d. Experiments with fish exposed to zinc and sele-
nium for 5-d and 7-d indicated that sufficient growth
differences could distinguish the toxic effect even at 5-d
(Norberg-King, 1989). However, if this information is
needed in Phase III, it is important to correlate the
same type of data and terminating the test early may
require additional tests later on.
Because the chronic test is longer and requires
more laboratory work than the acute test, loss of toxic-
ity of any effluent sample is more troublesome when it
occurs. If the presence of toxicity is not measured in
the whole effluent before Phase I tests begin, much
work will be wasted if the sample is non-toxic initially.
On the other hand, to delay by waiting for the test may
also result in the loss of toxicity. The best approach is
to examine existing data sets for evidence of toxicity
loss due to storage of samples. If there are none then
start a baseline test, and upon the onset of chronic
toxicity (e.g., 60% mortality, no reproduction by day 5 in
high test concentrations of a 7-d test, absence of food
in the gut of the fishes), additional follow-up manipula-
tions of Phase I tests should be started. Toxicity
degradation can be a useful tool in identification and
confirmation (cf., Section 2). Once it has been deter-
mined that the sample toxicity degrades quickly, Tier 1
and Tier 2 steps should be started on the day of arrival.
Removal of headspace in effluent storage containers
may help minimize the loss of toxicity.
5.8 Test Endpoints and Data Analysis
For evaluating whether any manipulation changed
toxicity, the investigator should not rely on statistical
evaluations only. Some treatments may have a signifi-
cant biological effect that was not detected by the
statistical analysis. Judgement and experience in toxi-
cology should guide the interpretation.
Endpoints for the most commonly used freshwater
short-term chronic tests are growth, reproduction, and
survival. Historically, the effect and no effect concen-
trations have been determined using the statistical ap-
proach of hypothesis testing to determine a statistically
significant response difference between a control group
and a treatment group. The no effect level, called the
no observed effect concentration (NOEC), and the ef-
fect concentration, called the lowest observed effect
concentration (LOEC), are then statistically defined end-
points. The NOEC/LOEC are heavily affected by choice
of test concentrations and test design. For example,
these effect levels are dependent not only on the con-
centration intervals (dilution sequence) chosen, but the
number of organisms, the number of replicates used,
and the choice of the statistical analysis for the data
(i.e., parametric or non-parametric). The minimum sig-
nificant difference detected in hypothesis tests can be
quite variable (e.g., 10% or 50%; Stephan and Rogers,
1985) and yet this difference is used to determine the
NOEC. In the chronic testing manual (EPA, 1989C),
the minimum number of replicates (a relatively large
number), organisms, and dilutions for the C. dubia and
fathead minnow short-term tests are needed to meet
the hypothesis testing requirements. When less repli-
cates, fewer numbers of dilutions and fewer test organ-
isms are used (as in the chronic TIE) the hypothesis
tests will not be able to detect smaller differences that
are needed for chronic TIEs. Therefore, hypothesis
testing is not suitable for Phase I purposes and a point
estimation method must be used.
The linear interpolation method described in the
supplement to the freshwater chronic manual (EPA,
1989C) calculates a point estimate of the effluent con-
centration that causes a given percent reduction based
5-4
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on the organisms response. The inhibition concentra-
tion (ICp3) program (Norberg-King, 1989; DeGraeve et
al., 1988; EPA, 1989C) was developed for the purpose
of analyzing data from the short-term tests. This method
of analysis is not as dependent on the test design as
hypothesis analysis and is particularly useful for ana-
lyzing the type of data obtained from Phase I testing.
When analyzing data for the ICp estimates, only one
test endpoint is determined. For C. dubia all the data
are used. If all animals have died, the data are entered
as zeros and if some animals have some young but the
adult dies, the partial brood values are used. We have
found with some effluents that when the 4-d test is
routinely applied during a chronic TIE, often the first
brood is produced and then the adult dies. In other
cases we have observed no adult mortality in the 4-d or
7-d test, but at the same effluent exposure concentra-
tions the 7-d test animals will not produce any young
while the 4-d test animals produce their first brood.
The dose response from this 4-d test is not typical in
the 7-d test, and the production of young can be prob-
lematic in data interpretation and analysis since mortal-
ity also occurred. For example, when analyzing the
data using the ICp program, the effects of survival and
young production are incorporated into one estimate for
the IC50 and IC25. Yet there is no doubt that 0-40%
survival is a significant reduction in survival that indi-
The ICp program (Release 1.1) calculates confidence intervals which
are limiting when the sample size is <5 and these confidence intervals
are less than 95% in version 1.1 (R. Regal, personal communication,
University of Minnesota, Duluth, MM). This is being corrected in the
revision of the program now underway (for more information, contact
Teresa Norberg-King). The ICp program is available by sending a
formatted disk to Teresa Norberg-King, EPA, 6201 Congdon Boule-
vard, Duluth, MN 55804.
cates toxicity, and would cause a routine test to fail
(EPA, 1989C). Therefore when this occurs, to track
toxicity in the TIE, it may require calculating the IC25/
IC50 for young production and survival and then recal-
culating the IC25/IC50 for survival alone. For the
fathead minnow test in the routine monitoring test and
the TIE tests, the weights are calculated as mean
weight per original fish rather than mean weight per
surviving fish (EPA, 1992C). Also the program allows
direct comparison of results from tests conducted using
different concentration intervals. The level of inhibition
(p) used as an endpoint (e.g., 25 or 50%) is not critical,
although the IC25 is generally suggested as an equiva-
lent for the NOEC (EPA, 1991B). Confidence intervals
are calculated using a bootstrap technique, and these
confidence intervals can be used to determine the sig-
nificance of toxicity alterations observed in Phase I. A
"significant reduction" in toxicity must be determined by
each laboratory for each effluent and in combination
with the precision of reference toxicant tests that the
performing laboratory achieves. The use of the IC50
for Phase I TIEs may be more useful when trying to
correlate the characterization test results to the effluent
toxicity. However, an IC50 may not be able to be
estimated while the IC25 can; use of a consistent
endpoint effect level is important for subsequent TIE
work (EPA, 1992A: EPA, 1992B). We have observed
substantial toxicity reductions in characterization tests,
yet it does not always appear to be a significant reduc-
tion when only the IC25s are compared. When this
happens the sample size should be increased with
subsequent testing in order to more clearly differentiate
the toxicity and the dose response curve should be
studied. Once the toxicant is identified, the number of
replicates is increased and more dilutions are used
(Phase III; EPA, 1992B), which increases the confi-
dence in the IC25.
5-5
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Section 6
Characterization Tests
The chronic Phase I manipulations follow the same
approach and employ the same type of manipulations
used in the acute TIE (EPA, 1991 A). These include
aeration, filtration, C1$ SPE extraction and chromatog-
raphy, chelation with EDTA, oxidant reduction and/or
complexation with sodium thiosulfate, and toxicity test-
ing at different pH values (Figure 6-1). The main
differences between the acute and chronic techniques
are that the concentrations of additives must be lower
and the test conditions must be less severe in a chronic
TIE because the chronic test organisms are more sen-
sitive to these conditions. The pH adjustment proce-
dures in Tier 2 are changed from the acute Phase I
because we found that consistent, representative blanks
with reconstituted water could not be obtained at higher
pH's.
The following characterization steps are all based
on the use of Ceriodaphnia or fathead minnows. Obvi-
ously, use of other species will require consideration of
appropriate test volumes and additive concentrations.
As discussed in the acute manual, if the TIE is done
with species different from the species used in the
permit, one must demonstrate that both species are
sensitive to the same toxicant(s) (see Section 5).
More than one effect is measured in chronic tests
(reproduction or growth and survival) and because par-
tial effects are more frequent in short-term chronic tests
than in acute tests, a graded response with concentra-
tion is often seen. A graded response allows one to
better judge small changes in toxicity—an advantage
not often available in acute tests. Also, effects (initial
mortality, delayed mortality, aborted young, reduced
young, poor growth) can be observed and used in
interpreting the results as can the time to onset of
effect be used. Such effects can be useful in distin-
guishing the response to different toxicants.
For acute TIEs, tests are quick and relatively inex-
pensive, so the need to maximize their usefulness is
lessened. The chronic test is more work not only
because the test is longer and more complex, but also
because more sample volume is needed. For ex-
ample, for tests such as the sublation test (a subse-
quent step in the aeration test (Section 6.4)) sample
size can be very restricting. In addition, if an effluent is
not always toxic, a decision has to be made as to
whether to test for the presence of toxicity first, before
manipulations are started. If the effluent is not toxic
and all the manipulations are set up, the results may be
of no value. On the other hand, if the presence of
toxicity is first established, often a week will have passed
and by the time manipulations are tested, the toxicity
may have degraded. Unfortunately, there is no clear
answer to which way to proceed. When there are data
for effluent toxicity for preceding months, examination
of these data may assist in the decision.
In the acute TIE, the initial test (EPA, 1991C) is
used to set the range of concentrations to test. How-
ever in the chronic TIE, an equivalent of the initial test
is not practical, therefore historical data must be used
to make such judgements. Lacking historical data, a
judgement will have to be made to set the test range
and guidance for this is given in Section 5.4.
For chronic Phase I characterization, the use of two
tiers of characterization tests is suggested (Figure 6-1).
Tier 1 is done without major pH adjustments. Experi-
ence with acute TIEs has shown that major pH adjust-
ments are usually not needed. Tier 2 is performed
only when Tier 1 does not provide sufficient informa-
tion, and consists of filtration, aeration and the Cu
separation technique of Tier 1 with an effluent sample
adjusted to both pH 3 and pH 10. Therefore when the
characterization tests indicate Tier 2 is not required,
resources needed to conduct the TIE are significantly
reduced.4 Each characterization test used in the Tier 1
or Tier 2 has as its foundation the information in the
acute Phase I manual (EPA, 1988A; EPA, 1991 A).
The principles, methods, and interpretation of results
are based on the acute manual, and the tests for Tier 1
(Figure 6-2) are discussed in Sections 6.1-6.8. All tests
within a Tier (1 or 2) should be started on the same
day. Starting chronic tests involves more effort than
acute tests, and logistics must be planned (for in-
stance, available animals of the appropriate age for the
chronic test, sufficient food supply for more chronic
tests, adequate supply of dilution water for all test
renewals). Tests need to be started on the same day
in order to compare results of each manipulation test to
others and to the baseline test (Section 6.3) results
(Table 6-1). Once the Tier 1 data are generated, they
are compared, and interpretations are made to see
which inferences can be drawn concerning the nature
of the toxicants. Usually, multiple manipulations and a
retest of selected manipulations will be effective in
A recent estimate of the cost of the Tier 1, Phase I for chronic toxicity
was equivalent to the full Phase I acute TIE (Aquatic Habitat Institute,
1992).
6-1
-------
Figure 6-1. Overview of characterization tests.
Chronic Phase I Characterization Tests
TieM
Baseline whole effluent test
EDTA addition test
Sodium thiosulfate addition test
Filtration test
Aeration test
Post C|8 solid phase extraction (SPE) column
test
Methanol eluate test
Graduated pH test
T
Tier 2
Baseline whole effluent test
pH adjustment test
Filtration and pH adjustment test
Aeration and pH adjustment test
Post C1S SPE column and pH adjustment test
Methanol eluate test
yielding information concerning the nature of toxicants
before additional effluent samples are tested (see Sec-
tions 6.15, 6.16 and acute Phase I manual, EPA 1991 A).
Sample Preparation for the Characterization
Tests
As for acute TIE tests, we suggest doing certain
chemical measurements and the manipulations on one
day and then starting the tests the next day (Table 6-1).
This schedule balances the work load more evenly.
When the sample is received (day 1), various measure-
ments (Section 4) are taken and some preparatory
manipulations for the Tier 1, Phase I are done.
First, the routine chemical measurements are taken
as discussed in Section 4. DO, conductivity, and pH
should be measured on the 100% effluent to ensure
that the values are in the physiologically tolerable range
for the test species. If these are at levels that could be
toxic (EPA, 1989C), there is little point to test the
effluent sample without some sample manipulation. In
addition, the water hardness and alkalinity should be
measured so that the appropriate dilution water can be
selected (see Section 3, Dilution Water). As the TIEs
have progressed, we have begun to match both the
hardness and the alkalinity of the dilution water to
similar values for the effluent.
Figure 6-2. Tier 1 sample preparation and testing overview.
Effluent Sample
EDTA
Additions
Sodium Thiosulfate
Additions
Minor
pH adjstments
6-2
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Table 6-1. Outline of Phase I effluent manipulations Tier 1 and Tier 2.
Description Section
DAY 1 SAMPLE ARRIVAL:
Measure
4.0
• temperature
• conductivity
• pH
• DO
• alkalinity
• hardness
• total ammonia
• total residual chlorine
Perform Sample Manipulations
• filter effluent
• perform solid phase extraction (SPE)
• collect effluent
• collect methanol eluate
DAY2 TOXICITY TESTING:
Warm aliquot of whole effluent and aliquots
of filtered effluent, post C,, SPE column effluent,
and methanol eluates.
Initiate Tier 1 Tests
• baseline toxicity test
• EDTA addition test
• sodium thiosulfato addition test
• aeration test
• filtration test
• post Cw SPE column test
• methanol eluate test
• graduated pH test'
6.0
6.4
6.6
6.7
6.1
6.2
6.3
6.4
6.5
6.6
6.7
6.8
ADDITIONAL TESTING ON SUBSEQUENT DAYS1:
Tier 2 Tests
• pH adjustment test 6.10
• aeration and pH adjustment test 6.11
• filtration and pH adjustment test 6.12
• post Ctl SPE column and pH adjustment test 6.13
• methanol eluate test for pH adjusted samples 6.14
1 Experimentation may be needed for this test (see text for details).
2 Tier 2 is primarily for those effluents where the results from Tier 1
did not indicate any clear pattern of toxicity change following
manipulation (see text for details).
The initial pH of effluent upon arrival at the testing
laboratory is referred to as pH i, which is not necessar-
ily the pH of the effluent at air equilibrium5. The pH of
the sample after being warmed, may be selected as
EPA suggests that toxicity must be prevented under worst case
scenarios (EPA, 1991B) which may mean the routine monitoring tests
were conducted at high pH's.
pH i rather than the pH upon arrival. The important
point is to use the same pH < for all subsequent tests.
As an effluent warms to 25°C in an open container,
CO2 escapes and the pH may rise from 7.2-7.6 to 8-
8.5. In some tests, once the food is added the pH may
rise faster or in some cases (e.g., the fathead minnow
growth test), once the food has been in the test solution
for a period of time, the pH may be lower (e.g., 7.5-
7.6). These changes may be important for interpreting
the data in a chronic TIE, and pH should be measured
in the test dilutions that determine the test endpoint. Of
course, since the endpoint may be unknown, pH is
typically measured in all test concentrations.
Since samples are cooled for shipping and storage,
upon warming to 25°C, some of the samples are apt to
be supersaturated. Supersaturation can usually be
monitored by measuring DO. If DO is too high, it
should be reduced to acceptable levels as described by
EPA (1989C) for the routine monitoring test or by maxi-
mizing surface-to-volume ratio of the container to facili-
tate more rapid exchange of equilibrium of the sample
and atmospheric oxygen. Ceriodaphnia are less sensi-
tive to supersaturation than newly hatched fathead min-
nows. For chronic Phase I tests, routine water chemis-
try measurements (such as DO, pH, temperature) are
more important than in acute Phase I tests.
The manipulations performed the day the sample
arrives are filtering, extraction on the C18 SPE column,
and collection of the methanol eluates (see Sections
6.5 and 6.7 below). The aliquots of filtered effluent and
post-column effluent will be held until the next day (day
2) to start the tests. Of course these samples should
be stored in the refrigerator at 4 (± 2°C). This sample
preparation schedule is particularly convenient for labo-
ratories who rely on courier services to deliver samples,
which typically occurs late in the morning.
On day 2, the EDTA addition test should be pre-
pared first so that compounds that are EDTA chelat-
able, yet may require an equilibration time for complex-
ation, can be chelated (see Section 6.4). Then the rest
of the manipulations (aeration, sodium thiosulfate addi-
tions, graduated pH adjustments) should be started.
For the laboratory that is experienced in chronic toxicity
testing, the amount of time required to conduct the
Tier 1 sample manipulations and set up the toxicity
tests is about 6-10 h.
6.1 Baseline Test
General Approach: To determine the effects of
Phase I manipulations on the toxicity of the effluent, its
inherent toxicity must be determined. The toxicity mea-
sured in this test is used to gauge toxicity changes
caused by some manipulations and to detect changes
in the sample's toxicity during storage. Baseline tests
must be repeated each time additional manipulation
tests are started.
Methods: The baseline test will be initiated using
concentrations based on the historical data for each
particular discharger. For the TIE, use of four (and
6-3
-------
three) dilutions have been sufficient for defining toxicity
(Section 5.4). If the toxicity is low, in order to draw
distinctions between the concentrations used in the test
for the various characterization tests, the dilutions may
need to be set closer, for example, 40%, 60%, 80%,
100%. In this test, and all subsequent characterization
tests, the test concentrations, test volumes and number
of replicates should be kept the same as described in
Section 5, Toxicity Testing.
On day 2, an aliquot of the effluent is warmed
slowly in a warm water bath to test temperature (25°C).
The various test concentrations are prepared using the
appropriate hardness reconstituted water. Next, rou-
tine chemistries are measured (initial pH, temperature,
DO). The use of dilution water controls is not required
for every manipulation but at least two sets of controls
should be included to estimate reproducibility. In addi-
tion, the tests are conducted using one C. dubia per
one 10 ml test volume in a 1 oz plastic cup (or glass
beaker) and five animals per treatment. For the fathead
minnow tests, two replicates per treatment, 10 fish in
50 ml in a 4 oz plastic cup, or 100 ml in a 400 ml
beaker, are assumed.
