vvEPA
United States
Environmental Protection
Agency
Environmental Research
Laboratory
Duluth MN 55804
National Effluent Toxicity
Assessment Center
Research and Development
EPA/600/3-88/034 Sept 1988
Methods for Aquatic
Toxicity Identification
Evaluations
Phase I Toxicity
Characterization
Procedures
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EPA-600/3-88/034
September 1988
Methods for Aquatic
Toxicity Identification Evaluations
Phase I Toxicity Characterization Procedures
Donald I. Mount
U.S. Environmental Protection Agency
Environmental Research Laboratory
Office of Research and Development
Duluth, Minnesota 55804
Linda Anderson-Carnahan
U.S. EPA Region IV
Water Management Division
Atlanta, Georgia 30365
National EffluentToxicity
Assessment Center
Technical Report 02-88
U.S. Environmental Protection Agency
Region 5,1 'hnr- r-< s - ••,
77 WestJ&c-."'. •"' - - -.
Chicago, JL 60604-.,.. /' ' r"J^
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Notice
Mention of trade names or commercial products does not constitute endorsement or
recommendation for use.
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Foreword
This document is one in a series of guidance documents intended to aid dischargers
and their consultants in conducting aquatic organism Toxicity Identification Evaluations
(TIEs) as part of the Toxicity Reduction Evaluations (TREs). Such effluent evaluations
may be required as the result of an enforcement action or as a condition of a National
Pollutant Discharge Elimination System permit. It will also help provide U.S.
Environmental Protection Agency (EPA) and State Pollution Control Agency staff with
the background necessary to overview and determine the adequacy of effluent TIEs
proposed and performed by NPDES permittees.
The approach is divided into three phases. Phase I contains methods to identify the
physical/chemical nature of the constituents causing toxicity. Such characteristics as
solubility, volatility and filterability are determined without specifically identifying the
toxicants. Phase I results are intended as a first step in specifically identifying the
toxicants but the data generated can also be used to develop treatment methods to
remove toxicity without specific identification of the toxicants. Two EPA TRE manuals
in draft stage (EPA, 1988A; 1988B) use parts of Phase I in developing those
approaches.
Phase II (Mount and Anderson-Carnahan, 1988) describes methods to specifically
identify the toxicants if they are non-polar organics, ammonia, chlorine or metals. This
Phase is incomplete because methods for other specific groups, such as polar
organics, have not been developed. As additional methods are developed, they will be
added.
Phase III (Mount, 1988) describes methods to confirm the suspected toxicants. It is
applicable whether or not the identification of the toxicants was made using Phases I
and II. Phase III has been only infrequently done in TIEs completed to date. To avoid
Phase III may be to invite disaster because the suspected toxicants were not the true
ones.
Phases I and II depend on acute toxicity and cannot be used for effluents that do not
have it. Importantly, however, that limitation does not mean that effluents having
chronic limits cannot be evaluated using these methods. So long as there is acute
toxicity, even though it may be non-lethal toxicity, the methods can be used.
These methods are not mandatory but are intended to aid those who need to
characterize, identify or confirm the cause of toxicity in effluents or similar aqueous
samples such as leachates. Where we lack experience, we have so indicated and tried
to provide suggested avenues. All tests need not be done on every sample; the tests
are, in general, independent. Experience has taught us, however, that skipping tests is
likely to result in wasted time, especially during the early stages of Phase I. An
exception is when one only wants to know if a specific substance, for example
ammonia, is causing the toxicity or if there are other toxicants than ammonia.
Otherwise we urge the use of the whole battery of tests.
We welcome comments from users of these manuals so that future editions can be
improved.
in
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Abstract
This manual describes procedures for characterizing the physical/chemical nature of
toxicants in acutely toxic effluent samples. To detect the presence and potency of the
toxicants as the sample is manipulated the measurement of toxicity using organisms is
essential. The final step is to separate the toxicants from other sample constituents to
simplify the analytical process. Usually the toxicants must be concentrated for analysis.
