vvEPA
United States
Environmental Protection
Agency
Office of Research and
Development
Washington DC 20460
EPA/600/6-91/005
June 1991
Toxicity Identification
Evaluation:
Characterization of
Chronically Toxic Effluents,
Phase I
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EPA-600/6-91/005
June, 1991
Toxicity Identification Evaluation:
Characterization of Chronically Toxic Effluents, Phase I
T.J. Norberg-King
Environmental Research Laboratory
Duluth, Minnesota 55804
D.I. Mount
J.R. Amato
D.A. Jensen
J.A Thompson
AScI Corporation
Duluth, Minnesota 55804
National Effluent Toxicity
Assessment Center
Technical Report 05-91
U.S. Efwiwronental Protection Atenc»
Region 5, Library (PL-12J)
77 West Jackson Boulevard, 12th Flow
CMcafMl 60604-3590
Office of Research and Development
U.S. Environmental Protection Agency
Duluth, MN 55804
~>X.-- 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. This document is a preliminary draft. It
has not been formally released by the U.S. Environmental Protection Agency and
should not at this stage be construed to represent Agency policy. It is being circulated
for comments on its technical merit and policy implications. Mention of trade names
or commercial products does not constitute endorsement or recommendation for use.
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FOREWORD
This draft guidance document has been prepared to assist dischargers and/or their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 and details in the acute Phase I manual are 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 C18 solid phase extraction
(SPE) resin. For Tier 1, the tests are all done using the effluent sample without any
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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 C18 SPE steps of Tier 1 performed at either pH 3
or 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
characteristics 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
t
in aTRE (EPA, 1991 A).
These chronic TIE methods are not written as rigid, required protocols, but rather as
guidance for conducting TIEs with effluents. These 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 111; 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 to reflect additional
information in the revised acute Phase I manual (EPA, 1991 A).
We welcome your comments on the manual. Please send comments to T. Norberg-
King, NETAC, Environmental Research Laboratory, 6201 Congdon Boulevard, Duluth,
MN 55804.
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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
enforcement 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
i
Phase I TIE manual (EPA, 1988A; EPA, 1991 A). This chronic Phase I manual
describes procedures for characterizing the physical/chemical nature of toxicants in
effluents that exhibit chronic toxicity. Aliquots of effluent samples are manipulated and
the resulting effect on toxicity measured. 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 approach.
Tier 1 consists of filtration, aeration, use of additives to chelate or reduce the
toxicants, minor pH adjustments, and use of a separation technique with the C18 solid
phase extraction resin. Each effluent is characterized in Tier 1 by performing the
manipulations at the initial pH (pH i) of the effluent. Tier 2 consists of the Tier 1
manipulations combined with pH adjustments of additional aliquots of the effluent
sample. Aeration, filtration, and C18 solid phase extraction of effluent samples
adjusted to pH 3 and pH 10 are Tier 2 characterization steps.
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The characterization methods rely on short-term "chronic" test methods using two
species, Ceriodaphnia dubia and the fathead minnow (Pimephales promelas). 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 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
i
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 treatment 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 several of the
characterization tests combined.
VI
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CONTENTS
Page
Foreword iii
Abstract v
Contents vii
Figures ix
Tables x
Acknowledgements xi
1. Introduction 1-1
2. Quality Assurance, Health and Safety, and Facilities and Equipment 2-1
2.1 Quality Assurance 2-1
2.2 QC/QA Cost Considerations and Testing Requirements 2-2
2.3 QC/QA and Chronic Testing Considerations 2-4
2.4 QC/QA and Blanks and Artifactual Toxicity 2-7
2.5 Health and Safety Issues 2-9
2.6 Facilities and Equipment 2-10
3. Dilution Water 3-1
4. Handling Effluent Samples 4-1
5. Toxicity Testing 5-1
5.1 Principles 5-1
5.2 Test Species 5-2
5.3 Toxicity Test Procedures 5-3
5.4 Concentrations to Test 5-6
5.5 Renewals 5-7
5.6 Toxicity Blanks 5-8
5.7 Renewal of Manipulated Samples 5-10
5.8 Test Endpoints and Data Analysis 5-12
6. Characterization Tests 6-1
6.1 Baseline Test 6-9
6.2 EDTA Addition Test 6-11
6.3 Sodium Thiosulfate Addition Test 6-20
6.4 Aeration Test 6-27
6.5 Filtration Test 6-31
6.6 Post C18 Solid Phase Extraction Column Test 6-35
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6.7 Methanol Eluate Test 6-42
6.8 Graduated pH Test 6-46
6.9 Tier 2 Characterization Tests 6-55
6.10 pH Adjustment Test 6-57
6.11 Aeration and pH Adjustment Test 6-62
6.12 Filtration and pH Adjustment Test 6-64
6.13 Post C18 Solid Phase Extraction Column and pH Adjustment Test 6-66
6.14 Methanol Eluate Test (for pH Adjusted Samples) 6-68
6.15 Toxicity Characterization Summary 6-69
6.16 Use of Multiple Characterization Tests 6-73
7. References 7-1
VIII
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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. 6-5
6-3. Tier 2 sample preparation and testing overview. 6-56
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TABLES
Number Page
6-1 Outline of Phase I effluent manipulations. 6-6
6-2. Chronic toxicity of EDTA (mg/L) to C. dubia and P. promelas
in various hardness waters using the 7-d tests. 6-14
6-3. Concentrations of EDTA to add for chronic TIEs. Values given
are the final water concentration in mg/L 6-16
6-4. The chronic toxicity of zinc to C. dubia in very hard
reconstituted water and the toxicity of zinc when EDTA is added. 6-19
6-5. Chronic toxicity of sodium thiosulfate (mg/L) to C. dubia and
P. promelas in various hardness waters using the 7-d tests. 6-23
6-6. Concentrations of sodium thiosulfate to add for chronic TIEs.
Values given are the final water concentration in mg/L. 6-24'
6-7. Chronic toxicity of methanol (%) to C. dubia and P. promelas
using the 7-d tests. 6-44
6-8. Chronic toxicity of sodium chloride (g/L) to C. dubia
and P. promelas in various hardness waters using the 7-d tests. 6-61
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ACKNOWLEDGEMENTS
Many people at the National Effluent Toxicity Assessment Center 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 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,
Nola Englehorn, Doug Jensen, 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, Nola Englehorn, Shaneen
Murphy, Joe Amato, Mary Schubauer-Berigan, Greg Peterson, and Jim Jenson.
The skillful assistance and dedication of Debra Williams in producing this document is
gratefully acknowledged.
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.
XI
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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 Discharge 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
treatment technologies (best available technology). The technology-based limits are
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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 conjunction with
EPA's water quality criteria and other toxicity databases to determine the adequacy of
the technology 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 biological oxygen demand (BOD), suspended solids (SS), and color.
Additional components were added in subsequent amendments to the CWA, for
example, the list of 126 "priority pollutants" of which many or most were required
monitoring for the permittees. Water quality criteria were used to develop the water-
quality based limits for these pollutants. However, water quality criteria or discharge
limits exist for only a few of the thousands of chemicals in use.
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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 narrative 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 concentrations
that are toxic to aquatic life, wildlife or human health. Use of toxicity testing and whole
effluent toxicity 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 approach" for controlling toxic pollutants. This integrated approach is
referred to as the water quality-based approach and is described in detail in the
Technical Support Document (hereafter referred to as the TSD; EPA, 1985A; EPA,
1991B). The control regulations for EPA (Federal Register 23868, 1989) establish
specific requirements that the integrated approach be used for water quality-based
toxics control. This integrated approach 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
combination of chemical specific and whole effluent toxicity limitations are essential to
the control of toxic pollutants. Once the permit limits are set, compliance is
established through routine monitoring of effluent quality. In this manner, water
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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 (fertilization, growth,
reproduction) are measured in addition to lethality. The chronic test is used to
measure the effects of long-term exposure to chemicals, wastewaters, and leachates
to aquatic organisms. True chronic toxicity tests include the life-cycle of the organism.
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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 toxicity. Hereafter, chronic
tests refer to the short-term tests that are described in the EPA manuals (EPA, 1991D;
EPA, 1991E; EPA, 1989C; EPA, 1985C).
Toxicity is a useful parameter to protect receiving waters from impacts on water quality
and designated uses caused by the mixture of toxic pollutants in wastewaters. EPA
has published manuals which provide methods for use of freshwater and marine
organisms to determine acute and chronic toxicity of effluents. These manuals have
been available since 1978 and 1985, respectively (EPA, 1978; EPA, 1985B; EPA,
1985C; EPA, 1988B; EPA, 1989C) and are currently being revised (EPA, 1991C; EPA,
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1991D; EPA, 1991E). These methods are used by federal, state and local
governments to determine compliance of permitted point source discharges. Since the
late 1970's, toxicity has been measured in wastewaters; permit writers began using
toxicity limits in the early 1980's. With the increased use of toxicity testing, substantial
numbers of unacceptably toxic effluents have been identified. Now, some permittees
are required to perform toxicity reduction evaluations (TREs) as a condition of the
NPDES permit. The TSD defines a TRE as "a site specific study conducted in a
stepwise process designed to identify the causative agents of effluent toxicity, isolate
the sources of toxicity, evaluate the effectiveness of toxicity control options, and then
confirm the reduction in effluent toxicity"
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Methods to characterize (Phase I; EPA, 1988A; EPA, 1991A), identify (Phase II; EPA,
1989A), and confirm (Phase III; EPA, 1989B) the cause of acute toxicity in effluents
have been developed. These methods are generally referred to as toxicity
identification evaluations (TIEs), which are a part of the TRE.