Interpretation of Results/Subsequent Tests: The
baseline tests serve as the basis for determining the
effects produced by various characterization tests. This
test serves as the toxicity control lor some of the other
tests. If baseline tests done on subsequent days with
additional manipulations indicate that the toxicity of the
effluent is decreasing, either every effort should be
expended to characterize the toxicity more quickly (i.e.,
Phase II identification or Tier 2 tests) or another sample
should be obtained. The "shelf life" of the toxicity can
be determined after a few samples have been evalu-
ated.
Special Considerations/Cautions: The controls
in this test will provide information on the health of the
test organisms, the dilution water, the test glassware
and equipment used to prepare the test solutions and
the cleanliness of the test chambers. This baseline test
serves as the toxicity controller some subsequent Tier
1 or Tier 2 tests.
6.2 EDTA Addition Test
General Approach: This test is designed to detect
effluent toxicity caused by certain cationic metals. The
addition of EDTA to water and effluent solutions can
produce non-toxic complexes with many cationic met-
als. Loss of toxicity with EDTA addition(s) suggests
that cationic metals are causing toxicity.
EDTA is a strong chelating agent and because of
its complexing strength, it will often displace other soluble
forms (such as chlorides and oxides) of many metals.
The ability of EDTA to chelate any metal is a function of
pH, the type and speciation of the metal, other ligands
in the solution, and the binding affinity of EDTA for the
metal. And the complexation of metals by EDTA may
vary according to the sample matrix. The specific form
of metal that causes toxicity in the water matrix may be
more important than the total concentration of the metal.
Cations strongly chelated by EDTA include alumi-
num (3+), cadmium, copper, iron, lead, manganese (2*),
nickel, and zinc (Stumm and Morgan, 1981). EDTA
weakly chelates barium, calcium, cobalt, magnesium,
strontium, and thallium (Flaschka and Barnard, 1967).
EDTA can form relatively weak chelates with arsenic
and mercury and anionic forms of metals (selenides,
chromates and hydrochromates) will not be chelated.
For some cationic metals for which EDTA forms
relatively strong complexes, the acute toxicity to C.
dubia is reduced (Mount, 1991; Hockett and Mount, In
Preparation). EDTA was shown to chelate the metal
causing the acute toxicity (at 4x the LC50) for copper,
cadmium, lead, manganese (2+), nickel, and zinc to C.
dubia in both dilution water and effluents. However,
they also found that EDTA did not remove/reduce the
acute toxicity of silver, selenium (either as sodium se-
lenite or sodium selenate), aluminum (AI(OH)/), chro-
mium (either as chromium chloride or potassium di-
chromate), or arsenic (either sodium m-arsenite or so-
dium arsenate) when tested using moderately hard
water and C. dubia (Hockett and Mount, In Prepara-
tion).
In the acute Phase I manual (EPA, 1988A), the
recommended amount of EDTA to be added was high
because the authors thought calcium and magnesium
had to be complexed in order to complex toxic metals
(D. Mount, personal communication, NETAC, Duluth,
MN). The mass of EDTA required was approximated
by the amount needed for the titration of hardness or
the measurement of calcium and magnesium when
titration was not possible due to interferences. A third
choice was to use 0.5x the EDTA LC50 for the test
species (EPA, 1991 A). Ideally the amount of EDTA to
add would be just enough to chelate the toxicant(s)
without causing toxicity or otherwise changing the ma-
trix of the effluent. Without knowing how much toxicant
must be chelated, the amount of EDTA to add must be
estimated. Recently, the role of calcium and magne-
sium was tested in our laboratory. Acute toxicity tests
with C.dubia were conducted in moderately hard and
very hard reconstituted water using copper, cadmium,
and zinc at 4x, 2x, and 1x the LC50 of each. When
one metal and EDTA were present at approximately a
1:1 molar basis, all the toxicity was removed regardless
of water hardness (J. Thompson, personal communica-
tion, NETAC, Duluth, MN). These results indicate that
calcium and magnesium concentrations do not affect
the levels of EDTA needed to remove the acute cat-
ionic metal toxicity. Whether toxicity reduction using
the 1:1 molar ratio is true for chronic toxicity has not yet
been evaluated in a likewise manner (cf., Interpretation
of Results/Subsequent Tests below). However, EDTA
and nitrotriacetic acid (NTA) were effective in chelating
the toxicity of one concentration of either cadmium or
copper to C. dubia at molar ratios of less than 1:1
(Zuiderveen and Birge, 1991). However, NTA pos-
6-4
-------
sesses the characteristic of increasing the toxicity of
some metals therefore NTA is limited in its usefulness
for the TIE.
The threshold levels for C. dubia and fathead min-
nows to EDTA were determined using 7-d tests in
different hardness waters and the results are given in
Table 6-2. For C. dubia, the chronic toxicity of EDTA is
not water hardness dependent, but for fathead min-
nows the sublethal toxicity appears to be greater in
softer waters. This is in contrast to the acute toxicity of
EDTA to Ceriodaphnia which indicated that EDTA
toxicity decreased with increased water hardness (Phase
I; EPA, 1991 A). Natural waters and effluents have
many constituents in addition to those added to recon-
stituted waters, and the behavior of EDTA in effluents
(or receiving waters) could be different than in simple
reconstituted water.
Methods: The goal is to add enough EDTA to
reduce metal toxicity, without causing EDTA toxicity or
substantially changing the water quality. The toxicity of
EDTA as determined in clean reconstituted water is
Table 6-2. Chronic toxicity of EDTA (mg/1) to C. dubia and
P. promelas in various hardness waters using the 7-d
tests.
Species
C. dubia
P. promelas
Water
Type
VSRW
SRW
MHRW
HRW
VHRW
VHRW
SRW
MHRW
HRW
VHRW
IC50
95% C.I.
4.5
3.6-6.0
7.5
6.2-8.3
8.8
4.7-13
7.5
6.2-9.8
7.8
6.7-8.6
12
10-14
136
130-139
163
150-188
236
227-248
287
269-300
IC25
95% C.I.
3.0
2.1-3.9
4.9
3.7-5.7
5.9
3.4-10
5.5
0.98-6.9
6.1
4.0-6.8
8.3
4.2-10
103
94-110
132
123-144
1
230
203-247
NOEC
2.5
3.1
5.0
5.0
5.0
7.5
100
100
200
200
LOEC
5.0
6.3
10
10
10
15
200
200
400
400
1 Value could not be determined, value would be less than lowest
test concentration.
Note: C.I. = confidence interval; VSRW = very soft reconstituted
water; SRW = soft reconstituted water; MHRW = moderately
hard reconstituted water; HRW = hard reconstituted water;
VHRW = very hard reconstituted water.
likely to be higher than the toxicity of EDTA added to
an effluent. Therefore, the EDTA toxicity values con-
tained in Table 6-2 represent maximum toxicity in any
effluent. The toxic concentration of EDTA in one efflu-
ent will probably not be the same as the concentration
causing toxicity in a different effluent or even a different
sample of the same effluent. To be safe, the concen-
trations of EDTA added to any effluent should be less
than the expected effect concentration of EDTA in clean
water. For either species, two EDTA concentrations
are added to two sets of two effluent dilutions. EDTA
stock solution is added after the effluent dilutions are
prepared so that the EDTA concentrations for each
addition are constant across each set of effluent dilu-
tions. A stock solution of EDTA (ethylene-
diaminetetraacetic acid, disodium salt dihydrate) is pre-
pared in distilled water. This EDTA stock solution
should be prepared so that only microliter amounts of
the stock are needed to minimize effluent dilution. No
more than 5% dilution of the effluent aliquot by EDTA
stock should occur.
To perform the effluent dilution test, two sets of
effluent dilution concentrations are prepared (e.g, 100%,
50%, 25%,) and each set receives one of two addition
levels of EDTA (Table 6-3). By using non-toxic con-
centrations of EDTA, there is less chance for artifactual
toxicity; since the total amount of metal to be chelated
is probably low for most chronically toxic effluents,
there is no reason to add high levels of EDTA. The
additive levels are based on the assumption that the
calcium and magnesium need not be chelated in order
to chelate the toxic metals, although the amount of
EDTA added is most likely still an excess.
An EDTA stock solution of 2500 mg/l can be pre-
pared. For the C. dubia tests, 0.06 ml is added to three
separate 50 ml aliquots in the first effluent dilution set
(i.e., 25%, 50%, 100%) to obtain a 3.0 mg/l final EDTA
concentration. In the second dilution set, 0.16 ml is
added to the other set of 50 ml effluent aliquots for a
final concentration of 8.0 mg/l. For the fathead minnow
tests, the same concentration of an EDTA stock solu-
tion can be used but the volume of stock additions
must be doubled for the 100 ml test volume/concentra-
tion.
Table 6-3. Concentrations of EDTA to add for chronic TIEs. Values
given are the final exposure concentration in mg/l.
Species
Water Type'
Concentrations
(mg/l)
C. dubia
and
P. promelas
SRW, MHRW, HRW, VHRW 3.0
8.0
' In very soft water, the final concentrations of EDTA must be lower
in order to not have EDTA induced toxicity, for example 1.0 mg/l
and 5.0 mg/l.
Note: SRW = soft reconstituted water, MHRW = moderately hard
reconstituted water; HRW = hard reconstituted water; VHRW
= very hard reconstituted water.
6-5
-------
To allow the EDTA time to complex the metals,
solutions should be set up on day 2 and all solutions
containing EDTA are allowed to equilibrate while other
manipulations are being prepared before test organ-
isms are introduced. A minimum of 2 h equilibration
time should elapse before organisms are added.
Since EDTA is an acid, the pH of the effluent after
addition of EDTA should be checked, although addi-
tions at these low levels should not lower the pH of the
effluent. The amount of change in solution pH will
depend upon the buffering capacity of the effluent and
the amount of reagent added. If the pH of the effluent
has changed, readjustment of the test solution pH to
pH i should be performed.
The EDTA is not added to one batch of effluent on
day 2; rather at each renewal EDTA is added to the
renewal test solutions prior to dispensing into the test
chambers in the identical way that the test solution was
first made (allowing equilibration time).
Interpretation of Results/Subsequent Tests: Tox-
ic it y may be removed at all exposures provided the
addition of EDTA does not cause toxicity. If the effluent
is less toxic (i.e., EDTA addition IC50 (or IC25) shows
less toxicity than baseline test IC50 (or IC25)) in either
of the EDTA addition dilution tests, then EDTA re-
moved or reduced the toxicity and cationic metal toxic-
ity is probably present. If, in either test, the effluent is
more toxic than in the baseline test, EDTA itself may be
causing toxicity and the test should be repeated using
lower EDTA concentrations. If toxicity is not reduced
below the baseline test, the probability of cationic met-
als causing toxicity in the effluent is low and higher
concentrations of EDTA can be tried, although this may
or may not be useful.
Table 6-4 shows the results of a chronic zinc test
and the reduction of the toxicity by the addition of
EDTA. When C. dubia were tested in very hard recon-
stituted water, zinc was chronically toxic at 55 u.g/1 and
EDTA was chronically toxic at 15 mg/l. When EDTA
Table 6-4. The chronic toxicity of zinc (ng/1) to C. dubia in very hard
reconstituted water and the toxicity of zinc when EDTA
is added.
2nc'
Cone.
jig/1 0
0 19.2
Mean Young per Female
EDTA Additions (mg/l)
2.5
18.6
5.0
17.5
7.5
17.6
15
6.8
3.4 19.4
14
55
17.8
8.2
22.0
20.8
23.2
19.0
20.8
16.6
1.8
5.3
1 Measured values.
2 EDTA not added to this zinc concentration.
was added to solutions of 55 ng/l zinc at 2.5, 5.0, and
7.5 mg/l EDTA respectively, the toxicity of the zinc was
removed but at 15 mg/l EDTA, EDTA itself was toxic.
Such trends may be similar to the toxicity reduction
observed in effluents. If toxicity is reduced in a system-
atic manner, such as in the example, proceed to Phase
II methods for identification of those metal(s) which are
chelated by EDTA. Additions of EDTA at 3 mg/l and
8 mg/l removed the toxicity of copper to C. dubia in a 7-
d two-renewal test with hard reconstituted water at
levels of 210 u.g/1 and 105 u.g/1 of copper. In addition to
removing toxicity due to metals, EDTA reduces the
acute toxicity of some calionic surfactants. This reduc-
tion of toxicity may also occur in chronically toxic efflu-
ents, and the toxicity reduced by EDTA should not be
assumed to be due only to cationic metals. See Sec-
tion 6.4 Aeration Test for subsequent tests to conduct if
cationic metals are not present in the effluent at chroni-
cally toxic levels but EDTA reduced toxicity.
Special Considerations/Cautions: If pH in the
EDTA tests is greatly different from that in the baseline
test, the test might need to be redone. There is no way
to distinguish the effect of pH change on the toxicity of
a pH sensitive toxicant (e.g., ammonia) from toxicity
changes caused by EDTA. A change of 0.1 pH unit
can cause substantial errors if ammonia is involved.
Before the test is reinitiated, data from the graduated
pH test should be examined to evaluate whether the
toxicity is pH dependent. This test data may be useful
in deciding whether the EDTA addition test should be
redone. EDTA additions to dilution water are not rel-
evant controls for the EDTA additions to effluent; there-
fore, the toxicity control is the baseline test. The
control of the baseline test serves as the QC for the
health of the test organisms, the quality of the dilution
water, and general test conditions.
If all dilutions where EDTA is added should cause
mortality, one possibility is that the stock solution of
EDTA is contaminated and the stock solution should be
checked by conducting another test with a new EDTA
stock.
6.3 Sodium Thiosulfate Addition Test
General Approach: Oxidative compounds (such
as chlorine) and other compounds (such as copper and
manganese) can be made less toxic or non-toxic by
additions of sodium thiosulfate (Na2S2O3). Toxicity from
bromine, iodine, ozone, and chlorine dioxide is also
reduced. Sodium thiosulfate has been routinely used
to reduce the toxicity of substances such as chlorine
(EPA, 1989C).
Reductions in effluent toxicity observed with so-
dium thiosulfate additions may also be due to the for-
mation of metal complexes with the thiosulfate anion
(Giles and Danell, 1983). The ability of sodium thiosul-
fate to form a metal complex is rate dependent and
metal dependent (Smith and Mart ell, 1981) and sodium
thiosulfate is not a particularly strong ligand for metal
complexation. Cationic metals that appear to have this
potential for complexation, based upon their equilibrium
6-6
-------
stability constants, include cadmium, copper, silver, and
mercury (2*) (Smith and Martell, 1981). The rate of
complexation is specific for various metals and some
cationic metals may remain toxic in the 24-h or 48-h
renewal period of the chronic toxicity test due to the
slow rate of complexation or the stability of the com-
plex. The thiosulfate anion is not very stable, and the
ability of sodium thiosulfate to complex the compound(s)
causing chronic toxicity without daily renewals has not
been tested completely.
Recent findings have shown that the acute toxicity
of certain cationic metals may be reduced by levels of
sodium thiosulfate added in the acute Phase I tests
(EPA, 1988A; EPA, 1991 A). The acute toxicity of
several cationic metals was shown to be removed by
sodium thiosulfate in standard laboratory water. The
acute toxicity at 4x the LCSOs of copper, cadmium,
mercury, silver, and selenium (as selenate) to C.dubia
was removed by sodium thiosulfate additions at levels
suggested in the acute Phase I manual. However, for
zinc, manganese, lead, and nickel, the acute toxicity
was not removed by the sodium thiosulfate additions
(Mount, 1991; Hockett and Mount, In Preparation). The
toxicity of mercury with the addition of sodium thiosul-
fate was reduced for 24 h but not 48 h which indicates
it may not have been completely complexed by the
thiosulfate. If the acute toxicity of metals can be re-
duced or complexed by sodium thiosulfate, the same
may be true for chronic toxicity. However, for C. dubia
7-d tests with hard reconstituted water, sodium thiosul-
fate levels of 5 mg/l and 10 mg/l did not remove or
reduce the chronic toxicity of copper at the same con-
centrations where EDTA complexed the toxicity (cf.,
Section 6.2).
The test animals will probably tolerate more sodium
thiosulfate than would ever be needed to render oxi-
dants or metals non-toxic in effluent samples, espe-
cially the fathead minnows in comparison to the C.
dubia (Table 6-5). The presence of oxidants or
complexable metals will reduce the concentrations of
sodium thiosulfate below the nominal concentrations
added.
Table 6-5 gives the toxicity values in various recon-
stituted waters. The effect concentrations for C. dubia
and fathead minnows were measured in waters of dif-
ferent hardnesses (soft, moderately hard, hard, and
very hard water (EPA, 1989C)). For Ceriodaphnia, the
results indicate that the sublethal toxicity is unchanged
regardless of the water type (Table 6-5). The toxicity
tests with sodium thiosulfate and fathead minnows (7-d
growth test) indicate that the toxicity due to sodium
thiosulfate is greater in softer waters.
Methods: Two sets of effluent dilutions (such as
25%, 50%, 100%) each set with a different level of
thiosulfate concentration (Table 6-6) are prepared re-
gardless of whether C. dubia or fathead minnows are
used as the TIE test organism. The concentration of
thiosulfate remains constant across one set of effluent
concentrations within a series (identical to EDTA addi-
tion test). Small volumes (microliter) of the sodium
thiosulfate stock solution should be added to minimize
the dilution (5% of total volume). Non-toxic concentra-
tions of sodium thiosulfate are used to reduce the pro-
Table 6-5. Chronic toxicity of sodium thiosulfate (mg/l) to C. dubia and P. promelas in various hardness waters using the 7-d tests.
Species
C. dubia
P. promelas
Water
Type
SRW
HRW
VHRW
SRW
MHRW
HRW
VHRW
IC50
95% C.I.
39
30-42
36
26-44
43
37-44
1,070
1,041-1,1005
2,001
1,891-2,161
4,871
4,633-5,051
8,522
8,053-8,704
IC25
95% C.I.