Sample manipulations to alter toxicity include sparging, pH changes, filtration, solid
phase extraction and addition of chelating and reducing agents. The results will often
reveal information about the physical/chemical characteristics of the toxicants.
Subsequent manuals in preparation describe methods to specifically identify the
toxicants and to confirm that the suspected constituents are the true toxicants.
IV
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Contents
Page
Foreword iii
Abstract iv
Contents v
Figures vii
Tables ix
Acknowledgments x
1. Introduction 1-1
1.1 Background 1-1
1.2 Conventional Approach to TIEs 1-1
1.3 Toxicity Based Approach 1-3
2. Health and Safety 2-1
3. Quality Assurance 3-1
3.1 General 3-1
3.2 TIE Quality Control Plans 3-1
3.3 Cost Considerations/Concessions 3-1
3.4 Variability 3-2
3.5 mtra-Laboratory Communication 3-2
3.6 Record Keeping 3-2
3.7 Phase I Considerations 3-2
3.8 Phase II Considerations 3-3
3.9 Phase III Considerations 3-3
4. Facilities and Equipment 4-1
5. Dilution Water 5-1
6. Effluent Sampling and Handling 6-1
7. Toxicity Tests 7-1
7.1 Principles 7-1
7.2 Test Species 7-1
7.3 Toxicity Test Procedures 7-2
7.4 Test Endpoints 7-3
7.5 Feeding 7-4
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Contents (continued)
Page
8. Phase I Toxicity Characterization Tests 8-1
8.1 Initial Effluent Toxicity Test 8-4
8.2 Baseline Effluent Toxicity Test 8-4
8.3 pH Adjustment Test 8-6
8.4 pH Adjustment/Filtration Test 8-12
8.5 pH Adjustment/Aeration Test 8-18
8.6 pH Adjustment/Cis Solid Phase Extraction Test 8-22
8.7 Oxidant Reduction Test 8-26
8.8 EDTA Chelation Test 8-29
8.9 Graduated pH Test 8-33
9. Time Frame and Additional Tests 9-1
9.0 Time Frame for Phase I Studies 9-1
9.1 When Phase I Tests are Inadequate 9-1
10. References 10-1
VI
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Figures
Number Page
1-1. Conventional approach to TIEs 1-2
1 -2. Flow chart for toxicity reduction evaluations 1-4
6-1. Example data sheet for logging in samples 6-2
7-1. Schematic for preparing effluent test concentrations using simple
dilution techniques 7-4
8-1. Overview of Phase I effluent characterization tests 8-2
8-2. Example of data sheet for initial toxicity test 8 - 5,
8-3. Example of data sheet for definitive baseline toxicity data 8-7'
8-4. pe-pH diagrams for the C02, H2
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Tables
Number Page
6-1. Volumes Needed for Phase I Tests 6-3
8-1. Outline of Phase I Effluent Manipulations 8-3
8-2. Acute Toxicity of Sodium Chloride to Selected Aquatic Organisms 8-13
8-3. Toxicity of Methanol to Several Freshwater Species 8-26
8-4. Toxicity of Sodium Thiosulfate to Ceriodaphnia, Daphnia, and
Fathead Minnows 8-27
8-5. Toxicity of Disodium EDTA to Ceriodaphnia dubia and Fathead Minnows in
Water of Various Hardnesses and Salinities 8-30
IX
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Acknowledgments
The authors wish to acknowledge the help from many members of the Environmental
Research Laboratory (ERL-Duluth) staff for their help and advice in preparing this
manual. Throughout the text, we have referred to "experience" to suggest an approach
and we mean the collective experience of the ERL-D effluent research group. That
group consisted of Liz Durhan and Teresa Norberg-King of ERL-Duluth, Joe Amato,
Larry Burkhard, Art Fenstad, Jim Jenson, Marta Lukasewycz, Greg Peterson, Eric
Robert, and Jim Taraldsen of American Scientific International, Inc. (AScI), Duluth.