The acute TIE approach (EPA, 1988A; EPA, 1991 A) relies on the use of organisms to
detect the presence of toxicants in the effluent. The number of constituents in the
effluent is reduced before analyses begin, and information about the physical/chemical
characteristics of the effluent's toxicity is gained. Using this approach, analytical
problems can be simplified and the costs reduced. Toxicity throughout the TIE must
be tracked to determine if the toxicity is consistently being caused by the same
substance.
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Once the physical/chemical characteristics of toxicants are known, a better choice of
analytical methods can be made. Knowledge of physical/chemical characteristics are
used for the treatability approach to TRE's (EPA, 1989D; EPA, 1989E) as well.
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 characterization steps provides information on
whether the nature of compounds causing the chronic toxicity remains consistent. The
tests do not provide information on the variability of toxicants within a characterization
group. Samples should be subjected to Phase I until no additional responses are
found (usually at least three samples). From these data the toxicant characteristics
can be identified as pH sensitive, filterable, volatile, soluble, degradable, reducible, or
EDTA chelatable. Such information indicates how samples must be handled for
analyses and which analytical methods should be used. Following characterization, a
decision is made to proceed with identification (Phase II; EPA, 1989A) and
confirmation (Phase III, EPA, 1989B) or to conduct treatability studies where the
identification of the specific toxicants (cf., acute treatability procedures (EPA, 1989D;
EPA, 1989E)) is not made.
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Chronic toxicity must be present frequently enough so that an adequate number of
toxic samples can be obtained. Enough testing should be done on each effluent
before a TIE is initiated, 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 toxicity can assist in determining the cause of toxicity. One cannot assume
that if the effluent has acute toxicity and a TIE was done and the cause(s) of acute
toxicity determined, that the sublethal toxicity exhibited is due to the same compound.
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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) is primarily for analytical
analyses where standards to conduct performance evaluations can be obtained. A
QAP for toxicity testing can be developed, but determining the recovery of known
additions for toxicity testing is not possible. For TIEs the combination of chemistry
i
and biology requires a level of checks and balances not typically used under other
situations. A step-by-step QAP for a TIE is not practical because as a TIE
progresses, additional or different tests may be needed and many aspects of the TIE
cannot be foreseen. Adhering to the general guidelines of the QAP is important
however, and should increase the probability of the TIE succeeding. As additional
steps are recognized, the details should be added to the QAP.
Quality control (QC) procedures for aquatic toxicity tests are radically different than the
QC procedures for chemical analytical methods. The quality assurance (QA)/QC
guidance given by EPA (1989C) for the short-term tests lists numerous items of
concern for toxicity testing aspects. These are: (a) effluent sampling/handling, (b) test
organisms, (c) facilities, equipment and test chambers, (d) analytical methods, (e)
calibration and standardization, (f) dilution water, (g) test conditions, (h) test
acceptability, (i) test precision, (j) replication and test sensitivity, (k) quality of
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organisms, (I) quality of food, (m) control charts, and (n) record keeping and data
evaluation. Many of these should be closely followed, and the reader is encouraged
to review the guidance in relation to QA/QC in both the short-term effluent test manual
(EPA, 1989C; EPA, 1991D) and the acute Phase I manual (EPA, 1991 A).
2.2 QC/QA Cost Considerations and Testing Requirements
For the chronic TIE, cost considerations are important and concessions in the <
requirements of the QC 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, 1985C) 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 is
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
measurements are not pertinent for the methanol eluate collected from a solid phase
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extraction column. In contrast, frequent pH measurements on all test concentrations
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 were 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 (including maintaining control charts),
i
monitoring the preparation 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, to monitor 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).
Since toxicity tests in the early part of the chronic Phase I do not generally follow all
the effluent testing 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.
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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
responding as expected. Since only relative differences are needed at this stage
(Phase I), reference toxicant data are much less useful. 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
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chemical should become the reference toxicant.
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 will
aid in determining the appropriate control option in order that the final effluent is safe
upon discharge.
2.3 QC/QA and Chronic Testing Considerations
An inherent problem with effluents is that no effluent test can be repeated to assure
that the toxicity is the same and that the toxicants are the same. However, repeated
baseline tests (Section 6) can be done with the same effluent sample to determine
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how long that effluent sample can be used. The chemical and toxicological nature of
the effluent shifts as an effluent is discharged or as an effluent sample is stored.
Effluent 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. 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
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number of samples to evaluate and the number of tests to conduct must be weighed
against the cost of the effort and how representative each effluent sample is of the
effluent. Effluents that have low and non-persistent toxicity may need to be
approached with the Tier 1 and Tier 2 characterization steps applied simultaneously
(see Section 6).
In a chronic TIE, information obtained from a test should be maximized. This may
mean paying particularly close attention to details such as small differences in the
number of neonates the cladocerans are producing or the lack of food in the stomach
of the larval fish. Subtle indicators during a test may be quite informative about small
changes in toxicity. For example, 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 reduced the
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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. One animal must be chosen for 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 randomly 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 cleaning 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 Daphnia pulex where animals are caught at the surface of the test solution (called
"floaters") does not occur with C. dubia. All equipment to perform renewals (pipettes,
siphons) may need to be rinsed/cleaned between concentrations and the different
characterization tests to prevent solution contamination during organism transfer.
Randomization, careful exposure time readings, use of animals of uniform 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 measurements (temperature, DO, pH,
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conductivity, alkalinity, hardness, ammonia, chlorine), and other general routine
practices.
An important aspect of TIEs is accurate and thorough data recording. All observations
should be documented. Items that were not thought to be important at first may
actually assist in the confirmation that the toxicant(s) was present when the data is
later summarized. These observations can be as simple as large bubbles produced
during the aeration and filtration manipulations, large particles present in whole
effluent, and low pH upon arrival. It is best to record data such that any preconceived
ideas of the toxicants are avoided. Data records should include records of test
«
organisms (species, 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 for reference toxicant tests, 5% of the time the monthly reference toxicity
test results will be predicted to fall outside the acceptable limits if the 95% confidence
interval are used (EPA, 1991C). If TIEs are conducted during such a period, the TIE
data generated should be discarded.
2.4 QC/QA Blanks and Artifactual Toxicity
Throughout the TIE, dilution water samples are subjected to most of the procedures
and analyses performed 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 characterization manipulations.
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These manipulations 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) but to evaluate whether
a manipulation affected the toxicant(s), the results of all tests are not necessarily
compared to the baseline test. For instance, 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 C18SPE
column test) to the toxicity observed in the filtered effluent sample (filtration test) rather
t
than to the unfiltered whole effluent (baseline test).
Artifactual toxicity can occur in several of the manipulations, particularly from the major
pH adjustment manipulation (Tier 2). Toxicity results from tests relying on the addition
of the reagents (EDTA, sodium thiosulfate, 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 effluent 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 bacterial growth that will confound the
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results in the post-column blank and post C18 column tests. Originality and judgement
are needed to devise tests that will reveal artifactual toxicity.
2.5 Health and Safety Issues
For the toxicity identification work, hazards present in any effluent may not be known
until Phase II identification steps have been started. Therefore, safety requirements
for working with effluents (or other samples) of unknown composition must follow
safety procedures for a wide spectrum of chemical and biological agents. Knowledge
of the types of wastewater treatment applied to each effluent can provide some insight
i
for the possible hazards. For example, unchlorinated primary treatment plant effluents
containing domestic waste may contain pathogens. Chlorinated secondary effluents
are less likely to contain such agents. Effluents from activated sludge treatment plants
are less likely to contain volatile toxicants.
Because effluent characteristics are unknown, personnel should follow the guidelines
for hazardous materials (EPA, 1991 A; 1991C). Also, if any sample contains human
waste, personnel should be immunized 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 maintain 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
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(d) periodic inspections of emergency equipment and safety violations. Further
guidance on safety practices is provided in other documents (APHA, 1989; EPA,
1991 A; 1991C).
2.6 Facilities and Equipment
The laboratory facilities and equipment needed to conduct TIEs is discussed in the
acute Phase I manual (EPA, 1988A; EPA, 1991 A). Most of the equipment for
conducting the short-term tests is delineated elsewhere (EPA, 1989C; EPA, 1991D).
The reagents used for the chronic Phase I characterization are identical to those
»
described in the acute Phase I manual. Compressed air systems with oil-free
compressors and air filters to provide high purity are very important (EPA, 1991 A).
Glassware used for filtering should be rigorously cleaned to remove residual
contaminants from the glass frit(s). Filtering equipment may need to be made of
plastic to avoid leaching of metals or other toxicants from glass when acid washes are
used (see Section 6). Ultra pure acids and bases (e.g., Suprapur®, E. Merck,
Darmstadt, Germany) should be used to prevent impurities in the acids/bases from
interfering in the toxicity results.