26
15-33
27
20-36
34
21-37
820
785-859
720
550-1,528
3,590
3,226-3,800
6,780
6,065-7,073
NOEC
30
30
30
750
750
3,000
6,000
LOEC
60
60
60
1,500
1,500
6,000
12,000
Note: C.I. = confidence interval; SRW = soft reconstituted water; MHRW = moderately hard reconstituted water; HRW = hard reconstituted
water; VHRW = very hard reconstituted water.
6-7
-------
Table 6-6. Concentrations of sodium thiosulfate to add for chronic
TIEs. Values given are the final exposure concentration
in mg/1.
Species
Water Type1
Concentrations
(mg/1)
C. dubia
and
P. promolas
SRW, MHRW, HRW, VHRW 10 25
1 In very soft water, ttie final concentrations of sodium thiosulfate
must be lower in order to not have sodium thiosulfate induced
toxicity, for example 1.0 mg/1 and 5.0 mg/1.
Note: SRW = soft reconstituted water, MHRW = moderately hard
reconstituted water; HRW = hard reconstituted water, VHRW
- very hard reconstituted water.
bability of artifactual toxicity, yet sufficient concentra-
tions are needed to remove/reduce oxidants.
For a C. dubia test, to the first effluent dilution set
(i.e., 25%, 50%, 100%), 0.20 ml of sodium thiosulfate
stock (2500 mg/l) is added to each 50 ml dilution to
obtain final concentrations of sodium thiosulfate of 10
mg/l. To the second effluent dilution set, 0.50 ml of the
same stock solution is added to 50 ml of each test
dilution to obtain final concentrations of 25 mg/l (Table
6-6).
The fathead minnow test is similar except that
twice the volume of the same thiosulfate stock is needed
(because of 100 ml test volumes) to achieve the same
final concentrations (Table 6-6).
The sodium thiosulfate is not added to a batch of
the effluent on day 2; rather, at each renewal, sodium
thiosulfate is added to the renewal test solutions in a
manner identical to the way they were first prepared.
Interpretation of Results/Subsequent Tests: The
results of the sodium thiosulfate addition tests are
compared to one another and to the baseline test
results to determine whether or not toxicity reduction
occurred. Toxicity may be completely reduced, par-
tially reduced, or not reduced. If toxicity appears to be
reduced and/or removed, then more tests to determine
whether the toxicity is due to an oxidant or to some
metal should be performed. When chlorine concentra-
tions are ±0.1 mg/l total residual chlorine (TRC), there
may be a toxicity problem for C. dubia. A significant
drop in the chlorine level in the whole effluent may
occur in the first 24-h period after sample collection and
testing. Therefore, tests repeated on an aged sample
may give different results if an oxidant is involved but
may give the same results if a metal is involved.
For cases where oxidants account for only part of
the toxicity, sodium thiosulfate may only reduce, not
eliminate, the toxicity. Yet the sodium thiosulfate addi-
tion test is useful even when chlorine appears to be
absent in the effluent. Oxidants other than chlorine
occur in effluents, and even if the effluent is not chlori-
nated this test should not be omitted. Both thiosulfate
and EDTA reduce the toxicity of some metals and this
information can be helpful in identifying the toxicant.
(However, this effect of thiosulfate/metal complexation
has not been demonstrated for chronic toxicity.) In
cases where both the sodium thiosulfate addition test
and the EDTA addition test reduce the toxicity in the
effluent sample, there is a possibility that the toxicant(s)
may be a cat ionic metal(s). Many oxidants are reduced
by aeration but if aeration does not reduce toxicity,
Phase II methods for identification of cationic metal(s)
toxicants should be investigated. No change in toxicity
suggests either no oxidants or certain metals.
Special Considerations/Cautions: The general
test conditions, quality of the dilution water, and health
of the test organisms are tracked by the controls in the
baseline test. Additions of sodium thiosulfate to dilu-
tion water are not relevant controls for thiosulfate addi-
tions to effluent to determine if the thiosulfate was toxic.
Therefore the toxicity control is the baseline test.
If all dilutions where sodium thiosulfate is added
should exhibit mortality, one possibility is that the stock
solution of sodium thiosulfate is contaminated and this
phenomena should be checked by conducting another
test.
6.4 Aeration Test
General Approach: Changes in toxicity due to
aeration at pH / may be caused by substances that are
oxidizable, spargeable, or sublatable. The chemical/
physical conditions of the aeration process will also
affect whether or not the toxicity is reduced or re-
moved.
Sparging of samples is done using air which in-
cludes oxidation as a means of toxicity removal. In our
experience, typically volatile compounds that are highly
water soluble (such as ammonia) will not be air-stripped
at pH i by this method. If aeration is one of the
mechanisms that removes the toxicity, then additional
tests must be performed to identify which mechanism is
removing the toxicity. Subsequent tests with nitrogen
can be used to determine if toxicity reduction was due
to oxidation. Also, air or nitrogen sparging can cause
surface active agents to sublfte. As bubbles break at
the surface, sublatable compounds will be deposited on
the sides of the aeration vessel. Sublatable toxicity
identification requires special sample removal and rins-
ing (see below). A visible deposit does not indicate the
presence or absence of such toxicants.
Methods: For the aeration process, the volume
of effluent and dilution water aerated is kept the same
even though all of the dilution water volume is not
needed for the aeration blank. The flow rate, bubble
size, geometry of apparatus and time of aeration should
be consistent among treatments. Taller water columns
and smaller bubbles should ensure better stripping;
therefore, the aeration vessel should be half-full or
greater for this process. Each aliquot (effluent and
dilution water) should be moderately aerated for a stan-
dard length of time (60 min). Use of gas washing
bottles (Kontes Glass Co., Vineland, NJ) fitted with
6-8
-------
glass frit diffusers located at the bottom of the vessel
for aeration is suggested because they sparge the
sample effectively. During aeration, the pH of the
effluent is not maintained at "pH i."
The volume of effluent aerated should be the same
for either a 4-d C. dubia test or a 7-d C. dubia two
renewal test (four dilutions, five replicates for each
dilution; see Section 5), although there is excess of
solutions for the 4-d test. Use of 300 ml of effluent (or
dilution water) in a 500 ml gas washing bottle or 500 ml
in a 1 L bottle and a flow-rate of 500ml/min is sug-
gested. Any loss of volume and any formation of
precipitates should also be recorded.
Interpretation of Results/Subsequent Tests: If
the aerated effluent has less toxicity than the baseline
test, and the aeration blank is not toxic, aeration was
effective in reducing toxicity. If the toxicity of the
aerated effluent is less than the baseline test, even
though the aeration blank is toxic, the results indicate
that aeration is an effective removal technique. If the
effluent toxicity is not reduced or it is more toxic after
aeration than in the baseline test (and the aeration
blank was non-toxic, then either toxicity was concen-
trated during the aeration process or toxicity was added
or created during the aeration process (see Special
Considerations/Cautions below).
Typically, using this aeration technique, ammonia
is not air-stripped from the sample at pH /'. However, if
total ammonia is at least 10 mg/l or higher and the pH
is above 8.0, ammonia measurements in the aerated
sample may be useful if the aeration manipulation re-
sulted in a toxicity reduction.
If a substantial reduction in toxicity is observed,
then the mechanism for the toxicity removal must be
determined. To determine if the reduction is due to
oxidation, sparging, or sublation, the air should be re-
placed by nitrogen. The flow of nitrogen through the
sample must be the same as for air. If nitrogen sparging
as well as air sparging removes or reduces the toxicity,
then oxidation as the removal process is eliminated. If
aeration only succeeds in reducing toxicity, then oxida-
tion may be involved. It is possible that a toxicant can
be removed through sparging and oxidation in which
case air should reduce toxicity more than nitrogen.
The presence of sublatable substances can be
determined (whether air or nitrogen is used) by remov-
ing the aerated sample from the aeration vessel by
siphoning or pipetting without contact with the sides of
the aeration vessel. The geometry of the aeration
vessel (i.e., at least a half-full cylinder) must remain the
same as in the initial aeration experiment but the recov-
ery of sublated compounds can be difficult. Dilution
water added to the aeration vessel is used as a rinse to
remove the sublate residue on the walls. To attempt
this recovery, use of graduated cylinders with ground
glass stoppers has been successful for acute testing
(EPA, 1991 A) because the water can be shaken vigor-
ously to contact all surface areas to recover the
sublatables. This sublation procedure is effective for
dissolved surfactants, and while sewage particles ad-
sorb surface active particles tightly, the actual sublation
process may take some time (i.e., >1 h) (AHPA, 1989).
If toxicity is not recovered from the vessel walls, the
presence of such compounds cannot be ruled out.
Specific procedures, for the larger volumes needed in
the chronic tests, have not yet been developed.
In some instances, sublatable toxicants may not be
removed by dilution water, and the use of solvents
(e.g., methanol) may be needed for better recovery.
However, the solvent will have to be reduced in volume
(aired down) in order to have an adequate concentra-
tion factor in the test solution and a sufficiently low
concentration of solvent for the subsequent toxicity
tests (see Sections 6.7 and 6.8 for methanol toxicity
information). Of course, dilution water blanks must
also be subjected to all steps to check for artifactual
toxicity.
Special Considerations/Cautions: Removal of
compounds by precipitation can occur through oxida-
tion. However, the filtration test should not change
toxicity of the effluent if oxidation is involved but filtra-
tion might also remove the toxicity of some sublatable
compounds absorbed to particles and therefore the
results of the aeration test can be compared to the
filtration test.
Use of nitrogen to sparge the sample is likely to
drastically reduce the DO. For instance, 1 h of nitrogen
sparging has caused the DO to drop below 4 mg/l. To
increase the DO before initiating the test after a sample
has been sparged with nitrogen, transfer the sample to
a container with a large surface area to water volume
ratio. The DO should rise to >5 mg/l without additional
aeration.
The baseline test serves as the toxicity control and
the aeration of the dilution water (aeration blank) pro-
vides information on the system apparatus. The gen-
eral test conditions, quality of the dilution water, and
health of the test organisms are tracked by the controls
in the baseline test. No significant toxicity should occur
in the aeration blank. Toxicity in the aeration blank
implies toxic artifacts from the aeration process, the
glassware, or a dilution water problem. If the aeration
blank is toxic, check the results of the test of the
filtration blank. If both blanks are toxic, then most likely
there is a problem with the dilution water but if only the
aeration blank is toxic, artifactual toxicity arose during
that manipulation.
6.5 Filtration Test
General Approach: Filtration of the effluent sample
provides information on whether the toxicity is filterable
yet provides relatively little specific information about
which class of toxicant may be causing the toxicity.
Reductions in the toxicity caused by filtering alone may
imply toxicity associated with suspended solids or re-
moval of particle-bound toxicants. Whether compounds
in the effluent are in solution or sorbed to particles is
6-9
-------
dependent on particle surface charge, surface area,
compound polarity and charge, solubility, and the ma-
trix of the effluent. If particles are removed, other
compounds may be bound to them and are not avail-
able to cause toxicity. The way the toxicant is bound to
the particulates is probably more important when using
filter feeders as the toxicity test organism in short-term
chronic tests. This is primarily a route of exposure for
filter feeders as compared to the fathead minnow. Tox-
icity can also be reduced by filtering if a toxicant(s) is
not particle-associated; we have observed that some
chemicals in a dilution water stock are removed by
filtering (e.g., DDT).
The filtration step also serves an important purpose
for another Phase I manipulation, the solid phase ex-
traction (SPE) (Section 6.6), where aliquots of the efflu-
ent typically must be filtered before application to the
SPE sorbent (see Interpretation of Results/Subsequent
Tests below). If many particles are present in the
sample, the sorbent may act as a filter itself or the
column will become plugged.
Methods: The use of a positive pressure filtration
system is superior to the use of a vacuum filter be-
cause volatile compounds may be removed by vacuum
filtering and hence confuse the effect of filtering (see
Interpretation of Results/Subsequent Tests).
As in the acute Phase I, prepare the filters (typically
1 u.m glass fiber filters without organic binder) by pass-
ing an appropriate volume (approximately one-fourth of
effluent volume to be filtered) of high purity water over
the filter(s) in the filter housing. This water is dis-
carded, a small aliquot of the dilution water is filtered
(prepare excess, at least 500 ml for the C. dubia 7-d
test and 800 ml for the fathead minnow 7-d test) and
discarded (100 ml) and the rest collected. A portion of
the filtered dilution water is collected and used for
testing and a portion reserved for the post C1t SPE
column test blank (Section 6.6). For example, the last
400 ml of the filtrate is collected for the C. dubia 7-d
filtration blank and post C1t SPE column blank tests.
Next the effluent sample is filtered using the same
filter, and a portion of the filtrate is collected for toxicity
testing and a portion set aside that will be concentrated
on the C,, column. When filtering the effluent, filter
enough sample for this test and enough sample (>1 L)
to use for the SPE step described below. For some
effluents, one filter will not suffice. A technique we use
is to prepare several filters at once by stacking 5-8
filters together followed by rinses of high purity water
and dilution water using the same rinse volumes as
above. Then the filters are separated, and set aside,
using one at a time for the effluent sample. If the
samples measure quite high in total suspended solids,
pre-filtering using a larger pore size filter may help.
Again, appropriate blanks must be obtained for any
pre-filtering. Low levels of metals on the glassware or
the filters could cause interferences in toxicity interpre-
tation. Pre-rinsing the filters and glassware with high
purity water adjusted to pH 3 may provide consistently
clean blanks and possibly less contamination in effluent
samples. If the sample cannot be effectively/easily
filtered due to many fine particles, centrifuging may be
better (again blanks must be prepared).
The filter housing should be thoroughly cleaned
between effluent samples to prevent any particle build-
up or toxicity carryover. We have found large filter
apparatus (1 L), removable glass frits, or plastic filtering
apparatus (Millipore*) to be useful. The glassware
cleaning procedure that is described in the acute Phase
I TIE manual should be sufficient for chronic TIE work
(EPA, 1991 A). The glass frits may require rigorous
cleaning (i.e., soak in strong acid (10% v/v) for 20-40
min) to remove residuals that may remain after filtering,
since the glass frit may itself act as a filter.
Interpretation of Results/Subsequent Tests: If
toxicity in the whole effluent is reduced by filtration, a
method for separating the toxicants from other constitu-
ents in the effluent has been achieved. This should
advance the characterization considerably because any
subsequent analysis will be less confused by non-toxic
constituents. If appropriate, one should determine if
toxicity loss was due to volatilization. Comparisons of
pressure filtering and vacuum filtering should indicate if
volatilization is involved. For further characterization,
the mechanism of removal should be determined (pre-
cipitation, sorption, changes in equilibrium or volatiliza-
tion).
Identification efforts should be focused on the resi-
due on the filter after testing indicates that the toxicant(s)
is not volatile. To recover the toxicity from the filter(s),
use of acidic and basic water as well as various organic
solvents can be tried. The recovery achieved by these
various methods provides information about pK and
water solubility of the toxicants. Filtration has reduced
the quantity of total cationic metals present in some
effluents. The recovery of the metal and acute toxicity
was successful when dilution water adjusted to pH 3
was used to extract the filter (EPA, 1991 A). Filter
extraction into smaller volumes than that of the effluent
sample filtered will give a higher concentration of toxi-
cant, perhaps allowing the use of acute test endpoints.
However, evidence then must be gathered to be sure
the toxicants causing acute toxicity are the same as
those causing chronic toxicity. Use of solvents will
require solvent reduction or solvent removal (exchange)
before testing (see Phase II; EPA, 1992A). Sonication
of filters is another approach but the manipulation must
be accompanied by proper blanks in similar fashion to
those needed for the pH 3 extraction of the filter extrac-
tion step described above.
If large volumes of an effluent (~2 L over one 1 u.m
filter) can be readily filtered, the effluent should be
filtered for the filtration test and unfirtered effluent can
be passed over the Cie SPE column (see Section 6.6;
Post CI8 SPE column test). Once it has been demon-
strated that filtration does not reduce toxicity in the
effluent, and the toxicity is recovered in the methanol
eluate test the routine filtering can be eliminated. By
6-10
-------
this approach the amount of testing to be done is
decreased, yet the tracking of toxicity is possible. We
have infrequently experienced any effluents that have
low amounts of filterable solids where the effluent could
be concentrated without filtering. If any effluent sample
has reduced toxicity in the filtration test and toxicity is
not observed in the methanol eluate test, characteris-
tics of the toxicant(s) will be described as filterable and
not C.- recoverable.
1 B
If the toxicity cannot be recovered from the filter,
was not volatile (see Section 6.4 aeration test) and no
other manipulations changed toxicity, use of Tier 2 is a
good subsequent step. Toxicity could have been re-
moved by the glass frit, and use of a plastic filter
apparatus or stainless steel frits may assist in identify-
ing that the toxicant(s) removed is on the frit or filter.
Filter-removable toxicity in Tier 2 is more difficult to
identify (because of the radical pH adjustments) be-
cause of irreversible reactions and potential for artifac-
tual toxicity (see Section 6.12 below).
Special Considerations/Cautions: The filtered
dilution water and filtered effluent sample also serve as
the toxicity blank and toxicity control respectively for
the post C1t SPE column test (see Section 6.6). The
results of the effluent filtration test should be compared
with the filtration blanks and no major change in the
trend of young production, growth or survival should
occur in the filtration blanks in comparison to the con-
trols in the baseline test. If the filtration blanks are
acceptable, then the results of the filtration test and the
baseline test should be compared.
As a toxicity blank for the SPE tests, if the filtration
blank is either slightly or completely toxic, but the post
C1§ SPE column effluent is not toxic (and effluent toxic-
ity was unchanged after filtration), the filtration blank
toxicity can be ignored since the effluent toxicity was
removed. However, as work proceeds to identification,
the blank toxicity will have to be eliminated or else it
could introduce an artifact and lead to a misidentification
of the cause of toxicity.
6.6 Post C18 Solid Phase Extraction Column
Test
General Approach: The C1( SPE column is used
to determine the extent of the effluent's toxicity that is
due to compounds that are removed or sorbed onto the
column at pH / (cf., post C1t SPE column and pH
adjustment test. Section 6.13 below). By passing efflu-
ent through a SPE column, non-polar organics, some
metals, and some surfactants are removed from the
sample. In addition, these columns may also behave
as a filter (see filtration test above).