Special mention should be made of the invaluable help Dorette Gueldner (AScI) has
been, not only in typing but in figure preparation, "on-the-spot" corrections, and
generally seeing that details were in order. Joe contributed an immense amount of
work upon which we based many of the perceptions as well as specific
recommendations. Jim, Greg, and Gary Ankley (ERL-D) completed tests, generating
data for some of the tables. Larry and Joe helped in final reviews and last minute
changes. We gratefully acknowledge Teresa's work in diligently reviewing the
document, preparing the figures and tables, and making editing changes on the drafts
and final version.
Outside the effluent group, but still within ERL-D, Evelyn Hunt was very helpful in
refereeing the review comments. Without the always present backing and support of
Nelson Thomas (ERL-D), the effluent group would not have been able to complete
such a task.
Rick Brandes, U.S. EPA Permits Division, Washington, D.C., has been a strong and at
times a crucial voice in support of all the work upon which this manual is based. His
funding support has enabled more than a doubling of the staff of the National Effluent
Toxicity Assessment Center (NETAC) at ERL-Duluth.
Finally, we want to specifically recognize Bill Clemente of Battelle Laboratories,
Columbus, OH, for the most thorough, by far, and useful technical review comments
on the first draft of the manuscript and largely on his personal time! Burlington
Research, Inc., Burlington, NC, and EA Engineering, Science & Technology, Sparks,
MD reviewed the first draft.
The manual is truly the result of the efforts of many people. We welcome your
suggestions for improvement so that future revision can make the methods more
useful.
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Section 1
Introduction
1.1 Background
The Clean Water Act (CWA) provides the basis for
control of toxic substances discharged to waters of
the United States. The Declaration of Goals and
Policy of the Federal Water Pollution Control Act of
1972 states that "...it is the national policy that the
discharge of toxic pollutants in toxic amounts be
prohibited." This policy statement has been
maintained in all subsequent versions of the CWA.
It is the goal of the CWA that zero discharge of
pollutants to waters of the U.S. be achieved. Because
this goal is not immediately attainable, the CWA
allows for National Pollutant Discharge Elimination
System (NPDES) permits for wastewater discharges.
The five year NPDES permits contain technology-
based effluent limits reflecting the best controls
available. Where these technology-based permit
limits do not protect water quality, additional water
quality-based limits are included in the NPDES
permit in order to meet the CWA policy of "no toxic
pollutants in toxic amount." State narrative and
numerical water quality standards are used in
conjunction with EPA criteria and other toxicity data
bases to determine the adequacy of technology-
based permit limits and the need for additional water
quality-based controls.
To insure that the CWA's prohibitions on toxic
discharges are met, EPA has issued a "Policy for the
Development of Water Quality-Based Permit
Limitations for Toxic Pollutants" (Federal Register,
1984). This national policy recommends an integrated
approach for controlling toxic pollutants that utilizes
whole effluent toxicity testing to complement
chemical-specific analyses. The use of whole
effluent toxicity testing is necessitated by several
factors including a) the limitations presented by
chemical analysis methods, b) inadequate chemical-
specific aquatic toxicity data, and c) inability to predict
the aggregate toxicity of chemicals in an effluent.
To determine the toxicity of effluents to aquatic life,
standardized methods for measuring acute and
chronic toxicity have been developed by EPA (Peltier
and Weber, 1985; Horning and Weber, 1985). These
cost-effective methods facilitate the inclusion of
whole effluent toxicity limits and biomonitoring
conditions in NPDES permits for facilities suspected
of causing violations of state water quality toxicity
standards.
As a result of the increasing use of aquatic organism
toxicity limits and biomonitoring conditions in permits,
a substantial number of unacceptably toxic effluents
have been and continue to be identified. To rectify
these problems, permittees are being required,
through permit conditions and administrative orders or
other enforcement actions, to perform effluent toxicity
reduction evaluations (TREs). The object of the
Aquatic TRE is to determine what measures are
necessary to maintain the effluent's toxicity at
acceptable levels. Such evaluations, however, have
often proven to be very complicated.