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SECTION 3
DILUTION WATER
Dilution water used for chronic TIE'S must meet several requirements. Obviously it
must support adequate 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 alkalinity 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 produce
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.
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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 mixture will resemble those of the effluent. As information is
gained about the toxicant characteristics, the choice of dilution water can be improved.
The impact of dilution water choice depends on the IC25 (see Section 5.8)
concentration 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 concentrations vary over the course of the TIE period, the amount of
1 Foods added for the C. dubia tests are the yeast-cerophyll-trout food (YCT) and the algae
(Selenastrum capricomutum) at a rate of 0.1 mU15 ml (EPA, 1989C). Although at ERL-Duluth
the algae has been added at the rate of 0.05 mL/15 ml until May of 1991 when we switched to
EPA(1989C) levels.
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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 |ig/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 contains 480 u,g/L
of zinc. One would expect this sample to possess 3 TUs (480 (ig/L -*• 160 u,g/L). 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 CaCO3). The effect level would be near 20-
\
25% effluent where hardness would be <100 mg/L as CaCO3 and 1 TU of zinc would
be <160 (ig/L. In addition, if one were to use receiving water for the diluent, the
hardness might change dramatically and confound calculation of TU's in a like manner
if the effect concentration was <100% effluent.
2 Toxic unit (TU) is a means of normalizing the concentration term (i.e., LC50, NOEC, IC25--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 toxicant's 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 are calculated for either acute or chronic toxicity
endpoints. The acute TU for whole effluent is 100% + LC50 = TU. and the chronic TU for
whole effluent is 100% + NOEC = TUC 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 TU. or the chronic test NOEC or IC25 for the TUC. The assignment
of TUC is necessary for linear correlation (Phase III) when effluent toxicity TUs are compared to
suspect toxicant(s) TUs.
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SECTION 4
EFFLUENT SAMPLES
To determine whether an effluent sample is typical of the wastewater discharge
requires a number of samples to be tested. TIE work on atypical samples is not
useful, and TIE procedures do not apply to episodic events. Experience has shown
that the use of several samples spanning two to three months has been successful in
characterizing many effluents.
The acute Phase I manual discusses the quantitative and qualitative changes in
effluents (EPA, 1988A; EPA, 1991 A) that may affect toxicity. Varying concentrations '
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 involved 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, hardness, conductivity, total residual chlorine (TRC), total ammonia, alkalinity
and DO should be measured. Figure 4-1 provides a typical format to record such
information.
4-1
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Figure 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 Number.
Sampler:
Sample Type: Q Grab Q Composite
a Glass Q Plastic
Q Prechlorinated
Q Chlorinated
Q Dechlorinated
Sample Conditions Upon Arrival:
Temperature
pH
Total Alkalinity_
Total Hardness
Conductivity/Salinity,
Total Residual Chlorine,
Total Ammonia
Condition of treatment system at time of sampling:
Status of process operations/production (if applicable):
Comments:
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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. The TIE requires that
toxicity be present frequently in effluent samples, and that the toxicity in each sample
remain sufficiently long for testing to be done. For one chronic Phase I TIE, a typical
volume of effluent needed is 19 L but of course this will depend on the options chosen
for the TIE (Section 6). 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. Preliminary samples are useful to determine
which containers to use to provide samples that are the most representative of the
effluent (see Phase I, Section 6 (EPA, 1991 A) for more details).
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 consistent give
results that are easier to interpret and lead more rapidly to identification and
confirmation of the cause of toxicity. Grab samples can provide the maximum effluent
toxicity; however, although it is more difficult to catch intermittent peaks of toxicity
(such episodic events may not be caused by the same toxicant that causes routine
toxicity).
4-3
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Multiple effluent samples in each test should not be used in Phase I as is done for
permit testing (EPA, 1989C). Only one composite sample should be used for each set
of Phase I tests. The reason is that if several samples are used and the toxicants 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 it should be done
after at least one or preferably most of the toxicants are known.
Existing routine toxicity test data should be examined. 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.
4-4
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SECTION 5
TOXICITY TESTING
5.1 Principles
The test organism is used as the detector of chemicals causing chronic toxicity in
effluents and other aqueous 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 chemical
analytical methods be applied to identify and quantify the toxicants.
i
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, 1991D). However,
for the Phase I manipulations, conducting the tests strictly as detailed in those
manuals are 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) reduced number of test concentrations,
and (e) reduction in the frequency of the test solution renewal. In addition, 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. During Phase I the analyst is searching for an
obvious alteration in effluent toxicity, which may be obtained using abbreviated chronic
test methods. Confirmation testing (Phase III) conducted according to the standard
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methodologies will confirm whether the toxicant(s) detected 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 subjected to this evaluation because they
have been found to be chronically toxic to the cladoceran, C. dubia, or to the fish,
fathead minnow (Pimephales promelas), or possibly to the cladocerans, Daphnia
magna or Daphnia pulex. A TIE is best conducted using the species which detected
the toxicity triggering the TIE. 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 sensitivity 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. If the effluent possesses sufficient
variability, and the two species IC25's (see Section 5.8 below for a description of the
IC25) change in proportion one to 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
5-2
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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 conditions. Also, the
threshold levels for additives and reagents 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
chemistries, test duration, and volumes are not practical due to time constraints and
expense. Variations of the procedures 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 plastic cup (or
5-3
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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 in moving 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. Consistency of using the
same size test chambers and consistent volumes should be maintained in Phase I;
when Phase III is initiated, tests should be conducted following the test protocol that
\
was used to trigger the TIE.
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)
A shortened version of the 7-d C. dubia test, referred to as the 4-d test, can often be
used. 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
5-4
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similar results for single chemicals (Oris et al., 1990), 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. 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 pre-disposed to produce their first brood,
unlike the animals exposed as neonates (< 24 h old) and the exposure during a 4-d
test may miss their most life sensitive stage. However for the Phase I where the
purpose is to detect differences following various manipulations, 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 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
individually in test containers and fed daily until they are 72 h (± 6 h) old in a similar
test fashion (Oris et al., 1990). The animals are then transferred to the baseline test
solutions or the various characterization test solutions. 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 replicates is reduced. 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 dilutions and
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control, and so on until all test cups contain one young animal. By this technique,
animals from a given female that appears to be sick or produces no young can
legitimately be dropped from the data set without statistical bias (Norberg-King et al.,
1989). The ability to discard such data without bias improves precision.
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 for 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 choose 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 concentration 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 40%, 60%, 80%, and 100%. Choice of dilution factor and test range is a
matter of judgement and depends on needed precision and practicality.
5-6
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In nearly all examples in this document, the concentrations 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 toxicity 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, 1 x 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.
5.5 Renewals
For C. dubia, daily renewals of the test media (as required in the chronic manual,
EPA, 1989C) are not necessary as long as the toxicity of the effluent can be
measured in 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. For the fathead minnow test, the frequency
of sample replacement must be daily to maintain adequate water quality because the
live food (brine shrimp, Artemia salina) dies 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.
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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 measurements
and their usefulness. Initially, 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. But if a metal is suspected, then these measurements
are important. The pH measurement is frequently needed and for toxicants such as
ammonia it is extremely important. If an effluent contains greater than 5.0 mg/L of
%
ammonia, the pH should be carefully measured 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.
5.6 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 manipulations (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
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tests are more sensitive to artifactual toxicity, lower concentrations of additives or less
severe conditions must be used as compared to the acute test.
The presence of artifactual toxicity caused by contaminated acids, bases, air, filters
and columns and by intentional additives are detected by treatment blanks and toxicity
controls. A blank is dilution water manipulated the same as the effluent, and then it is
toxicity tested to determine if any toxicity was added. A 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 manipulation test. For example, the
toxicity control for the EDTA addition test is the baseline test while the toxicity control ^
for the post-C18SPE test is the filtration test (filtered whole effluent). Treatment blanks
for either the EDTA addition test or the sodium thiosulfate addition test are not
appropriate as the testing of these additives in clean dilution water is not
representative of the effluents' characteristics. The toxicity control must be
distinguished from the control treatment (animals in standard culture or dilution water),
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 apparatus, the C18 SPE column, and
other apparatus used.
Although artifactual toxicity may appear in the dilution water blanks, artifactual toxicity
in the effluent matrix may not be observed. One must decide whether the test results
from that manipulated sample are meaningful. For example, if the aeration
manipulation caused toxicity in the dilution water blank but aeration removed the
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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.
5.7 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 general guideline, we have chosen to discuss these Phase I
steps as though one aliquot of effluent samples prepared for the characterization tests
is used for all renewals. However for either daily or batch samples, the same
techniques should be used for all the manipulations. 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 C18
5- 10
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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 TIE work is often concerned with the qualitative evaluation of toxicity, rather
than quantitative, there is no reason why a test could not be terminated 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 inhibition of C. dubia reproduction
by day 5 of a 7-d test, and one of the manipulated samples (i.e., aeration) shows
normal reproduction, there may be little point in continuing that test, because toxicity
was altered. This type of judgmental decision is harder to make in a chronic fathead
«
minnow test based on growth; however, 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 selenium for 5-d and 7-d indicated
that sufficient growth differences could distinguish the toxic effect even at 5-d
(Norberg-King, 1989).