Compounds in effluent samples interact with the
C1t and depending upon the polarity and solubility of
the compounds, the sorbent may extract the chemicals
from the water solution/effluent onto the column. Ex-
traction occurs when the compounds have a higher
affinity for sorbent than for the aqueous phase. Non-
polar organic chemicals are extracted because the C1g
sorbent is very non-polar in comparison to the polar
water phase; this extraction process is referred to as
reverse phase chromatography.
The effluent that passes over the column is col-
lected and the post-column effluent is toxicity tested in
order to determine if the column removed toxicity. If
the toxicity of the post-column sample is decreased,
removal of toxicant(s) by the column is probable but if it
is not, artifactual toxicity may be obscuring the removal.
Steps to deal with this are given below in Interpretation
of Results/Subsequent Tests. If the post-column sample
is highly toxic, the capacity of the column to extract the
toxicants may be exceeded or the column may have
been inadequately conditioned.
Because toxicity may be retained by the C1§ col-
umn, efforts to recover the toxicity are necessary. After
a sample is passed over the C1§ column, many of the
compounds extracted by the sorbent at a neutral pH
should be soluble in less polar solvents than water (i.e.,
hexane, methylene chloride, methanol, chloroform).
However, most of the non-polar solvents are highly
toxic to aquatic organisms. Sorbed non-polar organics
are eluted from the column because they have higher
affinity for the non-polar solvent than the C1t sorbent.
The methanol eluate test (Section 6.7) is designed to
determine if toxicants are non-polar.
Methods: The toxicity of the effluent, the type of
test to be conducted, and the frequency of the solution
renewal affect how much effluent must be filtered and
passed over the C18 SPE column. First, the concentra-
tions and the volume of the eluate needed for the
methanol eluate test (Section 6.7) to test at 2x or 4x
the whole effluent concentrations should be determined
(keeping in mind that the methanol test level must be
below the chronic threshold level for the species used;
Section 6.7). However, limiting factors of the maximum
volume to apply to a column, the minimum elution
volume required, and the concentration that can be
obtained within these confines must be calculated
(Tables 6-7 and 6-8).
For example, our procedure has been to pass 1000
ml of 100% effluent over a 1 g (6 ml) column and elute
with 3 ml of methanol which results in a theoretical
333x concentrate. The 1000 ml is the limit of sample
volume over a 1 g (6 ml) column and the 3 ml methanol
elution is slightly more than the minimum elution vol-
ume required (Table 6-7). However to test C. dubia at
4x, and to have the methanol concentration at a non-
toxic chronic level (Table 6-9), the 3 ml must be further
concentrated to 1.5 ml (now 666x whole effluent con-
centration). At present 3 ml of the eluate is concen-
trated in graduated centrifuge tubes to 666x by using a
gentle stream of nitrogen gas over the surface of the
methanol eluate in a warm water bath (25-30°C) to
concentrate the 333x eluate to a final volume of 1.5 ml.
For five replicates of 10 ml each, 0.30 ml of the eluate
can be added to 50 ml of dilution water and the result-
ant effluent concentration is 4x and the methanol con-
centration is 0.6%. However the 1.5 ml eluate from the
6-11
-------
Table 6-7. Factors to consider for the size of available pre-packed Clt SPE columns. Appropriate volumes of sample to apply to each
column with respect to maximum volumes of sample and minimum elution volumes, and elution volumes frequently used in the
TIE process.
Maximum Minimum Methanol No.
Columns Available' Conditioning Volume (ml) Elution Elution Methanol Eluate
Size (ml) g of Sorbent Volume (ml) of Effluent Volume2 Used (ml)3 Fractions'4 Concentration
6
12
20
60
1
2
5
10
10
24
40
120
1,000
2,000
5,000
10,000
2.0, 2.4
4.8
12
24
3s, 2.4
3
6
12
3
3
3
3
333x5, 41 7x
417X
41 7X
41 7x
1 g columns are available from J.T. Baker Chemical Company, Phillipsburg N.J. (1 g, 6 ml columns have been extensively used at ERL-
Duluth). 1 g, 2 g, 5 g, and 10 g columns are available from Analytichem International, Mega Bond Elut™, Harbor City, CA. Pumping rates
for each column are proportional to volume based on 1 L at 5 ml/min; therefore 2 L at 10 ml/min, 5 L at 25 ml/min, and 10 L at 50 ml/min.
We are currently evaluating the minimum elution volumes to determine if less eluting solvent can be used. Pumping rates for 5 L and 10 L
may need to be slower when eluting each column. Yet how much the pump should be slowed will be a function of the toxicants. The
contact time of the elution solvent with Cu sorbent may need to be increased if toxicity is not recovered in the methanol eluates.
Minimum elution volume as recommended by the manufacturers. For the 1 g column, J.T. Baker recommends 2.0 ml and Mega Bond
Elut™ recommends 2.4 ml, but 2.0 ml is probably adequate.
Elution of two one-half volume aliquots is better for optimizing the elution efficacy
For each fractionation of any size column, collect three separate 100% methanol fractions to use in methanol eluate test to attempt
recovery of the non-polar toxicants (see text for more details).
This procedure has been routinely used for acute TIEs. To maximize concentration and minimize methanol levels in concentration and
minimize methanol levels in toxicity tests it is best to use the minimum elution volumes recommended by the manufacturer.
1 L fractionation will allow testing of 4x, 2x, 1x only if
two solution renewals are used (Table 6-8). Daily
renewals for a 7-d C. dubia test require a total of 3.7 ml
at a water concentration of 0.6% methanol (which means
3 L of effluent must be fractionated to obtain 9 ml of
333x eluate which is concentrated to 4.5 ml to test at
4x) (Table 6-8).
To test at 2x using a 417x eluate from a 2.4 ml
elution, 0.048 ml in 10 ml will result in the 2x test
concentration. For a 7-d, daily renewal test at 2x, 1x,
0.5x, 3.0 ml is needed (5 replicates of 10 ml each)
which will require 1 L of effluent to be concentrated
(Table 6-8). By this procedure the final melhanol con-
centration is 0.48%. The 417x concentrate can also be
concentrated to 834x and use 0.048 ml/10 ml to test
the eluate at 4x.
For the 7-d fathead minnow test using 50 ml per
replicate and two replicates, a total of 7.4 ml of a
methanol eluate is needed for test initiation and six
renewals, which requires fractionation of 3 L of effluent.
This assumes the methanol test concentration between
species are kept the same. Actually the fathead min-
nows could probably be tested at methanol concentra-
tions of -1%, and using 0.96 ml of the 417x eluate per
100 ml will result in 4x effluent test concentration and a
1% methanol concentration (Table 6-9).
The methods below assume one effluent volume
(usually the 100%) is concentrated and the post col-
umn effluent sample collected and used for all solution
renewals during the test (Table 6-8). The procedure
described below is an overview of the steps needed to
prepare the column, collect methanol blanks, recondi-
tion the column, collect post-column effluent, and col-
lect methanol eluate (steps needed for this test and the
next test—Section 6.7). All steps are detailed in the
acute Phase I manual (EPA, 1991 A), and the major
difference for the chronic Phase I is that fewer post-
column samples (one or two versus three) are col-
lected.
The general technique for conditioning and using
the prepackaged SPE columns is as follows. Using a
pump system with a reservoir for the effluent sample
and teflon tubing, first pump 10-120 ml of HPLC grade
methanol over the column to condition the sorbent
(Table 6-7). This methanol is discarded. Without
letting the column go to dryness, 10-120 ml of high
purity water is passed over the column and discarded.
Next, before the melhanol blank is collected, the col-
umn is allowed to go to dryness. For 1 L of sample and
a 1 g (6 ml) column, two 1.5 ml aliquots of 100%
methanol are collected, combined, and tested as the
blank. The elution is more efficient when two aliquots
of 1.5 ml are collected in contrast to one elution of 3 ml.
The collection of three 100% methanol eluates (2.4 or 3
ml each) has been more helpful for tracking toxicity
than only one 100% methanol eluate sample. The use
of three 100% methanol elutions is replaced when the
Phase II fractionation procedures are applied. These
100% methanol eluates may need to be concentrated
prior to testing (see Section 6.7). The containers to
collect the methanol should be acid leached, hexane
and acetone rinsed, and allowed to dry before use.
After the methanol blank is collected, the column must
6-12
-------
Table 6-8. Test volume of eluate needed for methanol eluate test with C. dubia or P. promelas. Volumes described are based on minimum
elution volumes recommended (Table 6-7) and the highest test concentration possible with the methanol level at an acceptable
concentration.
Test
Species
C. dubia
C. dubia
C. dubia
C. dubia
C. dubia
C. dubia
C. dubia
C. dubia
P. promelas
P. promelas
P. promelas
P. promelas
Test
Duration
4-d
4-d
7-d
7-d
4-d
4-d
7-d
7-d
7-d
7-d
7-d
7-d
No. Renewals
& Original
Sample
2
4
3
7
2
4
3
7
7
7
7
7
High
Test Cone.
2x
2x
2x
2x
2x
2x
2x
2x
2x
2x
4x
4x
No.
Rep.
5
5
5
5
10
10
10
10
2
4
2
4
Volume of Eluate
Needed for Testing at:
333x' 41 7x2
1.05
2.10
1.58
3.68
2.10
4.10
3.16
7.35
7.35
14.70
14.70
29.40
0.84
1.6B
1.26
2.94
1.68
3.36
2.52
5.88
5.88
11.76
11.76
23.52
Test
Concentrations
2x, 1x, 0.5x
2x, 1x, 0.5x
2x, 1x, 0.5x
2x, 1x, 0.5x
2x, 1x, 0.5x
2x, 1x, 0.5x
2x, 1x, 0.5x
2x, 1x, O.Sx
2x, 1x, O.Sx
2x, 1x, O.Sx
4x. 2x, O.Sx
4x, 2x, O.Sx
Minimum
Volume (L)
of Effluent3
1
1
1
2
1
2
2
3
3
5
5
10
For the 333x eluate concentration, this volume is based on the assumption that the C. dubia test solutions are prepared as 300 )il of 333x
into 50 ml for 2x, 150 ul into 50ml for 1x, and 75 uJ into 50 ml for O.Sx. More volume will be needed if serial dilutions are prepared (600 \i\
vs 525 nl). For the fathead minnow tests this assumes test solutions are prepared as 600 nl into 100 mL for 2x, 300 |il into 100 ml for 1x,
and 150 ul into 100 mL for O.Sx. More volume will be needed if serial dilutions are prepared (1200 (il vs 1050 ul).
For the 417x eluate concentration, this volume is based on the assumption that the C. dubia test solutions are prepared as 240 |il of 333x
into 50 ml for 2x, 120 nl into 50ml for 1X, and 60 ^ into 50 ml for O.Sx. More volume will be needed if serial dilutions are prepared. For the
fathead minnow tests this assumes test solutions are prepared as 480 |il into 100 ml for2x, 240 nl into 100 mL for 1x, and 120 ul into 100
ml for O.Sx. More volume will be needed if serial dilutions are prepared. For the 4x fathead minnow test, 960 \i\ per 100 ml must be
prepared for the 4x solution.
Volume is based on high test concentration (2x or 4x) tested without concentration to obtain eluate twice as concentrated. If further
concentration is needed, twice as much effluent will be needed.
Table 6-9. Chronic toxicity of methanol (%) to C. dubia and P. promelas using the 7-d tests.
Species
C. dubia
Water
Type
SRW
SRW2
SRW
SRW2
Test
Renewal
daily
twice
twice
twice
IC50
95% C.I.
1.2
1.1-1.2
1.4
1.2
0.69-1.7
1.3
IC25
95% C.I.
0.45'
0.35-1.0
0.45'
0.36-0.70
0.59
0.29-0.95
0.83
0.34-1.0
NOEC
<0.5
<0.5
0.75
0.75
LOEC
—
—
1.5
1.5
P. promelas
SRW
1 Value is extrapolated.
2 Tests all conducted independently.
Note: C.I. = confidence interval; SRW = soft water
aily 2.1
2.0-2.2
1.34
0.27-1.5
1.3
2.5
6-13
-------
be reconditioned with 10-120 ml of methanol (which is
discarded). Without allowing the column to go to dry-
ness, follow the methanol with an aliquot (10-120 ml) of
high purity water, immediately followed by an aliquot of
filtered dilution water. The amount of filtered dilution
water needed will be dependent on the species and
type of test to be conducted. The initial aliquot of the
post-column water should be discarded (-200 ml) and
the remainder of the post column dilution water should
be collected. This post-column dilution water sample
will serve as the dilution water blank for the post CJt
SPE column test.
In order to optimize concentration of an effluent
sample and not exceed the specifications of the sor-
bent capacity, when the maximum volume (Table 6-7)
of a sample is passed over a column, the sorbent must
be reconditioned following the collection of the post
column dilution water. For example if 1.2 L of dilution
water is needed and 5 L of effluent is to be concen-
trated on a 5 g column, without reconditioning the
column between the dilution water and the effluent, the
sorbent's capacity is likely to be exceeded. Toxicity
might be observed in the post C SPE column test
because of the excessive volume of dilution water and
5 L of effluent. The procedures for conditioning the
column are similar to those above. The appropriate
amount of methanol (Table 6-7) is used to condition the
sorbent and the methanol is discarded. Before the
column goes to dryness, follow the methanol with an
aliquot (10-120 ml) of high purity water, immediately
followed by the volume of filtered effluent to be concen-
trated. Again, collect about 200 ml of the post-column
effluent and discard it. This is discarded to reduce the
possibility of higher background concentrations of metha-
nol in the post-column sample which might contribute
to artifactual toxicity. Collect remainder of post-column
effluent as a batch or in aliquots. If small quantities
(<500 ml) of post-column effluent are needed for toxic-
ity testing, separate post-column effluent samples may
help determine if toxicity breakthrough occurred, and
concentration factors will be different for the lower vol-
umes.
Interpretation of Results/Subsequent Tests: The
extraction efficiency of the column is evaluated by com-
paring the toxicity in the post C SPE column test to
the filtration test data. This post Cie SPE column test is
most useful when there is no post-column toxicity, and
filtration did not reduce toxicity.
When toxicity in the post-column effluent is re-
duced or removed, then the next step is to compare the
results with the methanol eluate test. If toxicity was
recovered in the methanol eluates (see Section 6.7
below), then efforts to identify the toxicants (Phase II)
should be initiated immediately.
If the post-column effluent toxicity was removed or
reduced, but toxicity was not recovered in the methanol
eluates (see below), it is possible that the toxicant is
not eluted into 100% methanol and the C16 SPE column
contains the toxicant. Use of the gradient of methanol
and water fractions should be tried as well as testing
the eluate at higher concentrations than 2x (i.e., 4x or
8x). If those tests do not indicate toxicity present in the
eluates (see below) alternate elution schemes (EPA,
1992A) must be tried to recover the toxicant. It is
important to recognize that the toxicity removed by the
C1t SPE column is not necessarily due to non-polar
compounds. Metals can be removed from some efflu-
ents via the Cu SPE sorbent. However, metals are not
efficiently eluted in methanol or other organic solvents.
Acid adjusted (pH 3) dilution water may be needed to
elute toxicant(s) from the column. If this is done, the
pumping rate of the pH-adjusted water should be slowed
(perhaps by one-fourth of original pumping rate) to
allow adequate contact time to elute the compound
from the sorbent. In addition, compounds such as
polymers or surfactants may be sorbed onto the col-
umn and some will elute with methanol while others do
not.
The column can act as a filter itself and the various
solvents used do not elute the toxicant. To check
whether the Clt column is acting as a filter, unfiltered
effluent can be passed over the C18 column and toxicity
test results compared to those from the filtered effluent
sample simultaneously. When effluent samples are
readily filtered (e.g., >1.5 L for one 1 u.m filter) filter the
effluent to conduct the filtration test and use unfiltered
effluent for the post C SPE column test and the
methanol eluate test. When toxicity can be recovered
in the methanol eluate, the toxicant(s) is most likely to
be non-polar and since filtration can be eliminated for
subsequent identification steps the amount of testing is
subsequently reduced.
If the post-column toxicity was reduced and/or re-
moved but not recovered in the methanol eluate test,
the possibility exists that the toxicant has degraded or
decomposed during the manipulation and the toxicant(s)
was not concentratable.
As mentioned above, when no toxicity occurs in the
post-column effluent (or the toxicity is reduced), and yet
the methanol eluate test did not exhibit toxicity, metals
may be involved or a non-polar that was not recovered
in the solvent may be involved (discussed above). To
check for cationic metal toxicity, the post C}l SPE
column test should be combined with the EDTA addi-
tion test and the sodium thiosulfate addition test to
characterize the post-column toxicity (see Section 6.16,
multiple characterization tests).
For effluents that have shown that the toxicant is
C recoverable, but the degradation of toxicity occurs
fairly rapidly (i.e., the effluent sample is non-toxic in 1-2
weeks), it may be prudent to concentrate additional
volumes of effluent immediately after the effluent ar-
rives at the testing laboratory. Non-polar toxicants may
not degrade in the methanol fractions as quickly in the
effluent samples. Collect the methanol fractions (three
100% fractions) or the various methanol/water fractions
as described in Phase II (EPA, 1992A) and hold them
at 4°C for analysis as the TIE proceeds. Similarly,
6-14
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once the cause of toxicity has been determined to be
non-polar (C extractable) it might be more appropriate
to immediately concentrate 10 to 20 L of effluent and
for the elution step, replace the three 100% methanol
elutions with the methanol/water procedures (EPA,
1992A). For chronic work, we have been using seven
water/methanol fractions (50%, 75%, 80%, 85%, 90%,
95%, and 100%) rather than the eight used in acute
TIEs because the toxicity has never recovered in the
25% fraction and by eliminating it the testing workload
is reduced. It may be prudent to try two additional
100% methanol fractions following the seven fractions
as well or follow it with alternate elution schemes (cf.,
Phase II; EPA, 1992A). By immediately concentrating
the effluent, it is possible to optimize the amount of
methanol available for testing and subsequent concen-
tration for analysis and the post-column samples can
be tested at one time. This eliminates duplication of
effort that is required when additional methanol eluate
is needed for subsequent work in Phase II.