The goal of the TRE will be set by the State
Regulatory Agency or EPA and will be dependent on
state standards that define acceptable levels of
toxicity in the receiving water and effluent. Because
of this, and because specific TRE actions may also
be required, communication between the regulators
and TRE investigators is crucial.
This document provides NPDES permittees with
procedures to assess the nature of effluent toxicity to
aquatic organisms. It is intended for use by those
permittees having difficulty meeting their permit whole
effluent aquatic organism toxicity limits or permittees
required, through special conditions, to reduce or
eliminate effluent toxicity. This document does not
address human health toxicity concerns such as from
bioconcentration, water supply and recreational uses.
Neither are the methods applicable to identifying the
cause of chronic toxicity except for those effluents
which also display acute toxicity.
1.2 Conventional Approach to TIEs
In order to appreciate the complexities involved in a
typical effluent toxicity identification evaluation (TIE),
one must first understand the drawbacks in what can
be considered the conventional approach to the
problem. The following discussion is meant to
exemplify the need for a logical approach which
builds on the effluent data as it is being collected.
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Traditionally, when an effluent has been identified as
toxic or is suspected of being toxic to aquatic
organisms, a sample of the wastewater is analyzed
for the 126 "priority pollutants". The concentration of
each priority pollutant present in the sample is
subsequently compared to literature toxicity data for
the pollutant, or is compared to EPA's Ambient Water
Quality Criteria or state standards for aquatic life
protection. The goal of this exercise is to determine
which pollutants in the wastewater sample are
responsible for effluent toxicity (Figure 1-1).
Unfortunately, determining the source of an effluent's
toxicity is rarely this straightforward.
Figure 1-1. Conventional approach to TIEs.
Evaluate Effluent
Constituents and
Their
Concentrations
Search Literature
for Aquatic
Organism Toxicity
Data on Effluent
Constituents
The first problem encountered in this course is one of
effluent variability. Because toxicity is a generic
response, there is no way to determine whether the
toxicity observed over time is consistently caused by
a single constituent or a combination of constituents
or a number of different constituents, each
periodically acting to cause effluent toxicity.
Experience has shown that the latter may be a
frequent occurrence especially in privately owned
treatment works (POTW) effluents. To further
complicate the problem, the variability in conventional
effluent monitoring parameters may not coincide with
variability in the effluent toxicant(s). Monitoring
methods for conventional parameters such as
biological oxygen demand (BOD) frequently are not
responsive to shifts in the toxicants because they are
at relatively low concentrations in the effluent or
simply because the toxicants are not amenable to
analysis by these procedures. For this TIE approach
to be successful, it is crucial that the same sample be
analyzed using both chemical and biological
techniques, and that a number of samples over time
be studied to assess the variability in the toxicant(s).
A second problem with the conventional approach
involves the focus on the priority pollutants. These
have become known as the "toxic pollutants,"
conveying an implication that they constitute the
universe of toxic chemicals. The priority pollutants are
only a tiny fraction of all chemicals. Limiting the
search to these 126 compounds will result in failure to
identify the cause of toxicity in most cases.
On the surface, solving this difficulty may seem
inconsequential; the effluent analysis must include
monitoring techniques for "non-priority" as well as
priority pollutants. To analyze an effluent for every
chemical would cost tens of thousands of dollars and
there would be no assurance that the detection levels
would be low enough. Determination of the
composition of an effluent is limited to the analyses
used. Gas Chromatography/Mass Spectrometry
(GC/MS) will not identify cadmium. Inductively
Coupled Emission Spectroscopy (ICP) may not detect
it when the concentration is low. The absence of a
measurable quantity of any substance is often
interpreted as meaning that it is not present in the
effluent.