Because the chronic test is longer and requires more laboratory work than the acute
test, loss of toxicity 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
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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 manipulations 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 determined 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 significant biological
effect that was not detected by the statistical analysis. Judgement and experience in
toxicology should guide the interpretation.
Endpoints for the most commonly used freshwater short-term chronic tests are
survival, growth, and reproduction. Historically, the effect and no effect concentrations
have been determined using the statistical approach 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 effect concentration, called the lowest observed effect concentration (LOEC), are
then statistically defined endpoints. The NOEC/LOEC are heavily affected by choice
of test concentration and test design. For example, these effect levels are dependent
5-12
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not only on the concentration 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 significant differences
detected in hypothesis tests can be quite variable (e.g., 10% or 60%) 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 replicates, fewer numbers of dilutions and
fewer test organisms 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,
t
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 concentration that
causes a given percent reduction based on the organisms response. The inhibition
concentration (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 analyzing the type of data obtained from Phase I testing.
When analyzing data for the ICp estimates, only one test endpoint is determined. For
3 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 the current edition. This
is being corrected in the revision of the program now underway (R. Regal, personal
communication).
5-13
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C. dubia all the data is used. If all 10 animals have died, the data is entered as zeros
and if some animals have some young but the adult dies, the partial brood values are
used. For the fathead minnow test, the weights are calculated as mean weight per
original fish rather than mean per surviving. 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 equivalent for the NOEC (EPA, 1991B).
Confidence intervals are calculated using a bootstrap technique, and these confidence
intervals can be used to determine the significance of toxicity alterations observed in
Phase I. A "significant reduction" m toxicity must be determined by each laboratory for
each effluent and in combination with the precision of reference toxicant tests the
performing laboratory achieves.
5- 14
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SECTION 6
CHARACTERIZATION TESTS
The chronic Phase I manipulations follow the same approach and employ the same
manipulations used in the acute TIE (EPA, 1991 A). These include aeration, filtration,
C18 SPE extraction and chromatography, chelation with EDTA, oxidant reduction and/or
complexation with sodium thiosulfate, and toxicity testing 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 chronic TIE because the chronic test is more sensitive to these conditions. The pH
adjustment procedures are changed 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. Obviously, use of other species will require consideration of 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).
Because more than one effect is measured in chronic tests and because partial effects
are more frequent than in acute tests, a graded response with concentration 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 as well as the
6-1
-------
Figure 6-1. Overview of characterization tests.
Chronic Phase I Characterization Tests
Tier!
Baseline whole effluent test
EDTA addition test
Sodium thiosulfate test
Filtration test
Aeration test
Post-CIS SPE column test
Methanol eluate test
Graduated pH test (2 pH's)
Tier 2
Baseline whole effluent test
pH adjustment test
Filtration and pH adjustment test
Aeration and pH adjustment test
Post-CIS SPE column and pH adjustment test
Methanol eluate test
6-2
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time to onset of effect. Such effects can be useful in distinguishing the response to
different toxicants.
For acute TIEs, tests are quick and relatively inexpensive, 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
example, for tests such as the sublation test (a subsequent 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
toxicity is first established and even though toxicity is measured, 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. However 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 (see
Section 5.4).
6-3
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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. Experience
with acute TIEs has shown that major pH adjustments are usually not needed. Tier 2
is performed only when Tier 1 does not provide sufficient information, and consists of
filtration, aeration and the C18 separation technique of Tier 1 with an effluent sample
adjusted to both pH 3 and pH 10. For effluents not requiring Tier 2, resources to
conduct the TIE are reduced. 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
i
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 instance,
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 before additional
effluent samples are tested (see Sections 6.15, 6.16 and acute Phase I manual, EPA
1991 A).
6-4
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Figure 6-2. Tier 1 sample preparation and testing overview.
o>
en
Baseline
Toxicity
Test
EDTA
Toxicity
Tests
Thiosulfate
Toxicity
Tests
Graduated pH
Toxicity
Tests
Effluent Sample
EDTA
Additions
Sodium Thiosulfate
Additions
Minor
Filter
pH Adjustments
Methanol
Elution
Toxicity
Test
Post-Column
Sample
-------
Table 6-1. Outline of Phase I effluent manipulations
Description Section
DAY 1 SAMPLE ARRIVAL:
Measure 4.0
• temperature
• conductivity
• pH
• DO
• alkalinity
• hardness
• total ammonia
• total residual chlorine
Perform Sample Manipulations 6.0
• filter effluent 6.4
• collect solid phase extraction 6.6
• collect methanol eluate 6.7
DAY 2 TOXICITY TESTING:
Warm up aliquot of whole effluent and aliquots
of filtered effluent, post-C18 SPE column effluent,
and methanol eluates.
Initiate Tier 1 Tests
• baseline toxicity test 6.1
• EDTA addition test 6.2
• aeration test 6.3
• filtration test 6.4
• sodium thiosulfate addition test 6.5
• post-C18 SPE column test 6.6
• methanol eluate test 6.7
• graduated pH tesf 6.8
ADDITIONAL TESTING ON SUBSEQUENT DAYS.2
Tier 2 Tests
• pH adjustment test 6.10
• aeration and pH adjustment test 6.11
• filtration and pH adjustment test 6.12
• post C18 solid phase extraction 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).
6-6
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Sample Preparation for the Characterization Tests
As for acute TIE tests, we suggest doing certain chemical measurements and the
manipulations on one day and starting the test on the next day (Table 6-1). This
schedule balances the work load more evenly. When the sample is received (day 1),
various measurements (Section 4) are taken and some preparatory manipulations for
Phase I are done.
First, the routine chemical measurements are taken as discussed in Section 4. DO,
conductivity, and pH should be measured to ensure that the values are in the
physiologically tolerable range for the test. If these are at levels that could be toxic '
(EPA, 1989C), there is little point to further 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).
The initial pH of effluent upon arrival at the testing laboratory is referred to as pH i,
which is not necessarily the pH of the effluent at air equilibrium. The pH of the
sample after being warmed, may be selected as pH i rather than the pH upon arrival.
The important point is to use the same pH i 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
6-7
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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 it is too high, it should be reduced to acceptable levels as
described by EPA (1989C) for the routine monitoring test. Ceriodaphnia are less
sensitive to supersaturation than newly hatched fathead minnows. For chronic
Phase I tests, routine water chemistry 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
SPE column, and collection of the methanol eluate (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 test. Of course these samples should be stored in the refrigerator
at (4 ± 2°C). This sample preparation schedule is particularly convenient for
laboratories who rely on courier services to deliver samples, typically late in the
morning.
On day 2, the EDTA addition test should be prepared first so that compounds that are
EDTA chelatable, yet require an equilibration time, can be chelated (see Section 6.4).
Then the rest of the manipulations (aeration, sodium thiosulfate addition, graduated pH
adjustments) should be started. For the laboratory that is experienced in chronic
6-8
-------
toxicity testing, the amount of time required to conduct the Tier 1 sample
manipulations and set up the toxicity tests is about 6-10 hours.
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 measured 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 oh the
historical data for each particular discharger. For the TIE use of four (and 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, routine chemistries are measured
6-9
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(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 addition, 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 for 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 evaluated.
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 control for some subsequent Tier 1 or Tier 2 tests.
6-10
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6.2 EDTA Addition Test
General Approach: This test is designed to direct effluent toxicity caused by certain
cationic metals. The addition of EDTA to water and effluent solutions can produce
non-toxic complexes with many cationic metals. 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 thet
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 typically strongly chelated by EDTA are aluminum (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).
6-11
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EDTA has been 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 selenite or
sodium selenate), aluminum (AI(OH)4"), chromium (either as chromium chloride or
potassium dichromate), or arsenic (either sodium m-arsenite or sodium arsenate)
when tested using moderately hard water and C. dubia.
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). 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 matrix of
the effluent. Without knowing how much toxicant(s) must be chelated, the amount of
EDTA to add must be estimated. Recently, the role of calcium and magnesium 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, NET AC, personal communication). These results indicate
that calcium and magnesium concentrations do not affect the levels of EDTA needed
6- 12
-------
to remove cationic metal toxicity. Whether toxicity reduction using the 1:1 molar ratio
is true for chronic toxicity has not yet been evaluated.
The threshold levels for C. dubia and fathead minnows 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 minnows 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
reconstituted waters. 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 likely to be higher than the toxicity of EDTA
added to an effluent. Therefore, the EDTA toxicity values contained in Table 6-2
represent maximum toxicity in any effluent. The toxic concentration of EDTA in one
effluent 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
concentrations of EDTA added to any effluent should be less than the expected effect
concentration of EDTA in clean water.
6-13
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Table 6-2. Chronic toxicity of EDTA (mg/L) to C. dubia and P. promelas In
various hardness waters using the 7-d tests.
Water
Species Type
Ceriodaphnia dubia VSRW
SRW
MHRW
HRW
VHRW
VHRW
Pimephales promelas 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
t
10
15
200
200
400
400
1 Value could not be determined.
Note: VSRW = very soft reconstituted water; SRW = soft reconstituted water;
MHRW = moderately hard reconstituted water; HRW = hard reconstituted water;
VHRW = very hard reconstituted water.