Artifactual toxicity in the test containers may ap-
pear as a biological growth in the 100% post-column
effluent and the effluent dilutions during the test. Efflu-
ents from biological treatment plants may develop this
characteristic more readily than physical-chemical treat-
ment plant effluents. This growth can negate actual
toxicant removal by the column. While this growth
does not occur in all effluents, when it does occur with
one post-column effluent sample, the growth often oc-
curs in each subsequent post-column effluent sample.
The growth appears as a filamentous growth and gives
a milky appearance in the test vessel. This growth has
been linked to methanol stimulation of bacterial growth.
Methanol is present in the post-column samples be-
cause methanol is constantly released from the sorbent
during the sample extraction. Additional filtering of the
post-column effluent sample through a 0.2 u.m filter
before testing to remove bacteria and eliminate the
growth, has not been particularly successful. Artifac-
tual toxicity from the post-column effluent may be
avoided if the tests with the post-column samples are
initiated on the same day the effluent is concentrated.
To date, when we have collected the post-column
samples and tested them on the same day, we have
not experienced less artifactual toxicity than we found
in those effluents where artifactual toxicity consistently
has been a problem. However, less time elapses
before animals are exposed to the test solution, there-
fore less time is available for bacteria to cause prob-
lems in the post-column sample matrix. Another option
is to perform daily concentration of the effluent and
extraction of the column during the 7-d test, as fresh
post-column samples may minimize the artifactual tox-
icity.
When post-column artifactual growth is not readily
eliminated, then a different solvent (acetonitrile) to pre-
pare the column (but not for eluting) may be useful in
reducing the post-column artifactual bacterial growth.
Acetonitrile causes narcotic effects in toxicity tests, and
is recommended only to condition the columns to avoid
toxic concentrations. This technique has been suc-
cessful on a limited number of effluents.
Special Considerations/Cautions: Careful ob-
servations and judgement must be exercised in detect-
ing problems in the post C1t SPE column test. Low DO
levels can occur in these samples. Through testing
experience, the investigator will know whether toxicity
appears as artifactual (i.e., growth, low DO) as op-
posed to the presence of the sample toxicity. If artifac-
tual toxicity is not recognized, then a conclusion that
the C SPE column did not remove toxicity can errone-
ously be made. For this reason if the post-column
effluent is toxic, the methanol eluate must be tested
(Section 6.7). This avoids the artifactual toxicity issue
and the error can be avoided by determining the toxic-
ity of the eluate.
The methanol elution process does not always pro-
duce predictable results with the same effluent sample.
When toxicity is removed by the column but no toxicity
occurs in the 100% methanol eluates, it does not indi-
cate that the toxicity is not due to a non-polar toxicant(s).
To check this possibility, immediately test the series of
methanol/water fractions at concentrations of 4x or 8x.
Not all non-polar organic compounds elute into 100%
methanol as well as they do into lower methanol/water
concentrations. Also toxicants may smear across the
fractions and when <100% recovery of toxicity from the
column is not 100%, toxicity may not be observed at 2x
or 1x.
General test conditions will be tracked (dilution wa-
ter, health of test animals) by the controls in the baseline
test. The post-column dilution water blanks should be
compared to those controls to determine if the column
imparted toxicity. If the post-column dilution water
blank was toxic, but no toxicity or artifactual toxicity
occurred in the post-column effluent sample, the toxic
blank can be ignored.
Results of the post Cig SPE column effluent test(s)
must be compared to the results of the filtration test to
determine if the manipulations effectively reduced tox-
icity. When the post C(l SPE column test is plagued by
artifactual toxicity, the importance of the methanol elu-
ate test increases. The results of the post Cia SPE
column test must also be compared to the baseline
test to determine if toxicity was removed by the Cu
SPE column.
6.7 Methanol Eluate Test
General Approach: In order to elute toxicants
from the C SPE sorbent, a relatively non-polar solvent
is used. Hexane, one of the most non-polar solvents,
can be used to remove highly non-polar compounds
from the C]a SPE column. Yet hexane is one of the
most toxic solvents to aquatic organisms and has a low
miscibility with water. Methanol is more polar than
hexane, but is much less toxic and will elute many
6-15
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compounds. The use of methanol has been adopted
as the eluant for the acute TIE (EPA, 1991 A; EPA,
1989A) and the chronic TIE because of its low toxicity
(Table 6-9) and its usually adequate ability to elute
chemicals from the C1§ SPE column.
Methods: The conditioning and elution steps are
described in detail in the post C1t SPE column test
above (see Section 6.6). For this test, we assume that
the column extraction efficiency and elution efficiency
are 100%.
If a 1 g (6 ml) SPE column was used with 1 L of
100% effluent, and a 3 ml methanol eluate was col-
lected, the methanol eluate is a 333x concentrate of the
original effluent (Table 6-7). Depending on the amount
of effluent toxicity, this eluate may have to be concen-
trated further in order to test at a sufficient concentra-
tion (i.e., 4x) and have methanol concentrations in the
test lower than the methanol effect concentration. In
Table 6-9 the toxicity data for methanol toxicity to
C. dubia and fathead minnows are given. The toxicity
of methanol is slightly greater for C. dubia when the
test solutions were renewed daily but not significantly
for this characterization stage of the TIE. From these
data, one can decide how much methanol can be
added and how concentrated the eluant must be to
achieve 2x or 4x the original effluent concentration.
The choice of test concentration depends on the toxic-
ity of the effluent; for example, if the effluent is toxic at
-25%, one may not need to achieve a 4x concentra-
tion. Some methanol toxicity can be present, as long
as sufficient toxicity from the effluent is present to be
measurable. As discussed in the post C SPE column
test, the fathead minnows can be tested at 4x using
only 0.96 ml of a 417x methanol eluate but the metha-
nol concentration is about 1%, which cannot be toler-
ated by C. dubia.
Interpretation of Results/Subsequent Tests: If
toxicity occurs in the methanol eluate test at any con-
centration tested, Phase II should be initiated. This
step would include the use of a gradient of methanol/
water eluant solutions to elute additional columns and
conduct the toxicity tests on each fraction (Phase II;
EPA 1989A; EPA, 1992A). Toxicants other than non-
polar compounds may be retained by the SPE column
but they are less likely to be eluted sharply or eluted at
all (see Section 6.6). Non-polar toxicity can in some
instances be distinguished from post-column artifactual
toxicity if the eluate is checked for toxicity. Some
toxicants (such as some surfactants) may not elute
from the SPE column with methanol, but if toxicity is
not recovered in the eluate, it does not exclude the
possibility of a non-polar toxicant or metal (see Section
6.6 for additional discussion). Dilution water adjusted
to pH 3 or pH 9 may be useful in eluting a toxicant(s)
from the column. Some experimentation will be needed
to determine the volumes of water to pump over the
column. The pumping rate should be slowed consider-
ably to allow sufficient contact time on the column (see
details in Section 6.6 and Table 6-8).
At this time, we have not been successful in track-
ing chronic non-polar toxicity using the acute test end-
point with the methanol eluates, rather chronic tests
have been needed to track the chronic toxicity.
A subsequent test that may be useful is to assess
whether the toxicant must be metabolically-activated by
the test organism before exhibiting toxicity. These
activation reactions consist of oxidative metabolism by
a family of enzymes collectively known as cytochrome
P-450. Some toxicants require cytochrome P-450 acti-
vation before expressing toxicity. Piperonyl butoxide
(PBO) is a synthetic methylenedioxyphenyl compound
that effectively binds to, and blocks the catalytic activity
of cytochrome P-450. When a non-toxic amount of
PBO is added to an effluent test solution which con-
tains a toxicant(s) that requires metabolic activation,
the toxicity of the effluent can be reduced or completely
blocked (EPA, 1991 A). The relative specificity of PBO
for blocking the toxicity of metabolically-activated or-
ganic compounds makes this test a useful part of the
subsequent testing in the TIE. For example in the
acute Phase I (EPA, 1991 A) as a subsequent test, we
suggest that PBO may be added directly to the effluent
before adding the organisms. The 48 h LC50 of PBO
is 1 mg/l for C. dubia and we have used 0.250 to
0.500 mg/l to effectively block the acute toxicity of
metabolically-activated compounds for C. dubia in the
effluent and the methanol eluate. The NOEC and the
IC25 for PBO and C. dubia was determined as 63 u,g/l
and 89 ug/l, respectively. Low concentrations of PBO
have reduced the chronic toxicity in the methanol elu-
ate test and levels of 100 or 50 |ig/l have been useful in
chronic tests with C. dubia. The PBO should be added
using a minimal amount of methanol as a carrier sol-
vent since the level of methanol present in conjunction
with the methanol eluate is present. Since PBO is not
readily soluble in water, a superstock of 20 g/l is pre-
pared by dissolving PBO in reagent grade methanol.
An aliquot of the superstock is mixed in the standard
laboratory dilution water to produce a stock solution at
a concentration of 25 mg/l and aliquots of this stock
solution are added to the test cups after addition of the
methanol eluate, and the solution thoroughly mixed.
This test should be conducted in similar fashion to the
EDTA addition test. Appropriate blanks must be used,
for example both the methanol blank and the methanol
eluate must be tested with and without PBO. If toxicity
occurs in the methanol blank fraction with the PBO
additions, either PBO was present at toxic concentra-
tions or the methanol concentration in the test was too
high. If toxicity is observed in the methanol eluate with
the PBO addition, but not in the methanol eluate with-
out the PBO or either of the blank eluates (with PBO
and without PBO), this result is not very informative. It
is possible that the PBO has interacted in a synergistic
fashion with another compound present in the test
effluent that normally would not be toxic.
Compounds that are sparingly soluble in water may
not be eluted from the column with methanol. If this
6-16
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occurs, less polar solvents will have to be tried, but this
technique will require solvent exchanges to avoid toxic
solvent concentrations and other solvents may recover
chemicals not toxic in the effluent due to solubility
problems. At this time, we have not used solvent ex-
changes for chronic toxicity tests, but are exploring the
use of methylene chloride. The 48 h LC50 of methyl-
ene chloride to C. dubia is 0.13% and the chronic
toxicity to C. dubia is <0.03%. Therefore it cannot
readily be used as the primary solvent, but rather as
the exchange solvent and may be of limited use for this
effort. Additional work on the appropriate solvent ex-
change for chronic TIEs is on-going (EPA, 1992A).
Special Considerations/Cautions: The baseline
test serves as the toxicity control, and the methanol
blank serves as a comparison of the effects of metha-
nol alone in water. The health of the test animals, the
viability of the dilution water and general test conditions
are evaluated by the baseline controls. If the effluent
methanol eluate is non-toxic at 2x or 4x but the metha-
nol blank is toxic, the blank toxicity can be ignored
since no non-polar toxicity is recovered.
If effluent dilutions are set at 100%, 80%, 60%, and
40%, it might be useful to test the eluate at a multiple of
these concentrations, i.e., 2x, 1.6x, 1.2x, 0.8x or con-
centrate them to 4x, 3.2x, 2.4x, or 1.6x to compare the
baseline toxicity with the toxicity in the methanol eluate
tests. The artifactual growth observed in the post C]t
SPE column test from the methanol has not occurred in
our methanol eluate tests. This is most likely due to
the differences in how the methanol degrades/behaves
in dilution waters which are low in methanol-oxidizing
bacteria and other organic matter in contrast to effluent
samples (even post-column effluents).
6.8 Graduated pH Test
General Approach: This test will determine
whether effluent toxicity can be attributed to compounds
whose toxicity is pH dependent. The pH dependent
compounds of concern are those with a pKt that allows
sufficient differences in dissociation to occur in a physi-
ologically tolerable pH range (pH 6-9). The toxicity
depends on the form that is toxic (ionized versus un-
ionized). Metal toxicity can be affected by pH differ-
ences through changes in solubility and speciation. pH
dependent toxicity is likely to be affected by tempera-
ture, DO and CO2 concentrations, and total dissolved
solids (TDS). The graduated pH test is most effective
in differentiating substantial toxicity related to ammonia
from other causes of toxicity.
Ammonia is an example of a chemical that exhibits
different ionization states and subsequently pH depen-
dent toxicity. Ammonia is also frequently present in
effluents at concentrations of 5 mg/l to 200 mg/l (or
higher). Measuring the total ammonia in the sample
upon its arrival will be helpful to assess the potential for
ammonia toxicity. pH has a great effect on ammonia
toxicity. For many effluents (especially with municipal
effluents) the pH of a sample rises upon contact with
air, typically the pH of effluents at air equilibrium ranges
from 8.0 to 8.5. Literature data on ammonia toxicity
(EPA, 1985D) can be used only as a general guide
because the pH values for most ammonia toxicity tests
as reported in the literature are usually not measured
or reported fully enough to be useful in TIE tests.
Additional data on ammonia toxicity for C. dubia and P.
promelas is provided in the revised Phase II (EPA,
1992A). The acute Phase I manual has a lengthy
description of the toxicity behavior of ammonia (EPA,
1991 A) and Phase II provides additional information
(EPA, 1992A).
One might expect ammonia to be removed during
the Tier 2 aeration and pH adjustment test at basic pH
(described in Section 6.11). Based on our experience,
however, ammonia is not substantially removed by the
methods used to aerate the sample described in this
manual. (If a larger surface to volume ratio is used,
this manipulation can reduce ammonia levels; see In-
terpretation of Results/Subsequent Tests below and
Phase II; EPA, 1992A.) Other techniques which can be
used to remove ammonia may also displace metals or
other toxicants with completely different physical and
chemical characteristics. For example, ion exchange
resins (e.g., zeolite) remove ammonia, cationic metals,
and possibly organic compounds through adsorption.
Toxicity related to metals may also be detected by
the graduated pH test, although these effects are less
well documented in effluents (and for chronic toxicity)
than those associated with ammonia toxicity. The tox-
icity may change for both pH increases and decreases
from neutral pH (pH 7). Such behavior is characteristic
of aluminum and cadmium. Acute toxicity test experi-
ments with C. dubia in clean dilution waters indicate
lead and copper were more acutely toxic at pH 6.5 than
at pH 8.0 or 8.5 (in very hard reconstituted water),
while nickel and zinc were more toxic at pH 8.5 than at
6.5 (EPA, 1991 A). In recent experiments during a
chronic TIE, we have found that chromium is pH de-
pendent on an acute basis for C. dubia, but not water
hardness dependent. The pH dependence was not
observed in acute tests unless food (YCT) (EPA, 1992C)
was added during the 48 h acute test at test initiation.
Therefore, caution must be exercised in interpreting the
chronic toxicity results with effluents, because the
toxicant(s) may behave in certain ways that are not
documented in the literature.
By conducting tests at different pHs, the effluent
toxicity may be enhanced, reduced or eliminated. For
example (at 25°C) where ammonia is the primary toxi-
cant, when the pH is 6.5, 0.180% of the total ammonia
in solution is present in the toxic form (NH3). At pH 7.5,
1.77% of the total ammonia is present as NH3 and at
pH 8.5, 15.2% is present as NH3. This difference in the
percentages of un-ionized ammonia is enough to make
the same amount of total ammonia about three times
more toxic at pH 8.5 as at pH 6.5. Whether or not
toxicity will be eliminated at pH 6.5 and the extent to
which toxicity will increase at pH 8.5 will depend on the
total ammonia concentration. If the graduated pH test
is done at two pHs using the same dilutions, one
6-17
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should see toxicity differences between pH 6.5 and 8.5.
The effluent effect level (expressed as percent effluent)
should be tower at pH 8.5 than pH 6.5 if ammonia is
the dominant toxicant.
The most desirable pH values to choose to test for
the graduated pH test will depend upon the characteris-
tics of the effluent being tested. The graduation scheme
that includes the air equilibrium (the pH the effluent
naturally drifts to) will allow a comparison of treatments
to unaltered effluent (i.e., baseline test). For example,
if the air equilibrium pH of the effluent is pH 8.0, it may
be more appropriate to use pHs 6.5, 7.3 and 8.0. The
pHs of many municipal effluents rise to 8.2 to 8.5 (or
higher), so pHs such as 6.5, 7.5 and 8.5 may be more
appropriate. In any case, it will be necessary to con-
duct the test at more than one effluent concentration
(e.g., 100%, 50%, 25%) to determine what role, if any,
the pH dependent compounds play in toxicity.
The challenge of the graduated pH test is to main-
tain a constant pH in the test solution. This is a
necessity if the ratio of ionized to the un-ionized form of
a pH sensitive toxicant is to remain constant and the
test results are to be valid. However, in conducting
either acute or chronic toxicily tests on effluents, it is
not unusual to see the pH of the test solutions change
1 to 2 pH units over a 24-h period.
Methods: To lower the pH of the samples, either
CCX/air mixtures or HCI additions (or the combination of
both) are used. The pH should be maintained through-
out the 4-d or 7-d test with little variation (±0.2 pH
units).
When CO /air (without any acid addition) is used to
control the pn, the pH of the effluent samples is ad-
justed by varying the CO2/air content of the gas phase
over the water or effluent samples. By using closed
headspace test chambers, the CO2 content of the gas
phase can be controlled. The amount of CO2/air needed
to adjust the pH of the solution is dependent upon
sample volume, the test container volume, the desired
pH, the temperature, and the effluent characteristics
(e.g., dissolved solids). The exact amount of COj/air to
inject for a desired pH must be determined through
experimentation (on day 1) with each effluent sample
before the graduated pH test begins. Therefore, the
test may have to be set up later than the other Phase I
tests (e.g., day 3) unless experimentation was initiated
on day 1. The amount of CO2 added to the chamber
assumes that the liquid volume to gas volume ratio
remains the same. Generally, as the alkalinity in-
creases, the concentration of CO2 that is needed to
maintain the pH also increases. For adjusting pH's
downward from pH 8.5 to 6, 0.5-5% CO., has been
used. If more than 5% CO is needed, adjust the
solutions with acids (HCI) and then flush the headspace
with no more than 5% CO/air. With appropriate vol-
umes of effluent, experiments with variable amounts of
CO^air and equilibrated for about 2 h, are used to
select the needed CO concentration. More than 5%
CO2 is not recommended as CO2 toxicity is likely to be
observed. When dilutions of an effluent have the same
hardness (or alkalinity) and initial pH as the effluent,
the same amount of CO is usually needed for each
dilution, but sometimes different amounts are needed
in the higher effluent concentrations. Use of a dilution
water of similar hardness (or alkalinity) as the effluent
makes the CO2 volume adjustments easier. When
tests are conducted in these CO2 controlled environ-
ments, dilution water controls for each pH should be
included.