6- 14
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For either species, three EDTA concentrations are added to three sets of three
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 dilutions. A stock solution of EDTA (ethylenediaminetetraacetic acid,
disodium salt dihydrate) is prepared 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, three sets of effluent dilution concentrations are
*
prepared (e.g, 100%, 50%, 25%,) and each set receives one of three addition levels of
EDTA (Table 6-3). By using non-toxic concentrations 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 2.5 g/L can be prepared. For the C. dubia tests, then
0.01 ml is added to three separate 50 ml aliquots in the first effluent dilution series
(i.e., 25%, 50%, 100%) to obtain a 0.5 mg/L final EDTA concentration. In the second
effluent dilution series, 0.06 ml of stock is added to three separate 50 ml_ aliquots
(25%, 50%, 100%) to achieve a final concentration of 3.0 mg/L in each dilution, and in
6-15
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Table 6-3. Concentrations of EDTA to add for chronic TIEs. Values given are
the final water concentration in mg/L.
Species Water Type Final Concentrations (mg/L)
C. dubia, SRW, MHRW, HRW, VHRW 0.5 3.0 8.0
Fathead
minnow
Note: SRW = soft reconstituted water; MHRW = moderately hard reconstituted water;
HRW = hard reconstituted water; VHRW = very hard reconstituted water.
6-16
-------
the third dilution series, 0.16 mL is added to the last 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 solution can be used but the volume of stock
additions must be doubled for the 100 mL test volume.
To allow the EDTA time to complex the metals, solutions should be set up on day 2
and solutions allowed to equilibrate while other manipulations are being prepared
before test organisms are introduced. A minimum of a 2 h equilibration time should
elapse before organisms are added.
t
Since EDTA is an acid, the pH of the effluent after addition of EDTA should be
checked, although additions at these 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 in the identical way test solution was first
made (allowing equilibration time).
Interpretation of Results/Subsequent Tests: Toxicity may be removed at all exposures
if EDTA alone 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 any of the
three EDTA addition dilution tests, then EDTA removed or reduced the toxicity and
6-17
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cationic metal toxicity is probably present. If, in all three tests, 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 metals causing toxicity in the effluent is low.
Higher concentrations of EDTA can be tried although this usually is not 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 reconstituted water,
zinc was chronically toxic at 55 |ig/L and EDTA was chronically toxic at 15 mg/L
When EDTA was added to solutions of 55 |ig/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 systematic manner, such as in the example, proceed to
Phase II methods for identification of those metal(s) which are chelated by EDTA.
In addition to removing toxicity due to metals, EDTA reduces the acute toxicity of
some cationic surfactants. This reduction of toxicity may also occur in chronically toxic
effluents, and the toxicity reduced by EDTA should not be assumed to be due only to
cationic metals, (see Section 6.4 Aeration Test for subsequent tests to conduct if
cationic metals are not present in the effluent at chronically 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 should be redone. There is no way to distinguish the
6- 18
-------
Table 6-4. The chronic toxicity of zinc to C. dubla in very hard reconstituted
water and the toxicity of zinc when EDTA is added.
Zinc1
Cone.
H9/L
0
3.4
14
55
1 Measured
2 crvrA n«+
0
19.2
19.4
17.8
8.2
values.
r*r4f4r\rl +f\
Mean Youna oer
EDTA Additions
2.5 5.0
18.6 17.5
— 2
22.0 23.2
20.8 19.0
Female
(ma/L)
7.5
17.6
~
20.8
16.6
15
6.8
~
1.8
5.3
6- 19
-------
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. EDTA additions to dilution water are not relevant
controls for the EDTA additions to effluent; therefore, 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 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 (Na^Oa). Toxicity from bromine, iodine, ozone, and chlorine
dioxide is also reduced. Sodium thiosulfate has been routinely used to reduce the
toxicity compounds such as chlorine (EPA, 1989C).
Reductions in effluent toxicity observed with sodium thiosulfate additions may also be
due to the formation of metal complexes with the thiosulfate anion (Giles and Danell,
1983). The ability of sodium thiosulfate to form a metal complex is rate dependent
and metal dependent (Smith and Martell, 1981). Cationic metals that appear to have
6-20
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this potential for complexation, based upon their equilibrium 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 complex. The thiosulfate anion is not very stable,
and the ability of sodium thiosulfate to complex the compound(s) causing 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;
t
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 thiosulfate 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 reduced or
complexed by sodium thiosulfate, the same may be true for chronic toxicity.
The test animals will tolerate more sodium thiosulfate than would ever be needed to
render oxidants or metals non-toxic in effluent samples. The presence of oxidants or
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complexable metals will reduce the concentrations of sodium thiosulfate below the
nominal concentrations added.
Table 6-5 gives the toxicity values in various reconstituted waters. The effect
concentrations for C. dubia and fathead minnows were measured in waters of different
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.
i
Methods: Three sets of effluent dilutions (such as 25%, 50%, 100%) each set with a
different level of thiosulfate concentration (Table 6-6) are prepared regardless 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 addition test). Small volumes (microliter) of the
sodium thiosulfate stock solution should be added to minimize the dilution (Ł5% of
total volume). Non-toxic concentrations of sodium thiosulfate are used to reduce the
probability of artifactual toxicity, yet sufficient concentrations are needed to
remove/reduce oxidants.
For a C. dubia test, to the first effluent dilution set (i.e., 25%, 50%, 100%), 200 \iL of
sodium thiosulfate stock (2.5 g/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,
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Table 6-5. Chronic toxicity of sodium thiosulfate (mg/L) to C. dubla and
P. promotes in various hardness waters using the 7-d tests.
Species
Ceriodaphnia dubia
Pimephales promelas
Water
Type
SRW
HRW
VHRW
SRW
IC50
95% C.I.
39
30-42
38
26-44
43
37-44
1,070
1,041-1,1005
IC25
95% C.I.
26
15-33
27
20-36
34
21-37
820
785-859
NOEC LOEC
30 60
30 60
30 60
750 1,500
t
MHRW 2,001 720
1,891-2,161 550-1,523
750 1,500
HRW 4,871 3,590 3,000 6,000
4,633-5,051 3,226-3,800
VHRW 8,522 6,780 6,000 12,000
8,053-8,704 6,065-7,073
Note: SRW = soft reconstituted water; MHRW = moderately hard reconstituted water;
HRW = hard reconstituted water; VHRW = very hard reconstituted water.
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Table 6-6. Concentrations of sodium thiosulfate to add for chronic TIEs.
Values given are the final water concentration in mg/L.
Species Water Type Final Concentrations (mg/L)
C.dubia, SRW, MHRW, HRW, VHRW 1 5 10
Fathead
minnow
Note: SRW = soft reconstituted water; MHRW = moderately hard reconstituted
water; HRW = hard reconstituted water; VHRW = very hard reconstituted
water.
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100 \iL of the same stock solution is added to 50 mL of each test dilution to obtain
final concentrations of 5 mg/L. To the third set of effluent dilutions, 50 \iL is added to
each to obtain final concentrations of 1 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 the identical way
\
as they were first prepared.
Interpretation of Results/Subsequent Tests: The results of the sodium thiosulfate
addition tests are compared to each other and to the baseline test results to determine
whether or not toxicity reduction occurred. Toxicity may be completely reduced,
partially 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 some metal
should be performed.
When chlorine concentrations are >0.1 mg/L total residual chlorine (TRC), they 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.
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For cases where oxidants account for only part of the toxicity, sodium thiosulfate may
only reduce, not eliminate, the toxicity. Yet the thiosulfate addition 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 chlorinated 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 EDTA addition test reduce the toxicity in the
effluent sample, there is a possibility that the toxicant(s) may be a cationic 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 dilution water are not relevant controls for
thiosulfate additions to effluent to determine if the thiosulfate was toxic. Therefore the
toxicity control is the baseline test.
If all dilutions where sodium thiosulfate is should cause mortality, one possibility is that
the stock solution of sodium thiosulfate is contaminated and should be checked by
conducting another test.
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6.4 Aeration Test
General Approach: Changes in toxicity due to aeration at pH i 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 removed.
Sparging of samples is done using air which includes 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 sublate. 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 rinsing. 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
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moderately aerated for a standard length of time (60 min). Use of gas washing bottles
(Kontes Glass Co., Vineland, NJ) fitted with 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 and a flow-rate of
500 mL/min is suggested. 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 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 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 blank was non-toxic, then either toxicity was concentrated
during the aeration process or toxicity was added or created during the aeration
process (see Special Considerations/Cautions below).
Typically, using this aeration techniques, ammonia is not air-stripped from the sample
at pH i. However, if total ammonia is at least 10 mg/L or higher and the pH is above
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8.0, ammonia measurements in the aerated sample may be useful if the aeration
manipulation resulted 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 replaced 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 oxidation may be
involved. It is possible that a toxicant can be removed through sparging and oxidation
t
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 removing 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 recovery 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 vigorously to contact all surface areas to recover the sublatables. 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.
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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
concentration 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 oxidation. However, the filtration test should not change toxicity of the effluent
t
if oxidation is involved 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
(blank) provides information on the system apparatus. The general 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 blank implies toxic artifacts from the aeration process, the glassware, or
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a dilution water problem. If the 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 removal of
particle-bound toxicants. Whether compounds in the effluent are in solution or sorbed
to particles is dependent on particle surface charge, surface area, compound polarity
and charge, solubility, and the matrix of the effluent. If particles are removed, other
compounds may be bound to them and are not available 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. This is primarily a route of exposure for filter
feeders as compared to the fathead minnow. Toxicity can also be reduced by filtering
if 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 extraction (SPE) (Section 6.6), where aliquots of the effluent must be
6-31
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filtered before application to the SPE sorbent. If many particles are present in the
sample, the sorbent will become plugged or may act as a filter itself.
Methods: The use of a positive pressure filtration system is superior to the use of a
vacuum filter because 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 urn glass fiber filters without
organic binder) by passing 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 discarded and an aliquot of the dilution water is filtered (at least 400 ml;
dependent on the species used) and a portion of the dilution water is collected for
testing and a portion reserved for the solid phase extraction test blank (Section 6.6).
For example, the last 300 mL of the filtrate is collected.
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 C18
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. 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.
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Low levels of metals on the glassware or the filters could cause interferences in
toxicity interpretation. 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 will needed to be prepared).
The filter housing should be thoroughly cleaned between effluent samples to prevent
any particle build-up or toxicity carryover. We have found the use of 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
t
manual should be sufficient for chronic TIE work (EPA, 1991 A). The glass frits may
require more rigorous cleaning 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 constituents 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 (precipitation, sorption, changes in equilibrium or volatilization).
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Identification efforts should be focused on the residue on the filter after testing
indicates 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 pKa 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 toxicant, 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. Sonication of filters is
another approach but the manipulation must be accompanied by proper blanks.
If the toxicity cannot be recovered from the filter, was not volatile (see Section 6.4
Aeration Tesf) and no other manipulations changed toxicity, use of Tier 2 is a good
subsequent step. Toxicity could have been removed by the glass frit, and use of a
plastic filter apparatus or stainless steel frits may assist in identifying 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) because of irreversible reactions and
potential for artifactual 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 C18
6-34
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SPE column test (see Section 6.6). The effluent filtration results should be compared
with the filtration blanks and no change in trend of young production, survival or
growth should occur in the blanks in comparison to the controls in the baseline test If
the 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 C1B SPE column effluent is not toxic (and effluent toxicity
was unchanged after filtration), the blank toxicity can be ignored since the effluent
toxicity was removed. However, as work proceeds to identification, the blank toxicity
i
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 C18 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 i (cf., Post C18SPE column and pH Adjustment Test, Section 6.13
below). By passing effluent 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.
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Compounds in effluent samples interact with the C18 and depending upon the polarity
and solubility of the compounds, the sorbent may extract the chemicals from the water
solution/effluent onto the column. Extraction occurs when the compounds have a
higher affinity for sorbent than for the aqueous phase. Non-polar organic chemicals
are extracted because the C18 sorbent is very non-polar in comparison to the polar
water phase; this extraction process is referred to reverse phase chromatography.
The effluent that passes over the column is collected and the post-column effluent is
toxicity tested in order to determine if the column removed toxicity. If the toxicity of
the sample is decreased, removal 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.
Because toxicity may be retained by the C1B, efforts to recover the toxicity are
necessary. After a sample is passed over the C18 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 C18 sorbent. The methanol eluate test (Section 6.7) is designed to determine
if toxicants are non-polar.
6-36
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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 concentrations and the volume of the
eluate needed for the Methanol Eluate Test (Section 6.7) to test at 4x the whole
effluent concentrations should be determined (with the methanol test level below the
chronic threshold level for the species used). 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. For
example, 1000 mL of 100% effluent over a 6 mL (1 gm) column, eluted with 3 mL of
methanol results in a theoretical 333x concentrate. The 1000 ml is the limit of
t
sample volume over a 6 mL (gm) column and the 3 mL methanol elution is slightly
more than the minimum elution volume (2.4 mL of solvent) required. However to test
C. dubia at 4x, and have the methanol concentration at a safe chronic level, the 3 mL
must be further concentrated to 1.5 mL (now 666x whole effluent concentration).
Then 0.30 mL can be added to 50 mL and the resultant effluent concentration is 4x
and the methanol concentration is 0.6%. The 1.5 mL (from 1 L) will allow testing of
4x, 2x, 1 x with two solution renewals. Daily renewals for a 7-d C. dubia test require a
total of 3.7 mL (which means 4 L of effluent must be fractionated). For the 7-d
fathead minnow test, a total of 7.4 mL of a 666x methanol fraction is needed for seven
renewals, which requires fractionation of 5 L of effluent. The methods below assume
one effluent volume (usually the 100%) is concentrated and collected for all the
sample renewals. The procedure described below is an overview of the steps needed
to prepare the column, collect methanol blanks, recondition the column, collect post-
column effluent, and collect methanol eluate (steps needed for this test and the next
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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 collected.
The general technique for conditioning and using the SPE prepackaged columns is as
follows. Using a pump system with a reservoir for the effluent sample and teflon
tubing, first 12-120 ml4 of HPLC grade methanol is pumped (at 5 mUmin for a 6 ml
column4) over the column to condition the sorbent. This methanol is discarded.
Without letting the column go to dryness, 12-120 mL of high purity water is passed
over the column and discarded. Before the methanol blank is collected, the column is
i
allowed to go to dryness. For 1 L of sample and a 6 mL column, 2-1.5 mL aliquots of
100% methanol are collected, combined, and tested as the blank. This methanol will
be concentrated prior to testing however (see Section 6.7). The containers to collect
the methanol should be acid leached, hexane and acetone rinsed, and allowed to dry
before use.
4 We most frequently use 6 mL columns containing 1 gm of C18 packing (J.T. Baker, Phillipsburg,
NJ) for 1 L of sample and elute with two 1.5 ml fractions of methanol. Larger columns for
larger sample volumes are now available. Sample volumes of 5 L can be concentrated on 20
mL (5 gm) columns, and 10 L^can be concentrated on 60 mL (10 gm) columns (available from
Analytichem Mega Bond But™, Minneapolis, MN). Elution volumes for each of the larger
volumes are proportional to the volumes for the 1 gm column, but minimum elution volumes for
effective elution are 2.4 mL (1 gm), 12 mL (5 gm), and 24 mL (10 gm). The amount of
solvent/water used for preparing the column is determined by the volume of column. Usually
two column volumes are used (i.e., for 20 mL columns, use 40 mL). The pumping rate is
based on 5 mL/min tor the 6 mL column and higher flow rates for larger columns is set
dependent on the surface area. While we have limited experience with the faster pumping rate
and larger columns, for the 20 mL column, 12 mL/min should be sufficient.
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After the methanol blank is collected, the column must be reconditioned with
12-120 ml of methanol (which is discarded). Without allowing the column to go to
dryness, follow the methanol with an aliquot of high purity water, immediately followed
by an aliquot of filtered dilution water, which should be collected post-column. This
post-column dilution water sample will serve as the dilution water blank for the post
C18SPE column test.
Immediately following the dilution water the effluent sample is passed over the same
column and the post-column effluent is collected for testing. If small quantities (<500
ml_) of post-column effluent are needed for toxicity testing, two separate post-column
%
effluent samples may help determine if toxicity breakthrough occurred, and
concentration factors will be different for the lower volumes.
Interpretation of Results/Subsequent Tests: The extraction efficiency of the column is
evaluated by comparing the toxicity of the post C18SPE column effluent to the filtration
test data. This post C18 SPE column test is most useful when there is no post-column
toxicity.
When toxicity in the post-column effluent is reduced or removed, then the next step is
to compare the results with the methanol eluate test If toxicity was recovered in the
methanol eluate (see Section 6.7 below), then efforts to identify the toxicants (Phase
II) should be initiated immediately.
6-39
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If the post-column effluent toxicity was removed or reduced, but toxicity was not
recovered in the methanol eluate (see below), it is possible that the column may still
contain the toxicant and that alternate elution schemes must be tried to recover the
toxicant. The toxicity removed by the C18 SPE column is not necessarily due to non-
polar compounds. Metals can be removed from some effluents via the C18SPE
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
i
polymers or surfactants may be sorbed onto the column 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. Finally the possibility exists that the toxicant
has decomposed or degraded during the manipulation, and 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 in the toxicity. The post C13SPE column test should be combined
with the EDTA addition test and the sodium thiosulfate addition test to characterize the
presence of cationic metals.
Artifactual toxicity in the test containers may appear as a biological growth in the
100% post-column effluent and the effluent dilutions during the test. Effluents from
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biological treatment plants may develop this characteristic more readily than physical-
chemical 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 occurs 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 because methanol
is constantly released from the sorbent during the sample extraction. Additional
filtering of the post-column effluent sample through a 0.2 jim filter before testing to
remove bacteria and eliminate the growth, has not been particularly successful.
*
When post-column artifactual growth is not readily eliminated, then a different solvent
(acetonitrile) to prepare 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 successful on a limited number of effluents.
Special Considerations/Cautions: Careful observations and judgement must be
exercised in detecting problems in the post C18SPE 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 opposed to the
presence of the sample toxicity. If artifactual toxicity is not recognized, then a
conclusion that the C1B SPE column did not remove toxicity can erroneously be made.