Acid is used first to adjust pH's when the amount of
CO./air needed to adjust to the desired pH is greater
than 5% COJaw. Again experimentation is needed to
determine how much CO^air is needed. Techniques
for acid adjustment are described in Section 6.10 below
and also in the acute Phase I manual (EPA, 1991 A).
For adding a mixture of COj/air to the headspace
of the test compartments, a 1 L gas syringe (Hamilton
Model S-1000, Reno, NV) is used. In most instances,
the amount of CO2 produced by the invertebrates has
not caused further pH shifts, but with larval fathead
minnows, the pH may drop from the additional amount
of CO2 respired by the fish bacterial metabolic CO2
released.
For the pH controlled tests, the pH should be mea-
sured at least two to three times for each 24 h period
when readings of survival and/or young production are
made. If samples are not renewed daily (as may be
the case for the C. dubia tests), then the headspace
should be re-flushed with COj/air after the animals are
fed. Again, some experimentation may be needed to
determine the amount of CQjaw needed for this step.
In all graduated pH tests, the pH should be measured
in all the chambers. If the pH drifts as much as 0.2 pH
units, the results may not be usable and better pH
control must be achieved. However, if pH fluctuates
more than 0.2 pH units and toxicity is gone at one pH
and not another, the toxicity results may be useful (see
Interpretation of Results/Subsequent Tests below).
Measurements of pH must be made rapidly to mini-
mize the CO2 exchange between the sample and the
atmosphere. Avoid vigorous stirring of unsealed
samples because at lower pH values, the COf loss
during the measurement can cause a substantial pH
rise. In addition, measure the DO because toxicants
such as ammonia have different toxicities when DO is
decreased (EPA, 1985D). Keep in mind that if the test
animals have been dead for awhile, the pH and/or DO
of the test water most likely will have changed. There-
fore, pH measurements should be made as soon as
possible if animals die rapidly.
Methods that use continuous flow of a COJa'n
mixture, such as tissue cell incubators, may be prefer-
able and give better pH control. A pH feedback system
can be used to control the CO2-mix to the incubators.
At this time we have not attempted to use a continuous
flow of CO2 and cannot recommend a system to use.
Maintaining pH above the air equilibrium pH (gen-
erally above pH 8.3) is difficult to achieve because the
6-18
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concentration of CO2 must be very low, and microbial
respiration can increase the CO2 levels in the test
chamber. Frequently we use a dilution water that has a
higher pH (i.e., very hard reconstituted water) to pre-
vent pH drift downward.
Interpretation of Results/Subsequent Tests: For
the graduated pH test, the pHs selected must be within
the physiological tolerance range for the test species
used (which generally is a pH range of 6 to 9). In this
pH range, the amount of acid or base added is negli-
gible, and therefore the likelihood of toxicity due to
increased salinity levels is low.
When ammonia is the dominant toxicant, the toxic-
ity at pH 6.5, should be less than in the pH 8 test.
However, ammonia is not the only possible cause of
toxicity. Using the pH of the baseline test, the relative
toxicity of each pH adjusted solution can be predicted if
ammonia is the sole cause of toxicity (EPA, 1989A;
EPA, 1992A).
However, if ammonia is only one of several toxi-
cants in an effluent, this procedure will be hard to
interpret. For this reason, if total ammonia concentra-
tions in the 100% effluent are greater than 20 mg/l,
include a pH 6 (rather than 6.5) and pH 7.3 (±0.2)
effluent treatment interfaced with other Phase I tests.
Complicating effects of metal toxicity may be reduced
by adding EDTA to the test solutions. However, the
ability of EDTA to detoxify metals may also change with
pH, although we have not experienced this effect yet.
Other metals may exhibit some degree of pH de-
pendence, but these are not as well defined. Whether
the metal toxicity can be discerned will depend in large
part on the concentration of other toxicants in the
sample. In order to detect metal toxicity, one must be
cautious when selecting a dilution water if the test
solutions are low effluent concentrations. Artifactual
toxicity due to metals may be created if the hardness of
the dilution water is much different from that of the
effluent (see Section 3). This effect may be magnified
for metals when coupled with the pH change. A dilu-
tion water similar in hardness to that of the effluent
must be used for this test to reveal metal-caused toxic-
ity. If more than one pH dependent toxicant is present,
the pH effects may either cancel or enhance one an-
other.
In the acute TIEs, we have suggested the use of
hydrogen ion buffers to maintain the pH of effluent test
solutions and to compare these test results to those
from CO2 adjusted samples. Three hydrogen ion buff-
ers were used by Neilson et al. (1990) to control pH in
toxicity tests in concentrations ranging from 2.5 to 4.0
mM. These buffers were chosen based on the work
done by Ferguson et al. (1980). These buffers are: 2-
(N-morpholino) ethane-sulfonic acid (Mes) (pKm = 6.15),
3-(N-morpholino) propane-sulfonic acid (Mops)
(pKf=7^5), and piperazine-N,N'-bis (2-hydroxypropane)
sulfonic acid (Popso) (pKt=7.8). We have replaced the
Popso buffer with another buffer which is more readily
soluble in order to achieve better pH control around the
pH 8.0 range. This buffer is N-tris(hydroxymethyl)
methyl-3-amino propanesulfonic acid (Taps) (pKa = 8.4)
and has been used primarily for the chronic C. dubia
tests at this time.
The acute toxicity of these Mes, Mops, and Popso
buffers is low to both C. dubia and fathead minnows
(Phase I; EPA, 1991A) (48-h and 96-h LC50s for all
buffers are <25 mM for both species). Sublethal levels
of the buffer are added to hold the pH of test solutions
for the acute Phase I tests (see EPA, 1991 A). Chronic
toxicity results using these three buffers indicated that
16 mM did not cause reduced survival or growth for the
fathead minnow 7-d test. For C. dubia, 4mM of all four
buffers has not caused reduced survival or reproduc-
tion in either the 4-d or 7-d tests. Use of the buffers is
preliminary and the effects due to interferences from
the buffers themselves have not been studied. It is
possible that the buffers may reduce the toxicity of
some toxicants.
The buffers must be weighed and then added to
aliquots of the effluent dilutions and control water as
batches. Then adjust to desired pH with acid and base
to the selected values and add the test organisms.
Solutions should be left for several hours to equilibrate,
especially for the Popso buffer which has low solubility
in water (in contrast to other buffers). While our experi-
ence with the buffers is limited, we have found the
amount of any buffer needed to hold a pH is effluent
specific. Once the pH is adjusted to the desired pH,
the test solutions need not be covered tightly to main-
tain pH; however pH should be measured at each
survival reading at all dilutions. The test results with
the buffers should mimic those of the earlier graduated
pH test if ammonia is the suspect toxicant.
The methods described in Phase II can be used to
add identify ammonia as the pH sensitive toxicant. Use
of the air-stripping method to remove ammonia from
the sample at high pH's should help evaluate whether
toxicant(s) other than ammonia are present (Phase II,
1992A). The results of this air-stripping test should be
compared with the aeration test results of Phase I, the
baseline effluent test and the graduated pH test. If the
ammonia concentration is decreased and the toxicity is
reduced or absent, more evidence that ammonia is
playing a role in the toxicity of the effluent has been
generated. Other compounds could precipitate with the
pH adjustment and concentration during air-stripping
and when water is added back into the solution, they
may not be available.
Special Considerations/Cautions: The controls
in the CO2 controlled chambers for each pH and the
baseline test act as checks on the general health of the
test organisms, the dilution water and most test condi-
tions. If the effluent pH in the baseline test is close to
that of the pH adjusted test solutions, the toxicity ex-
pressed in the two tests should be similar. Significantly
greater toxicity may suggest interference from other
factors such as the ionic strength related toxicity (if the
6-19
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pH was adjusted with HCI) or CO toxicity. Dilution
water tested at the various pH's does not serve as
blanks, as the effluent matrix may differ from that of the
dilution water. However, if acids and bases are added
(with or without CO2 additions) then toxicity blanks with
the same amounts of acid/base added need to be
tested to determine the cleanliness and effects of the
acids and bases. Other compounds with toxicities that
increase directly with pH may lead to confounding re-
sults or may give results similar to ammonia. Monitor-
ing the conductivity of the effluent solutions after the
addition of the acids and bases may also be helpful in
determining artifactual toxicity.
6.9 Tier 2 Characterization Tests
Two tiers are used in the chronic TIE approach
primarily because in our experience, radical pH adjust-
ment often is not needed. Only when the manipula-
tions in Tier 1 do not indicate clear patterns is Tier 2
conducted. Tier 1 manipulations do not involve the use
of drastic pH manipulations to characterize the toxicity
of the sample. The pH adjustments are used to affect
toxicity when the Tier 1 tests are not adequate or to
assist in providing more information on the nature of
the toxicants (Figure 6-3).
Changes in pH can affect the solubility, polarity,
volatility, stability, and special ion of a compound. These
can change the bbavailability of the compounds, and
also their toxicity. The Phase I acute manual (EPA,
1991 A; EPA, 1988A) discusses the effect of pH on
groups of compounds at length, therefore only an ab-
breviated discussion of pH effects will be covered in
this document.
Un-ionized forms of chemicals are generally less
polar than the ionized form, and the ionized forms
interact with water molecules to a greater extent. Com-
pounds may be more toxic in the un-ionized form, as
was discussed above in Section 6.8 graduated pH test.
Un-ionized forms may be easily stripped from water
using aeration, or extracted with SPE techniques and
subsequent elution with non-polar solvents. Also,
changes in solubility with pH change may cause com-
pounds to be removed by filtration. The form of metals
can be altered by pH and organic compounds can be
degraded at extreme pH values.
Even if the chemical species are unchanged,
changes in the pH of the solution may affect the toxicity
of a given compound. The cell membrane permeability
and the chemistry of the toxicant may be affected.
Rgure 6-3. Tier 2 sample preparation and testing overview.
pH Adjustments of the
Effluent Sample
6-20
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Changing pH and returning it to pH i after a short time
(-1 h) will not always change the toxicrty. However,
this adjustment may result in a reduction, loss or in-
crease in the toxicity. Sometimes only the pH adjust-
ment in combination with a manipulation (e.g., filtering,
solid phase extraction) changes toxicity when the same
pH unadjusted manipulation test did not.
6.10 pH Adjustment Test
General Approach: For this Tier 2 test, the efflu-
ent is adjusted to either pH 3 or pH 10, and left at those
pHs until other manipulations (aeration, filtration, and
CUSPE post-column effluent samples) are ready to be
readjusted to pH i. The pH adjustment alone may not
change toxicity, if equilibrium is slow. Satisfactory
blanks in chronic tests with various reconstituted wa-
ters adjusted to pH 11 have not been consistently
produced, but acceptable blanks have been obtained at
pH 10 (and pH 3), while pH 11 adjustments have not
been problematic in some effluent matrices. Since
pH 11 was subjectively chosen, we recommend adjust-
ment to pH 10 for chronic TIE'S. The pH adjustment
test serves as a toxicity control for the pH adjustments
combined with aeration, filtration and the C1t SPE col-
umn manipulation. As described in Tier 1 and the
acute Phase I manual, pH may drift very differently
during the toxicity tests following these more severe pH
manipulations. Therefore, monitoring and control of
test pH is necessary.
Methods: An aliquot of effluent is pH adjusted to
pH 3 and another aliquot is adjusted to pH 10, along
with dilution water samples which will serve as blanks.
Enough sample and dilution water are pH adjusted to
provide the necessary volumes for the aeration and pH
adjustment test, the filtration and pH adjustment test,
and the post C1t SPE column and pH adjustment test.
Minimal dilution of the effluent should occur, and the
use of 0.01 N, 0.1 N, and/or 1.0 N solutions of acids/
bases (Suprapur®, E. Merck, Darmstadt, Germany) to
adjust pH are suggested. The volumes and strengths
of the acid/base additions should be recorded as this
information may be useful in determining if artifactual
toxicity should be expected. This information can be
helpful when subsequent testing is conducted and knowl-
edge of the volumes of acid/base added to the previous
samples assists in making the pH adjustments more
rapidly.
Interpretation of Results/Subsequent Tests: A
decrease in toxicity compared to the baseline test should
be pursued to detect the mechanism of toxicity reduc-
tion. Often precipitation occurs after drastic pH change.
If precipitation does occur, then the filtration and pH
adjustment test will likely remove the toxicant and ef-
forts should be focused on recovery and identification
from the filter. Similarly, if the Clt SPE column or
aeration changes toxicity, these manipulations should
be pursued. If toxicity is only reduced by pH change,
(which is not common) not much can be made of the
information, and clustering of several manipulations as
well as adding additional techniques such as ion ex-
change should be explored. Dilution from the acid and
base additions should also be checked. Degradation of
•toxicity is a possibility also, but is nearly impossible to
detect at this stage.
The adjustment of pH (to pH 3 or pH 10 and back
to pH i) may cause toxicity problems. Just the addition
of the NaOH or HCI may be the cause of the toxicity
and may also occur in the dilution water blanks or only
in the effluent sample. The effect on effluent toxicity of
the Na+ and Cr additions, depends on the TDS concen-
tration of the effluent. The acid/base additions are
typically more toxic in dilution water than in effluent,
unless the effluent TDS concentration is high, and the
additional concentrations of acid/base result in toxic
TDS concentrations. These effects are of more con-
cern in chronic TIE's. The effect of NaCI additions on
TDS can be tracked by measuring conductivity. Appre-
ciable increases in conductivity should be a warning to
evaluate TDS toxicity caused by acid and base addi-
tion.
Increases in toxicity compared to the baseline test
may be a result of either an increase in TDS or toxicant
changes. TDS as a toxicant may be eliminated by
calculating the TDS at the ICp value. Effluents that
have high toxicity require high dilution to determine the
ICp, and at such great dilution the TDS is subsequently
diluted sufficiently to remove TDS as a candidate. If
this is not the case, NaCI can be added to an aliquot of
effluent to see if the acid/base additions could have
caused the increased toxicity. Table 6-10 provides
chronic toxicity information for NaCI in various hard-
ness waters for C. dubia and fathead minnows.
Precipitates can remove toxicity through sorption of
such chemicals as non-polar organics. In this case the
precipitate is only the mechanism of removal, not the
toxicant itself. The C1t SPE column is likely to remove
the toxicity in such cases; however, in Tier 2 a pH
change can also desorb toxicants from particles and
make them bioavailable and therefore toxic.
Different pH drift during the baseline toxicity test
and those after manipulations has been discussed (EPA,
1991 A). For a valid test, the pH during the test must be
known and maintained the same as in the pH / test. If
the drift of the pH varies considerably, confusion in
interpreting the results can arise if a compound whose
toxicity is pH dependent is present in the sample. If
good pH control is not maintained incorrect conclusions
are likely to be made and mislead the TIE process.
Special Considerations/Cautions: The addition
of acids and bases to the effluent does not give compa-
rable results of acids and bases added to the dilution
water. The amount of acid and base added to each
sample will more than likely be dissimilar. However,
dilution water toxicity blanks to assess the additions of
the acid and base are needed to determine whether
toxic concentrations of ions have been reached and to
determine the cleanliness of the acid and base solu-
tions that are used in this manipulation and subsequent
pH manipulation tests. The controls from the baseline
test provide information on the health of the test organ-
6-21
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Table 6-10. Chronic toxicity of sodium chloride (g/l) to C. dubia and P. prome/as in various hardness waters using the 7-d tests.
Species
C. dubia
P. promotes
Water
Type
SRW
MHRW
HRW
VHRW
SRW
SRW
MHRW
HRW
VHRW
ICSO
95% C.I.
1.3
1.2-1.5
1.6
1.4-1.7
1.5
1.3-1.6
1.4
1.1-1.6
0.84
0.76-1.1
1.3
1.2-1.5
1.5
1.4-1.6
3.2
2.9-3.3
4.5
3.9-4.9
IC25
95% C.I.
0.93
0.76-0.96
1.3
0.24-1.3
1.2
1.0-1.3
1.0
0.58-1.2
0.67
0.63-0.77
0.93
0.76-0.96
1.2
1.1-1.2
2.3
2.0-2.5
3.2
2.4-3.5
NOEC
0.63
1.0
1.0
1.0
0.50
0.63
1.0
2.0
2.0
LOEC
1.3
2.0
2.0
2.0
1.0
1.3
2.0
4.0
4.0
Note: C.I. - confidence interval; SRW = soft reconstituted water; MHRW « moderately hard reconstituted water, HRW - hard reconstituted
water, VHRW - very hard reconstituted water, laboratory test conditions. The pH adjustment test serves as the toxicity control (or
perhaps the "worst case" toxicity control) for the subsequent pH adjustment/characterization tests.
isms, dilution water, and laboratory test conditions. The
pH adjustment test serves as the toxicity control (or
perhaps the "worst case" toxicity control) for the subse-
quent pH adjustment/characterization tests.
6.11 Aeration and pH Adjustment Test
General Approach: Aeration at pH 3 or pH 10
may make toxicants oxidizable, spargeable or sublatable,
that are not so at pH i. If this does occur, avenues are
then available to characterize and identify, similar to
the procedures described for aeration at pH i in Tier 1.