For this reason if the post-column effluent is toxic, the methanol eluate must be tested
6-41
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(Section 6.7). This avoids the artifactual toxicity issue and the error can be avoided
by determining the toxicity of the eluate.
General test conditions will be tracked (dilution water, 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-column effluent test(s) must be compared to the results of the
t
filtration tesfto determine if the manipulations effectively reduced toxicity. When the
post C18 SPE column test is plagued by artifactual toxicity, the importance of the
methanol eluate test increases. The results of the post-column test must also be
compared to the baseline test to determine if toxicity was removed by the C18 column.
6.7 Methanol Eluate Test:
General Approach: In order to elute toxicants from the C18 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 C18 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
compounds. The use of methanol has been adopted as the eluant for the acute TIE
6-42
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(EPA, 1991 A; EPA, 1989A) and the chronic TIE because of its low toxicity (Table 6-7)
and its usually adequate ability to elute chemicals from the C18 SPE column.
Methods: The conditioning and elution steps are described in the post C18SPE
column test above (see Section 6.6). For this test, we assume that the column
extraction efficiency and elution efficiency are 100%.
If a 6 ml_ SPE column was used with 1 L of 100% effluent, and a 3 mL methanol
eluate was collected, the methanol eluate is a 333x concentrate of the original
effluent. Depending on the amount of effluent toxicity, this eluate may have to be
i
concentrated further in order to test at a sufficient concentration (i.e., 4x) and keep
methanol concentrations sublethal. In Table 6-7 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 4x whole
effluent concentration. If the effluent is rather toxic, one need not achieve a 4x
concentration. Some methanol toxicity can be present, as long as sufficient toxicity
from the effluent is present to be measurable.
Interpretation of Results/Subsequent Tests: If toxicity occurs in the methanol eluate
test at any concentration 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).
6-43
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Table 6-7. Chronic toxicity of methanol (%) to C. dubia and P. promotes using
the 7-d tests.
Species
Ceriodaphnia dubia
Pimephales promelas
Water
Type
SRW
SRW2
SRW2
SRW2
SRW
Test
Renewal
daily
twice
twice
twice
daily
IC50
95% C.I.
1.2
1.1-1.2
1.4
1.2
0.69-1.7
1.3
2.1
2.0-2.2
IC25
95% C.I.
0.451
0.35-1.0
0.451
0.36-0.70
0.59
0.29-0.95
0.83
0.34-1.0
1.34
0.27-1.5
NOEC LOEC
<0.5
<0.5
0.75 1.5
0.75 1.5
1.3 2.5
1 Value is extrapolated.
2 Tests all conducted independently.
Note: SRW = soft water
6-44
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Toxicants other than non-polar compounds may be retained by the SPE column but
they are less likely to be eluted sharply or 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 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. 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 considerably to allow sufficient contact time on the column (see
details in Section 6.6).
i
Compounds that are sparingly soluble in water may not be eluted from the column
with methanol. If this occurs, less polar solvents will have be tried, but this technique
will require solvent exchanges to avoid toxic solvent concentrations. At this time, we
have not used solvent exchanges for chronic toxicity tests.
Special Considerations/Cautions: The baseline test serves as the toxicity control, and
the methanol blank serves as a comparison of the effects of methanol 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 effluent methanol eluate is non-
toxic at 4x but the methanol blank is, the blank toxicity can be ignored since no non-
polar toxicity is recovered.
6-45
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The artifactual growth observed in the post-C18 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
i
concern are those with a pKa that allows sufficient differences in dissociation to occur
in a physiologically 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 differences
through changes in solubility and speciation. pH dependent toxicity is likely to be
affected by temperature, 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 dependent toxicity. Ammonia is also frequently present in effluents
at concentrations of 5 mg/L to 40 mg/L (or higher). See Phase II (EPA, 1989A) of the
acute TIE procedures for additional discussion of ammonia toxicity. 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
6-46
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(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. The acute Phase I
manual has a lengthy description on the toxicity behavior of ammonia (EPA, 1991 A).
One might expect ammonia to be removed during the 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
i
this manual. (If a larger surface to volume ratio is used, this manipulation can reduce
ammonia levels; see Interpretation of Results/Subsequent Tests below.) 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 toxicity may change for both pH increases and
decreases from neutral pH (pH 7). Such behavior is characteristic of aluminum and
cadmium. Acute toxicity test experiments 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
6-47
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very hard reconstituted water), while nickel and zinc were more toxic at pH 8.5 than at
6.5 (EPA, 1991 A).
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 toxicant, 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
t
toxicity will increase at pH 8.5 will depend on the total ammonia concentration. If the
graduated pH test is done at two pH's using the same dilutions, one should see
toxicity differences between pH 6.5 and 8.5. The effluent effect level (expressed as
percent effluent) should be lower 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 characteristics 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 pH's 6.5
and 8.0. The pH's of many municipal effluents rise to 8.2 to 8.5 (or higher), so pH's
such as 6.5 and 8.5 may be more appropriate. In any case, it will be necessary to
6 - 48
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conduct 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 maintain 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 toxicity 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 CCyair mixtures or HCI additions (or
\
the combination of both) are used. The pH should be maintained throughout the 4-d
or 7-d test with little variation (± 0.2 pH units).
When C02/air (without any acid addition) is used to control the pH, the pH of the
effluent samples is adjusted by varying the CCVair 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 CCVair 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
6-49
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same. Generally, as the alkalinity increases, 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% C02 has been used. If more than 5% CO2 is needed, adjust the solutions with
acids (HCI) and then flush the headspace with no more than 5% COj/air. With
appropriate volumes of effluent, experiments with variable amounts of COg/air and
equilibrated for about 2 h, are used to select the needed CO2 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 CO2 is usually needed for each dilution, but sometimes
different amount are needed in the higher effluent concentrations. Use of a dilution
t
water of similar hardness (or alkalinity) as the effluent makes the CO2 volume
adjustments easier. When tests are conducted in these CO2 controlled environments,
dilution water controls for each pH should be included.
Acid is used first to adjust pH's when the amount of COa/air needed to adjust to the
desired pH is greater than 5% CCyair. Again experimentation is needed to determine
how much CO2/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 CO^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 the bacterial metabolic CO2 released.
6-50
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For the pH controlled tests, the pH should be measured at least 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 CCyair after the animals are fed. Again, some experimentation
may be needed to determine the amount of COj/air 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 minimize the CO2 exchange between
the sample and the atmosphere. Avoid vigorous stirring of unsealed samples because
at lower pH values, the CO2 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.
Methods that use continuous flow of a COj/air mixture, such as tissue cell incubators,
may be preferable and give better pH control. At this time we have not attempted to
use a continuous flow of CO2 and cannot recommend a system to use.
6-51
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Maintaining pH above the air equilibrium pH (generally above pH 8.3) is difficult to
achieve because the concentration of CO2 must be very low, and microbial respiration
can increase the C02 levels in the test chamber. Frequently we use a dilution water
that has a higher pH (i.e., very hard reconstituted water) to prevent 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 negligible, and therefore the likelihood of toxicity due to increased
salinity levels is low.
%
When ammonia is the dominant toxicant, the toxicity 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).
However, if ammonia is only one of several toxicants in an effluent, this procedure will
be hard to interpret. For this reason, if total ammonia concentrations in the 100%
effluent are greater than 20 mg/L, include a pH 6 (rather than 6.5) effluent treatment
interfaced with other Phase I tests. Complicating effects of metals can be reduced by
adding EDTA to the test solutions. However, the ability of EDTA to detoxify metals
may also change with pH.
6-52
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Other metals may exhibit some degree of pH dependence, 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 dilution water
similar in hardness to that of the effluent must be used for this test to reveal metal-
caused toxicity. If more than one pH dependent toxicant is present, the pH effects
may either cancel or enhance one another.
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 buffers 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) (pK"8 = 6.15),
3-(N-morpholino) propane-sulfonic acid (Mops) (pK. = 7.15), and piperazine-N,N'-bis
(2-hydroxypropane) sulfonic acid (Popso) (pK, = 7.8).
The acute toxicity of these buffers is low to both C. dubia and fathead minnows
(Phase I) (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
6 -53
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indicated that 16 mM did not cause reduced survival or growth for the fathead minnow
7-d test. For C. dubia, 2 mM has not caused reduced survival or reproduction 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
t
contrast to other buffers). Our experience with the buffers is limited, but 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
maintain 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.
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 conditions. If the effluent pH in the
baseline test is close to that of the pH adjusted test solutions, the toxicity expressed 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 pH was adjusted
with HCl) or C02 toxicity. Dilution water tested at the various pH's does not serve as
6-54
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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 results or may give results similar to ammonia.
Monitoring 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
i
Two tiers are used in the chronic TIE approach primarily because in our experience,
radical pH adjustment often is not needed. Only when the manipulations 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 speciation of a
compound. These can change the bioavailability 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 abbreviated discussion of pH
effects will be covered in this document.
6-55
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Figure 6-3. Tier 2 sample preparation and testing overview.