For this test, two effluent aliquots which were adjusted
to pH 3 and pH 10 in the pH adjustment test are each
aerated for a period of time, for example, 1 h. The
aeration process can concentrate compounds due to
loss of volume, and caution should be exercised in this
aeration process and lost water may need to be re-
placed with dilution water.
Methods: The steps for this procedure should be
identical to those used in the non-pH adjusted sample
aeration (Section 6.4). The pH of the effluent may drift
during the aeration, and it should be checked at 30 min
intervals and readjusted to the original pH (pH 3 or 10)
if it has drifted more than 1 pH unit. The amount of
NaCI added from the acid/base additions may be differ-
ent in aerated samples than for pH adjustment test and
proper compensation for this difference must be made
as described above. The volume of effluent aerated
should be compared to the amount of original sample
volume prepared.
After aeration is completed, adjustments back to
pH ( should be made on all samples at the same time.
The formation of any precipitates should be noted, but
the importance of precipitates (if any) will not be known
at this point in the characterization.
Interpretation of Results/Subsequent Tests: If
aeration with either pH adjustment removes or reduces
the toxicity, additional tests must be performed to iden-
tify whether sparging, sublation, or oxidation removed
the toxicity, as described in Tier 1 (Section 6.4). If
toxicity is reduced because of precipitation, the results
for this test and the filtration and pH adjustment test
should be similar, but if oxidation is a problem, pH
adjustment and filtration will not affect the toxicity of the
effluent. At pH 10 the total ammonia levels can be
reduced by aeration. However, the geometry of the
aeration technique (i.e., small surface area) for this pH
adjustment and aeration test described here is not
particularly conducive to ammonia removal. However,
if aeration at pH (10) reduces toxicity compared to the
toxicity in the aeration testa\ pH /and the baseline test,
measure the total ammonia level in the sample to
determine if it was stripped from the effluent.
Special Considerations/Cautions: The results of
this test should be compared to the toxicity control ( pH
6-22
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adjustment test) and the baseline test. The aeration
and pH adjustment blank should be compared to the
pH adjustment blank. If the effluent toxicity is reduced
in the effluent following pH adjustment/aeration, and
the blank is toxic, the blank can be ignored and the
results indicate toxicity removal. However, if toxicity is
the same or greater, artifactual toxicity cannot be ruled
out and further tests must be conducted. Compare the
results of the aeration and pH adjustment blank to the
filtration and pH adjustment blank and the pH adjust-
ment blank (Sections 6.10 and 6.12). If all have toxic-
ity, then artifactual toxicity occurred from the pH adjust-
ment, while if only the aeration and pH adjustment
blank has toxicity, then the artifactual toxicity crept in
during the aeration manipulation and the test should be
repeated.
6.12 Filtration and pH Adjustment Test
General Approach: Since a pH change can
cause toxicants to precipitate or cause solubilized toxi-
cants to sorb on particles, filtration at altered pH values
can be used as a tool in characterizing the effluent.
Therefore, by filtering pH adjusted effluent, compounds
that were in solution without a pH adjustment may no
longer be in solution or any toxicants associated with
particles may be removed by the filtration process.
Differences in the toxicity caused by filtering (at pH /)
compared to the pH adjustment test (Section 6.10) may
imply toxicity associated with suspended solids. If pH
affects the filterability of the toxicants, solubility changes
are implied at those pH values. Once the toxicants are
filtered, the particles may be recoverable from the filter
if toxicity has not degraded.
Methods: Details of preparing filters are generally
the same as described in Tier 1 (Section 6.5), except
the high purity water used to rinse the filters must be
pH adjusted to the appropriate pH, as should the dilu-
tion water for the blank.
Effluent samples adjusted to pH 3 or pH 10 (Sec-
tion 6.10) are filtered, readjusted to pH i, and the filtrate
toxicity tested. Stainless steel filter housings are not to
be used for this step, because stainless steel will fre-
quently bleed metals when a pH 3 solution being
filtered is in contact with the stainless steel. An inert
plastic or properly cleaned glass housing should be
used.
Interpretation of Results/Subsequent Tests: The
results of the filtration and pH adjustment test are
compared to the toxicity controls—the baseline test and
the pH adjustment test. If the effluent is more toxic
after filtration and contamination is not the cause, the
breaking of an emulsion might be involved. If the
toxicity is removed or reduced by the filtration step and
dilution is not the cause, then toxicants have been
separated from the whole effluent and efforts should
focus on identifying the compounds filtered out. The
next step is to recover the toxicity as described in
Tier 1 filtration test. This may be accomplished using a
pH adjusted sample of water, perhaps using the pH
opposite of that used in the filtration process.
Special Considerations/Cautions: The pH ad-
justed and filtered dilution water serves as a blank and
the pH adjusted and filtered effluent sample serves as
a toxicity control for the solid phase extraction step
(Section 6.13). The results of the filtration and pH
adjustment test should be compared to the effluent pH
adjustment test and the baseline test. The filtration
blank should be compared to the baseline control, the
aeration blank, and pH adjustment blank. Toxicity in
the filtration blank implies toxic artifacts from the filtra-
tion process, the glassware, the pH adjustment or a
dilution water problem. If the baseline control perfor-
mance is acceptable, the blank toxicity was most likely
created during the pH adjustment or filtration. If the
aeration and pH adjustment blank is non-toxic, and if
the filtration blank is toxic, and the filtered effluent
sample is still toxic or more toxic, artifactual toxicity
cannot be ruled out. To check if it occurred during the
manipulation, the experiment must be repeated. If the
filtration blank is toxic, yet the filtered pH adjusted
effluent indicates that toxicity is reduced/eliminated, the
toxicity in the blank can be ignored.
6.13 Post C18 Solid Phase Extraction (SPE)
Column and pH Adjustment Test (pH 3
andpHG)
General Approach: Shifting the ionization equilib-
ria at high and low pHs may cause the C1t SPE column
to extract different compounds than at pH i. pH ad-
justed and filtered effluent is passed over a prepared
Cu SPE column to remove non-polar organic com-
pounds (cf., post C SPE column test, Section 6.6
above). Organic acids and bases may be made less
polar by shifting their equilibrium to the un-ionized spe-
cies. By adjusting the effluent samples to a low pH and
a high pH, some compounds that are in the un-ionized
form should sorb onto the column. However, the C
packing degrades at high pH, so pH 9 (rather than pH
10 or pH 11) is used in this manipulation. Specific
manufacturer's data should be checked for acceptable
pH range. We have had no experience in eluting
toxicants off the C1§ SPE column that would be sorted
only at an altered pH, and therefore we can only pro-
vide general rules to follow in these cases except those
inferred from how ionizable compounds behave in re-
gard to pH change.
Methods: All of the procedures for this manipula-
tion and the use of the C1t SPE column are the same
as is described in Tier 1 for the SPE extraction at pH i
(Section 6.6) with one exception. All water passed
through the column (rinse, blank and effluent) should
be acidified or rendered basic depending on which pH
is under investigation (see Section 6.12). The potential
for bacterial growth and artifactual toxicity in the post-
column samples remain the same as for pH /.
6-23
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Interpretation of Results/Subsequent Tests: The
extraction efficiency of the column is assessed by com-
paring the results of the post Ctl SPE column and pH
adjustment test (pH 3 and pH 10) to the filtration and
pH adjustment test, and the pH adjustment test. Again
post-column test results are the most interpretable when
there is no art if actual toxicity and toxicity was removed.
When the toxicity is removed, compare the results
of the test with the methanol eluate test below (Section
6.14). If toxicity is removed that was not removed
under pH i and recovered in the methanol eluate, ef-
forts to identify the toxicants should be started. If
methanol does not recover toxicity, a pH adjusted wa-
ter should be tried. For further discussions of the
interpretation of the results, see Section 6.6 above.
Special Considerations/Cautions: Careful ob-
servations and judgement must be exercised in detect-
ing problems in the post C1t SPE column and pH
adjustment test. Low DO levels can occur in these
samples (cf., Section 6.6). Through testing experience,
the investigator will know whether toxicity appears as
artifactual (i.e., growth, low DO) or as lack of toxicity
removal. If artifactual toxicity is not recognized, then
an erroneous conclusion that the C1S SPE column did
not remove toxicity can be made.
General test conditions (dilution water, health of
test animals) will be tracked by the controls in the
baseline test. The post-column dilution water blanks
should be compared to those controls to determine if
the column imparted toxicity. If the post-column dilu-
tion water blank was toxic, but no toxicity or artifactual
toxicity occurred in the post-column effluent sample the
toxic blank can be ignored.
Results of the post-column effluent test(s) must be
compared to the results of the filtration and pH adjust-
ment test to determine if the manipulations effectively
reduced toxicity. When the post Ctl SPE column test
data is plagued by artifactual toxicity, the importance of
the methanol eluate test increases.
6.14 Methanol Eluate Test for pH Adjusted
Samples
General Approach: This test is essentially the
same as the methanol eluate test in Section 6.7, except
that the columns were prepared with pH adjusted wa-
ters/effluents (see Section 6.13).
Methods: These are identical to those in Section
6.7, except the pH of the rinse water, blank and effluent
sample has to be adjusted to pH 3 or pH 9 (lowered
from pH 10).
Interpretation of Results/Subsequent Tests: If
the toxicity is recovered in the eluate, identification
should be initiated. Refer to Sections 6.6, 6.7, and
6.13 for more information.
Special Considerations/Cautions: The baseline
test serves as the toxicity control, and the methanol
blank (for pH adjusted samples) serves as the toxicity
control for the effects of methanol in water. The health
of the test animals, the viability of the dilution water and
general test conditions are evaluated by the controls.
The artifactual growth observed in the post C1t SPE
column test (with and without pH adjustments) from the
methanol has not occurred in methanol eluate tests.
This is most likely due to the differences in how the
methanol degrades/behaves in dilution waters which
are low in bacteria and other organic matter in contrast
to effluent samples (even post-column effluents).
6.15 Toxicity Characterization Summary
Phase I will not usually provide information on the
specific toxicants. If effluent toxicity is consistently
reduced, for example, through the use of the C1§ SPE
column, this does not prove the existence of a single
toxicant because several non-polar organic compounds
may be causing the toxicity in the effluent over time,
but use of the Clt SPE technique in Phase I detects the
presence of these compounds as a group. This lack of
specificity is very important to understand for subse-
quent Phase II toxicant identification. Efforts should
concentrate on those manipulations affecting toxicity in
which the toxicant is isolated from other effluent con-
stituents, such as the SPE column, filtration and aera-
tion.
After the Tier 1 group of Phase I tests has been
completed, the results will usually show that some
manipulations increased toxicity, some decreased it,
and others effected no change. In some instances,
Tier 1 results allow the researcher to proceed immedi-
ately into the Phase II identification, and sometimes
Phase I (Tier 1 and/or Tier 2) and Phase II combina-
tions are needed to determine the cause of toxicity (cf.,
EPA, 1992A). Of course, new approaches are fre-
quently devised as more Phase I TIEs are completed.
Toxicity may be changed by two or more tests, and
if so, then more conclusive inferences might be pos-
sible than when only one manipulation changed the
toxicity.
If all of the toxicity is not removed, it is possible that
other toxicants could be present in the effluent so that
only partial removal was obtained. Frequently more
than one manipulation affects toxicity but only infre-
quently is there no effect from any manipulation. Even
if toxicity is affected by only one manipulation, one still
does not know whether or not there are multiple toxi-
cants. When several manipulations affect toxicity, it
still does not ensure that there are multiple toxicants.
There is also no way to tell at this stage if there are
multiple toxicants, whether or not they are additive,
partially additive or independent. In our experience
with acutely toxic effluents, we have not found syner-
gism, but independent action has commonly been found.
Some toxicants identified in effluents have been addi-
tive, but more often these have been only partially
additive.
6-24
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The two objectives which usually move the TIE
along more rapidly are to separate and concentrate the
toxicant(s). Therefore, the first step in Phase II (EPA,
1989A) will often be to reduce the number of constitu-
ents accompanying the toxicants. These efforts may
reveal more toxicants than are suggested by Phase I
testing. In Phase II one may discover that toxicants of
quite a different nature are also present but were not in
evidence in Phase I and if this is the case, different
Phase I characterizations may then be needed. Once
the analytical methods to identify one or more of the
toxicants is found, efforts to confirm the cause should
be initiated immediately (EPA, 1989B; EPA, 1992B).
As discussed earlier, the amount of time necessary
to adequately characterize the physical/chemical na-
ture and variability of the toxicity will be discharge
specific. For a given discharge, the factors that will
affect the length of time it takes to move through Phase
I is the appropriateness of Phase I tests to the toxi-
cants, the existence of long- or short-term periodicity in
individual toxicants and the variability in the magnitude
of toxicity. An effluent which consistently contains toxic
levels of a single compound that can be neutralized by
more than one characterization test should be identified
and moved into Phase II more quickly than an ephem-
erally toxic effluent with highly variable constituents,
few of which or none of which are impacted by any of
the Phase I tests. Several samples should be sub-
jected to the Phase I characterization tests but not all
manipulations have to be done on all subsequent
samples. The decision to do subsequent tests on these
samples to confirm or further delineate initial results is
a judgement call and will depend on whether or not the
results of Phase I are clear-cut. Sometimes it may be
reasonable to start Phase II and Phase III on the first
sample.
If the Phase I characterization tests that remove or
neutralize effluent toxicity vary by the sample, the num-
ber of tested samples must be increased and the fre-
quency of testing should be sufficient to include all
major variability. The differences seen among samples
can be used to decide when further differences are not
being found. Phase I characterization testing should
continue until there is reasonable certainty that new
types of toxicants are not appearing. No guidance can
be given as to how long this may take—each problem
for every discharger is unique. While the toxicity of
samples can be very different, the same characteriza-
tion tests must be successful in removing and/or neu-
tralizing effluent toxicity.
Often the next step of the TIE is obvious; at other
times the outcome of Phase I will be confusing and the
next step will not be obvious. In our experience with
acutely and chronically toxic effluents, once one toxi-
cant is identified, identification of subsequent toxicants
becomes easier because: (a) the toxicity contribution
of the identified toxicant can be established for each
sample; (b) the number of Phase I manipulations that
will affect the toxicity of the known toxicant can be
determined; (c) one can determine whether the identi-
fied and the unidentified toxicant(s) are additive; (d) if
some manipulations affect the toxicity due only to the
unidentified toxicants, some of their characteristics can
be inferred; and (e) one can determine if the relative
toxicity contributions of identified and unidentified toxi-
cants varies by sample. Such information can be used
to design tests to elucidate additional physical/chemical
characteristics of the toxicants that cause chronic toxic-
ity.
6.16 Use of Multiple Characterization Tests
Type and amount of testing is dependent on the
toxicity persistence in the effluent, the nature of the
toxicity, and reassessment of previous Phase I results
(observed trends in the characteristics can be very
important). Several tests could each partially remove
the effluent toxicity because several compounds are
causing the toxicity, or that one toxicant can be re-
moved by several Phase I steps. R-r example, if
several toxicants are acting to cause the toxicity, then
the graduated pH test and the post C1t SPE column
test both might result in a partial toxicity reduction. If
sodium thiosulfate and EDTA both reduce toxicity, cat-
ionic metals might be suspect.
In the acute Phase I (EPA, 1991 A), the use of
multiple manipulations (combining two of the Phase I
tests) was advocated and this same concept is also
useful for the chronic TIE as well. For effluents with
multiple toxicants, especially if they are not additive,
multiple manipulations are helpful. Especially when no
single manipulation removes all the toxicity, multiple
manipulations should be tried.
When the Cia SPE column only partially removes
toxicity, Phase I manipulations with the post-column
sample should be tried. For this multiple manipulation,
the post C1g SPE column effluent can be treated as
whole effluent, and several of the Phase I steps can be
conducted on the post-column effluent such as the
EDTA addition test, the thiosulfate addition test, and
the graduated pH test. However, these combinations
are useful only with the post-column effluent provided
that no artifactual toxicity is present.
If the C1t SPE column partially removes toxicity,
pass an aliquot of the post-column effluent over an ion
exchange column to determine the characteristics of
the remaining toxicity. If a non-polar toxicant and
ammonia are suspected, then passing the sample over
the C18 SPE column and then over zeolite may assist in
accounting for all of the toxicity. Likewise, passing the
effluent over zeolite and then over the C1( SPE column
may provide additional insight. To gain this knowledge
toxicity tests must be performed after each manipula-
tion and not just on the multiple manipulated sample.
Effluent characterization must be approached with-
out any preconceived notion or bias about the cause of
6-25
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toxicity because many constituents are present in efflu- times the answer being sought is only whether or not a
ents and their chemistry is often unknown, resulting in specific substance is causing toxicity. Obviously in
circumstantial evidence that is frequently misleading, such cases testing is specifically selected to answer
Certainly all available information and experience should that question and therefore not all manipulations need
be used to guide the investigative effort but temptations to be performed.
to reach conclusions too soon must be resisted. Sorrte-
6-26
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Section 7
Interpreting Phase I Results
After Phase I on one sample or several samples is
completed, the investigator must carefully evaluate the
data, draw conclusions, and make decisions about the
next steps that are needed. Sometimes the next step
is obvious, at other times the outcome will be confusing
and the next step will not be obvious. Several general
suggestions, based on our experience to date, may
provide some help.
In this section, various examples of Phase I results
are given with interpretation suggestions. This discus-
sion is repeated from the acute Phase I characteriza-
tion manual (EPA, 1991 A), and not all aspects have
been evaluated for chronic TIEs yet. These examples
should be used only as guides to thinking and not as
definitive diagnostic characteristics. Since almost any
toxicant can be present in effluents, clear-cut logic is
not totally dependable in interpreting results. Rather,
one must use the weight of evidence to proceed, and
be aware that artifacts cannot at this point always be
identified.
One should avoid making categorical assumptions
to every extent possible. For example, to assume that
the toxicity is due to a non-polar toxicant(s) because
the toxicity in the post C18 SPE column effluent was
removed, often is an error. Metals may also be the
toxicant adsorbed by the SPE column; we have ob-
served zinc, nickel, and aluminum concentrations re-
maining on the C1B SPE column. However, if the
toxicity can be recovered in the methanol fraction, then
the theory that a non-polar toxicant(s) is causing the
toxicity is better substantiated, because metals do not
elute with methanol and therefore do not produce toxic-
ity in the methanol fraction toxicity test (cf., Phase II).