Ol
o>
Toxicity
Test
Post-Column
Sample
Methanol
Elution
-------
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. Compounds 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 compounds 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. Changing pH and returning it to pH i after
a short time (~1 h) will not always change the toxicity. However, this adjustment may
result in a reduction, loss or increase in the toxicity. Sometimes only the pH
adjustment 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 effluent is adjusted to either pH 3 or
pH 10, and left at those pHs until other manipulations (aeration, filtration, and C18SPE
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
6-57
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tests with various reconstituted waters adjusted to pH 11 have not been consistently
produced, but acceptable blanks have been obtained at pH 10 (and pH 3). Since
pH 11 was subjectively chosen, we recommend adjustment 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 C18 SPE column 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 adjusted to
pH 10, along with dilution water samples which will serve as blanks. Enough sample
is adjusted to provide the necessary volumes for the aeration and pH adjustment test,
the filtration and pH adjustment test, and the post C18SPE 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.
Interpretation of Results/Subsequent Tests: A decrease in toxicity compared to the
baseline test should be pursued to detect the mechanism of toxicity reduction. Often
precipitation occurs after drastic pH change. If precipitation does occur, then the
filtration and pH adjustment test will likely remove the toxicant and efforts should be
focused on recovery and identification from the filter. Similarly, if the C18 SPE column
6-58
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or aeration changed 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 exchange 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 in effluents. The effect on effluent
i
toxicity of the Na+ and CI" additions, depends on the TDS concentration 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 concern in
chronic TIE'S. The effect of NaCI additions on TDS can be tracked by measuring
conductivity. Appreciable increases in conductivity should be a warning to evaluate
TDS toxicity caused by acid and base addition.
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
6-59
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increased toxicity. Table 6-8 provides chronic toxicity information for NaCI in various
hardness waters for C. dubia and fathead minnows.
Precipitates can remove toxicity through sorbtion of such chemicals as non-polar
organics. In this case the precipitate is only the mechanism of removal, not the
toxicant itself. The C18 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 before (EPA, 1991 A). For a valid test, the pH during the test must be
known and maintained the same as in the pH i test. If the drift of the pH varies
considerably, confusion in interpreting the results can arise if a compound whose
toxicity is pH depended is present in the sample. Otherwise incorrect conclusions are
likely to be made and mislead the TIE process.
Special Considerations/Cautions: The addition of acids and bases to the effluent do
not give comparable results when added to the dilution water. The amount of acid
and base added to each 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 solutions that are used in the this manipulation and
subsequent pH manipulation tests. The controls from the baseline test provide
information on the health of the test organisms, dilution water, and laboratory test
6-60
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Table 6-8. Chronic toxicity of sodium chloride (g/L) to C. dubla and
P. promelas in various hardness waters using the 7-d tests.
Water
Species Type
Ceriodaphnia dubla SRW
MHRW
HRW
VHRW
Pimephales promelas SRW
SRW
MHRW
HRW
VHRW
IC50
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
Extrapolated value.
Note: SRW = soft reconstituted water; MHRW = moderately hard reconstituted
water; HRW = hard reconstituted water; VHRW = very hard reconstituted
water.
6-61
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conditions. The pH adjustment test serves as the toxicity control (or perhaps the
"worst case" toxicity control) for the subsequent 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
replaced.
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 different in aerated samples than for pH adjustment
test and proper compensation for this difference must be made as described above.
After aeration is completed, adjustments back to pH i should be done on all samples
at the same time. The formation of any precipitates should be noted, but the
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importance of precipitates (if any) will not be known at this point in the
characterization.
Interpretation of Results/Subsequent Tests: If aeration with any pH adjustment
removes or reduces the toxicity, additional tests must be performed to identify 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 can be reduced by aeration. The geometry of the
aeration technique described here is not particularly conducive to ammonia removal.
However, if aeration at pH (10) reduces toxicity compared to the toxicity in the
aeration test at pH i and the baseline test, measure the total ammonia 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 (the pH adjustment test) and the baseline test. The aeration and pH
adjustment blanks should be compared to the pH adjustment blanks. If the effluent
toxicity is reduced in the effluent following pH adjustment/aeration, and the blanks are
toxic, the blanks 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 done. Compare the results of the aeration and pH adjustment blankio the
filtration and pH adjustment blank and the pH adjustment blank (Sections 6.10 and
6.12). If all have toxicity, then artifactual toxicity occurred from the pH adjustment,
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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 toxicants 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 i) 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 dilution water for the blank.
Effluent samples adjusted to pH 3 or pH 10 (Section 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 frequently bleed metals when a pH 3 solution
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is 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 toxiclty confro/s--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
i
filtration test. This may be accomplished using a pH adjusted sample of water,
perhaps of the opposite pH of the filtration process.
Special Considerations/Cautions: The pH adjusted 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 and pH adjustment
blank. Toxicity in the blanks implies toxic artifacts from the filtration process, the
glassware, the pH adjustment or a dilution water problem. If the control performance
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
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toxicity cannot be ruled out. To check if it occurred during the manipulation, the
experiment must be repeated.
6.13 Post-C18 Solid Phase Extraction (SPE) Column and pH Adjustment Test
(pH 3 and pH 9)
General Approach: Shifting the ionization equilibria at high and low pHs, may cause
the C18 SPE column to extract different compounds than at pH i. pH adjusted and
filtered effluent is passed over a prepared C18 SPE column to remove non-polar
organic compounds (cf., Post C18SPE Column Test, Section 6.6 above). Organic
acids and bases may be made less polar by shifting their equilibrium to the un-ionized
species. 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 C18 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 C18 SPE column that
would be sorbed only at an altered pH, and therefore we can only provide general
rules to follow in these cases except those inferred from how ionizable compounds
behave in regard to pH change.
Methods: All of the procedures for this manipulation and the use of the C18 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
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effluent) should be acidified or basified 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 i.
Interpretation of Results/Subsequent Tests: The extraction efficiency of the column is
assessed by comparing the results of the post C18SPE column tests (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 artifactual 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, efforts to identify the toxicants should be started. If
methanol does not recover toxicity, a pH adjusted water should be tried. For further
discussions of the interpretation of the results, see Section 6.6 above.
Special Considerations/Cautions: Careful observations and judgement must be
exercised in detecting problems in the post C18SPE 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 C18 SPE column did not remove toxicity can be
made.
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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 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-column effluent test(s) must be compared to the results of the
filtration and pH adjustment testlo determine if the manipulations effectively reduced
toxicity. When the post C18SPE column test date 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 waters/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
10).
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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 C18 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 C18 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 C18
SPE technique in Phase I detects the presence of these compounds as a group. This
lack of specificity is very important to understand for subsequent Phase II toxicant
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identification. Efforts should concentrate on those manipulations affecting toxicity in
which the toxicant is isolated from other effluent constituents, such as the SPE
column, filtration and aeration.
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
immediately into the Phase II identification, and sometimes Phase I (Tier 1 and/or 2)
and Phase II combinations are needed to determine the cause of toxicity. Of course,
new approaches are frequently 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 possible than when only one manipulation changes 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 infrequently 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 toxicants. 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 synergism but independent action has commonly been found. Some
toxicants identified in effluents have been additive, but more often these have been
only partially additive.
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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 constituents 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).
i
As discussed earlier, the amount of time necessary to adequately characterize the
physical/chemical nature 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 toxicants, 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 moved into
Phase II more quickly than an ephemerally toxic effluent with highly variable
constituents, none of which are impacted by any of the Phase I tests. Several
samples should be subjected 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.
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If the Phase I characterization tests needed to remove or neutralize effluent toxicity
vary by the sample, the number of tested samples must be increased and the
frequency 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 characterization tests
must be successful in removing and/or neutralizing effluent toxicity.
t
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
toxic effluents, once one toxicant 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 identified 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 toxicants varies by sample. Such information can be used
to design tests to elucidate additional physical/chemical characteristics of the toxicants
that cause chronic toxicity.
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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 removed by several Phase I steps. For example, if several
toxicants are acting to cause the toxicity, then the graduated pH test and the post-C18
SPE column test both result in a partial toxicity reduction. If sodium thiosulfate and
EDTA both reduce toxicity, cationic 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 C18 SPE column only partially removes toxicity, Phase I manipulations with
the post-column sample should be tried. For this multiple manipulation, the post C18
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.
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If the C18 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 C18 SPE column
may provide additional insight. To gain this knowledge toxicity tests must be
performed after each manipulation and not just on the multiple manipulated sample.
Effluent characterization must be approached without any preconceived notion or bias
about the cause of toxicity because many constituents are present in effluents and
t
their chemistry is unknown, circumstantial evidence is frequently misleading. Certainly
all available information and experience should be used to guide the investigative
effort but temptations to reach conclusions too soon must be resisted. Sometimes the
answer being sought is only whether or not a certain substance is causing toxicity.
Obviously in such cases testing is specifically selected to answer that question and
therefore not all manipulations need to be performed.
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SECTION 7
REFERENCES
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
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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.
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EPA. 1989B. Methods for Aquatic Toxicity Identification Evaluations: Phase III
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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
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EPA. 1989E. Generalized Methodology for Conducting Industrial Toxicity Reduction
Evaluations (TREs). EPA/600/2-88/070. Water Engineering Research
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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. 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. 1991E. 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 Intel-laboratory 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.
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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.
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
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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.
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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.
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583 p.
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