Example 1. Non-polar toxicant(s). The Phase I
results implicating non-polar toxicants are:
• Toxicity in the post Ctg SPE column test
was absent or reduced.
• Toxicity was recovered in the methanol
eluate test.
Toxicants other than non-polar compounds may be
retained by the SPE column but they are less likely to
be eluted sharply. Also, artifactual post-column toxicity
can occur, but non-polar toxicity is typically distinguished
from the artifactual toxicity when the eluate is checked
for toxicity. Some toxicants (metals, some surfactants)
may not elute from the SPE column with methanol and
so failure to recover the toxicity in the eluate does not
exclude the possibility of a non-polar toxicant. Recov-
ery of toxicity in the eluate at pH / is less likely to be an
artifact than recovery only at pH 3 or pH 9. For those
instances where methanol does not recover C1g-remov-
able toxicity, other solvents may be needed to elute the
toxicants (see Phase II; EPA, 1992A).
Example 2. Cationic Metals. This group of metals
has varied chemical/physical behaviors which result in
less definitive Phase I results. The following character-
istics can be used only in a general way to point to
metals as the cause of the toxicity:
• The toxicity is removed or reduced in the
EDTA addition test.
• The toxicity is removed or reduced in the
post C1t SPE column test.
• The toxicity is removed or reduced in the
filtration test, especially when pH
adjustments and filtration are combined.
• The toxicity is removed or reduced in the
sodium thiosulfate addition test.
• Erratic dose response curve observed.
No single characteristic is definitive, with the pos-
sible exception of EDTA. In addition, toxicity may be
pH sensitive in the range at which the graduated pH
test is performed but may become more or less toxic at
low or high pH depending on the particular metals
involved. This characteristic for chronic toxicity has not
yet been demonstrated to the extent it was for the
acute toxicity of several metals (EPA, 1991 A).
Example 3. Total dissolved solids (TDS). JDS
consists of a group of common cations and anions
(Ca2*, Mg2t, Na*, K*. SO4, NO3, Cr, CO ) and in parts
of the United States, this group is called salinity." TDS
is usually measured by conductivity, density or refrac-
tion, none of which measure specific compounds or
ions. The toxicity of any given amount of TDS will
depend on the specific composition. TDS behaves as
a mixture of toxicants, which do not cause toxicity
through osmotic stress. Evidence of this is that the
LCSOs of the individual salts expressed in moles, are
quite different. If osmotic stress were the mode of
action, the concentration in moles at the LCSOs would
be similar (EPA, 1991 A). One cannot use marine
organisms to circumvent TDS unless NaCI is by far the
7-1
-------
dominant IDS. Marine organisms regulate Na* and Cr
but like freshwater organisms, they too are sensitive to
non-NaCI IDS.
For these reasons, only very general relationships
exist between toxicity and TDS. Because of their
varied nature, they do not sort out clearly in Phase I.
Rather, unless conductivity is very high (e.g., 10,000
u.mhos/cm), one might suspect IDS when nothing else
is indicated. For example, if high TDS were present
and caused by calcium sulfate (CaSO4), toxicity is likely
to be removed in the pH adjustment test at pH 10 or in
the filtration and pH adjustment test at pH 10, whereas
if the TDS were due to NaCI, toxicity would likely not be
affected.
As a general guide, when conductivity exceeds
1,000 and 3,000 ^mhos/cm at the effect concentration
for Ceriodaphnia and fathead minnows, respectively,
TDS toxicity might be suspect. The conductivity of
100% effluent is not the relevant reading, but rather the
conductivity at the concentrations bracketing the efflu-
ent no effect and effect concentrations.
Following are some Phase I general indicators that
TDS might be a suspect:
• No pH adjustments changed the toxicity,
unless a visible precipitate occurs upon pH
adjustment, pH adjustment and filtration,
and pH adjustment and aeration.
• No loss of toxicity in the post C SPE
column test, or a partial loss of toxicity but
no change in conductivity measurements.
• No change in toxicity with the EDTA addition
test, sodium thiosulfate addition test or in
the graduated pH test.
In addition, there are two tests that can be used
that are not included in Phase I but may help to charac-
terize the toxicity:
• Use acid/base ion exchange resins (EPA,
1992A). When toxicity is removed or
reduced, the toxicity could be due to TDS.
• Use of activated carbon to remove toxicity
(EPA, 1992A). When no toxicity is removed
by passing the effluent over carbon, TDS
could be responsible for toxicity.
An additional caution is that where TDS is
marginally high, the addition of NaCI from pH manipula-
tions can increase TDS enough to produce art if actual
TDS toxicity. The conductivity of the solutions before
and after the pH adjustments should be monitored
closely to avoid this.
Example 4. Surfactants. There are three main
groups of surfactants and/or flocculants (anionic, cat-
ionic and nonionic) that may occur in effluents. The
Phase I behavior of these types of compounds may
vary depending on which particular groups are present.
The general Phase I results implicating a
surfactant(s) as the toxicant(s) are:
• Toxicity is reduced or removed in the
filtration test.
• Toxicity is reduced or removed by the
aeration test. In some cases, the toxicity is
recoverable from the walls of the aeration
vessel after removing the aerated effluent
sample.
• Toxicity is reduced or removed in the post
Cft SPE column test. The toxicity may or
may not be recovered in the methanol eluate
test.
• Toxicity is reduced or removed in the post
Ctl SPE column test using unfiltered effluent.
Toxicity reduction/removal is similar to that
observed in the filtration test and toxicity
may or may not be recovered in methanol
eluate test or the extraction of the glass
fiber filter.
• Toxicity degrades over time as the effluent
sample is kept in cold storage (4°C).
Degradation is slower when effluent is
stored in glass containers rather than plastic
containers.
Example 5. Ammonia. Ammonia concentrations
can be measured easily, and because it is such a
common effluent constituent, determining the total am-
monia concentration in the whole effluent is a good first
step (see Section 4). If more than 5 mg/L of total
ammonia is present, additional evaluations should be
done. Sole dependence on analyses is not advisable
because the chronic effects of ammonia and some
other toxicants (e.g., such as surfactants) is not well
known. Even though the ammonia concentration is
sufficient to cause toxicity, other chemicals may be
present to cause toxicity if the ammonia is removed.
Three indicators of ammonia toxicity are:
• The concentration of total ammonia is 5
mg/L or greater.
• In the graduated pH test the toxicity
increases as the pH increases.
• The effluent is more toxic to fathead
minnows than to Ceriodaphnia.
Example 6. Oxidants. In effluents, oxidants other
than chlorine may be present. Measurement of a
chlorine residual (TRC) is not enough to conclude that
the toxicity is due to an oxidant. In general, oxidants
are indicated by the following:
• The toxicity is reduced or removed in the
sodium thiosulfate addition test.
• Toxicity is removed or reduced in the
aeration test.
7-2
-------
The sample is less toxic over time when Of course, TRC greater than 0.1 mg/L in 100%
held at 4°C (and the type of container does effluent might indicate chlorine as the oxidant causing
not affect toxicrty). the toxicity. In addition, the dechlorination with SO2
_ . . , . ... provides evidence of chlorine toxicity in the same man-
Cenodaphma are more sensitive than ner as the sodium thiosulfate addition test.
fathead minnows.
7-3
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Section 8
References
Aquatic Habitat Institute. 1992. Proceedings of A
Workshop on Chronic Toxicity Identification
Evaluation (TIEs) in the San Francisco Bay Region.
Workshop sponsored by the San Francisco Bay-
Delta Aquatic Habitat Institute, Bay Areas
Discharger's Association and San Francisco Bay
Regional Water Quality Control Board, March 17
and 18, 1992, Richmond, CA.
APHA, 1989. Standard Methods for the Examination of
Water and Wastewater, 17th Edition. American
Public Health Association, Washington, D.C.
Clean Water Act, Public Law 92-500, October 18, 1972,
86 Stat. 816, U.S.C. 1251 et seq.
EPA. 1978. Methods for Measuring the Acute Toxicity
of Effluents to Aquatic Organisms. EPA/600/4-78/
012. Environmental Monitoring and Support
Laboratory, Cincinnati, OH.
EPA. 1979. Aqueous Ammonia Equilibrium - Tabulation
of Percent Un-ionized Ammonia. EPA/600/3-79/
091. Environmental Research Laboratory, Duluth,
MN.
EPA. 1985A. Technical Support Document for Water
Quality-Based Toxics Control. EPA/440/4-85/032.
Office of Water, Washington, D.C.
EPA. 1985B. Methods for Measuring the Acute Toxicity
of Effluents to Freshwater and Marine Organisms.
Third Edition. EPA/600/4-85/013. Environmental
Monitoring and Support Laboratory, Cincinnati, OH.
EPA. 1985C. Short-Term Methods for Estimating the
Chronic Toxicity of Effluents and Receiving Waters
to Freshwater Organisms. EPA/600/4-85/014.
Environmental Monitoring and Support Laboratory,
Cincinnati, OH.
EPA. 1985D. Ambient Water Quality Criteria for
Ammonia. EPA/440/5-85/001. Environmental
Research Laboratory, Duluth, MN, and Criteria and
Standards Division, Washington, D.C.
EPA. 1988A. Methods for Aquatic Toxicity Identification
Evaluations: Phase I Toxicity Characterization
Procedures. EPA/600/3-88/034. Environmental
Research Laboratory, Duluth, MN.
EPA. 1988B. Short-Term Methods for Estimating the
Chronic Toxicity of Effluents and Receiving Waters
to Marine and Estuarine Organisms. EPA/600/4-
87/028. Environmental Monitoring and Support
Laboratory, Cincinnati, OH.
EPA. 1989A. Methods for Aquatic Toxicity Identification
Evaluations: Phase II Toxicity Identification
Procedures. EPA/600/3-88/035. Environmental
Research Laboratory, Duluth, MN.
EPA. 1989B. Methods for Aquatic Toxicity Identification
Evaluations: Phase III Toxicity Confirmation
Procedures. EPA/600/3-88/036. Environmental
Research Laboratory, Duluth, MN.
EPA. 1989C. Short-Term Methods for Estimating the
Chronic Toxicity of Effluents and Receiving Waters
to Freshwater Organisms. Second Edition. EPA/
600/4-89/001 and Supplement EPA/600/4-89/001 A.
Environmental Monitoring and Support Laboratory,
Cincinnati, OH.
EPA. 1989D. Toxicity Reduction Evaluation Protocol
for Municipal Wastewater Treatment Plants. EPA/
600/2-88/062. Water Engineering Research
Laboratory, Cincinnati, OH.
EPA. 1989E. Generalized Methodology for Conducting
Industrial Toxicity Reduction Evaluations (TREs).
EPA/600/2-88/070. Water Engineering Research
Laboratory, Cincinnati, OH.
EPA. 1991 A. Methods for Aquatic Toxicity Identification
Evaluations: Phase I Toxicity Characterization
Procedures. Second Edition. EPA/600/6-91/003.
Environmental Research Laboratory, Duluth, MN.
EPA. 1991B. Technical Support Document for Water
Quality-Based Toxics Control. Second Edition.
EPA/505/2-90/001. Office of Water, Washington,
D.C.
EPA. 1991C.' Methods for Measuring the Acute Toxicity
of Effluents to Freshwater and Marine Organisms.
Fourth Edition. EPA/600/4-90/027. Environmental
Monitoring and Support Laboratory, Cincinnati, OH.
EPA. 1991D. Toxicity Identification Evaluation:
Characterization of Chronically Toxic Effluents,
Phase I. EPA/600/6-91/005. Environmental
Research Laboratory, Duluth, MN.
EPA. 1992A. Methods for Aquatic Toxicity Identification
Evaluations: Phase II Toxicity Identification
8-1
-------
Procedures. EPA/600/R-92/080.
Research Laboratory, Duluth, MN.
Environmental
EPA. 1992B. Methods for Aquatic Toxicity Identification
Evaluations: Phase III Toxicity Confirmation
Procedures. EPA/600/R-92/081. Environmental
Research Laboratory, Duluth, MN.
EPA. 1992C. Short-Term Methods for Estimating the
Chronic Toxicity of Effluents and Receiving Waters
to Freshwater Organisms. Third Edition. EPA/600/
4-91/002. Environmental Monitoring and Support
Laboratory, Cincinnati, OH.
EPA. 1992D. Short-Term Methods for Estimating the
Chronic Toxicity of Effluents and Receiving Waters
to Marine and Estuarine Organisms. Second
Edition. EPA/600/4-91/003. Environmental
Monitoring and Support Laboratory, Cincinnati, OH.
DeGraeve, G.M., J.D. Cooney, B.H. Marsh, T.L. Pollock
and N.G. Reichenbach. 1989. Precision of the
EPA-Developed Seven-Day Ceriodaphnia dubia
Survival and Reproduction Test: Intra- and
(nterlaboratory Study. Electric Power Research
Institute, (EPRI), Palo Alto, CA, Report EN-6469.
Denny, J.S. 1988. Guidelines for Culture of Fathead
Minnows (Pimephales promelas) for Use in Toxicity
Tests. EPA/600/S3-87/001. Environmental
Research Laboratory, Duluth, MN.
Federal Register. 1984. U.S. EPA: Development of
Water Quality Based Permit Limitations for Toxic
Pollutants; National Policy. EPA, Volume 49, No.
48, Friday, March 9, 1984.
Federal Register. 1989. U.S. EPA: National Pollutant
Discharge Elimination System; Surface Water Toxics
Control Program. EPA, Volume 54, No. 105, Friday,
June 2, 1989.
Ferguson, W.J., K.I. Braunschweiger, W.R.
Braunschweiger, J.R. Smith, J.J. McCormick, C.C.
Wasmann, N.P. Jarvis, D.H. Bell, and N.E. Good.
1980. Hydrogen Ion Buffers for Biological Research.
Anal. Biochem. 104: 300-310.
Flaschka, H.A. and A.J. Barnard, Jr. (Eds.) 1967.
Chelates in Analytical Chemistry. Marcel Dekker,
Inc., New York, NY. 418 p.
Giles, M.A. and R. Danell. 1983. Water Dechlorination
by Activated Carbon, Ultraviolet Radiation and
Sodium Sulphite. Water Res. 17(6): 667-676.
Hockett, J.R. and D.R. Mount. In Preparation. Use of
Metal Chelating Agent to Differentiate Among
Sources of Toxicity. Manuscript.
Masters, J.A., M.A. Lewis, D.H. Davidson and R.D.
Bruce. 1991. Validation of a Four-Day Ceriodaphnia
Toxicity Test and Statistical Considerations in Data
Analysis. Environ. Toxicol. Chem. 10:47-55.
McKim, J.M. 1977. Evaluation of Tests with Early Life
Stages of Fish for Predicting Long-Term Toxicity.
J. Fish. Res. Board Can. 34:1148-1154.
Mount, D.R. 1991. A Toxicity-Based Approach to
Pollutant Identification. In: Proceedings of the
Thirtieth Annual EPA Conference on Analysis of
Pollutants in the Environment, May 9 and 10, 1990.
21W-7005. Environmental Protection Agency, Office
of Water. Washington, D.C.
Neilson, A.J., A.S. Allard, S. Fischer, M. Malmberg, and
T.Viktor. 1990. Incorporation of a Subacute Test
with Zebra Fish into a Hierarchical System for
Evaluating the Effect of Toxicants in the Aquatic
Environment. Ecotox. and Environ. Safety 20: 82-
97.
Norberg-King, T.J. 1988. An Interpolation Estimate for
Chronic Toxicity: The ICp Approach. National
Effluent Toxicity Assessment Center Technical
Report 05-88, U.S. Environmental Protection
Agency, Environmental Research Laboratory,
Duluth, MN.
Norberg-King, T.J. 1989A. An Evaluation of Relative
Sensitivity of the Fathead Minnow Seven-Day
Subchronic Test for Estimating Chronic Toxicity.
Environ. Toxicol. Chem. 8(11):1075-1089.
Norberg-King, T.J. 1989B. Culturing of Ceriodaphnia
dubia: Supplemental Report for Video Training
Tape. EPA/505/8-89/002a. Office of Water,
Washington D.C.
Norberg-King T.J, and J.S. Denny. 1989. Culturing of
fathead minnows, (Pimephales promelas):
Supplemental Report for Video Training Tape. EPA/
505/8-89/002b. Office of Water, Washington D.C.
Oris, J.T., R.W. Winner, and M.V. Moore. 1991. A
Four-Day Survival and Reproduction Toxicity Test
for Ceriodaphnia dubia. Environ. Toxicol. Chem.
10:217-224.
Smith, R.M. and A.E Martell. 1981. Critical Stability
Constants. Volume 4: Inorganic Complexes.
Plenum Press, NY. p. 87.
Stephan, C.E. and J.W. Rogers. 1985. Advantages of
Using Regression Analysis to Calculate Results of
Chronic Toxicity Tests. Aquatic Toxicology and
Hazard Assessment: Eighth Symposium, ASTM
STP 891, R.C. Bahner and D.J. Hansen, Eds.,
American Society for Testing and Materials,
Philadelphia, pp. 328-338.
Stumm, W. and J.J. Morgan. 1981. Aquatic Chemistry
- An Introduction Emphasizing Chemical Equilibria
in Natural Waters. John Wiley & Sons, Inc., New
York, NY. 583 p.
Weltering, D.M. 1983. The Growth Response in Fish
Chronic and Early Life Stage Toxicity Tests: A
Critical Review. Aquat. Toxicol. 5:1-21.
8-2
-------
Zuiderveen, J.A. and W.J. Birge. 1991. A Comparison 12th Annual Meeting of the Society for Environ.
of Metal Chelators for Use in the TIE/TRE Chronic Toxicol. and Chem., November 1991.
Toxicrty Tests with Ceriodaphnia dubia. Poster,
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