The toxicants may be present at low concentrations
because only small concentrations of highly toxic
chemicals are needed to produce toxicity. If true, then
low concentrations must be measured. Such
chemicals are not easily found by examining system
loadings. For example, if a chemical has an LC50 of 1
ug/L, 380 grams, or less than a pound per day of the
compound is necessary to cause lethality in the
effluent of a 100 million gallons per day treatment
plant. Even with a removal efficiency of 99%, only
100 pounds per day loading would be needed to
produce a toxic effluent. Clearly then, large loadings
cannot be used to guide selection of analytical
techniques, and loads of a few pounds in a collection
system producing 100 million gallons per day may be
next to impossible to identify by usual methods of
establishing loadings.
Many analytical methods are relatively limited in their
applicability. Even GC/MS, an instrument heavily
relied upon in typical wastewater analyses, is
incapable of detecting about 80% of all synthetic
organic compounds (G. Veith, ERL-Duluth, personal
communication). This limitation is related to selection
and efficiency of solvent extraction techniques,
analyte volatility and thermal stability, detector
specificity and sensitivity, and analytical interferences
and artifacts. The percentage of organics detected
can be improved by derivatization but the results are
much more difficult to interpret. In general, the
broader spectrum methods are less sensitive and
require higher concentrations of analytes for
detection. To detect lower concentrations, more
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specific methods are usually more sensitive. To
choose specific methods one must have knowledge
of the toxicants-knowledge which does not exist
since that is the purpose of the analyses.
Surprisingly, even with these limitations, one usually
sees lengthy lists of effluent constituents when
analyses are performed on wastewater. In the case of
GC/MS chromatograms, large peaks of non-toxic
effluent constituents can overlap and hide smaller
peaks that may represent the toxicants of concern.
When many chemicals are present, the number of
peaks that can be identified may be small. Failure to
identify a component does not mean that the
chemical is not the toxic one. Using reference
spectra, many may be tentatively identified as several
different compounds which only serves to increase,
not decrease, the number of possibilities. No aquatic
toxicity data will be available for most of these
compounds and toxicity data must be generated.
Compounds often must be synthesized in order to
test them because they are not commercially
available. For those compounds for which aquatic
toxicity data are available, the data may not include
the species used for the TIE. Even if all this work is
done, trying to pinpoint the cause of toxicity in such a
complex mixture, is likely to fail because this
approach does not include matrix effects and toxicant
bioavailability. For example, several metals may be
present in an effluent sample at concentrations well
above the toxic threshold. These metals may not be
the source of the effluent's toxicity, however, because
they are not biologically available. Characteristics
such as total organic carbon (TOC), total suspended
solids (TSS), ionic strength, pH, hardness and
alkalinity, can change toxicity. The inability to
quantitate the effects these parameters have on
toxicity further decreases the chances for a
successful TIE.
1.3 Toxicity Based Approach
The approach described in this manual utilizes the
responses of organisms to detect the presence of the
toxicant during the first stages of the TIE. In this way,
the number of constituents associated with the
toxicants can be reduced before analyses begin and
some knowledge of physical/chemical characteristics
is gained. This approach simplifies the analytical
problems and reduces cost. Some of the problems
limiting the conventional approach can be used to
enhance the success of this alternate approach.
There are two main objectives in the first step of this
approach. First, characteristics of the toxicants (e.g.,
solubility, volatility) must be established. This allows
them to be separated from other non-toxic
constituents to simplify analyses and enhance
interpretation of analytical data. Secondly, throughout
the TIE, one must establish whether or not the toxicity
is consistently caused by the same substances.
Failing to establish the variability related to the
toxicants could lead to control choices that do not
correct the problem.
Knowledge of the physical/chemical characteristics of
the toxicants aids in choosing the appropriate
analytical method. Such information also may be
useful in selecting an effluent treatment method.
Figure 1-2 is a flowchart representation of a TRE.
This document details the toxicity characterization
procedures (Phase I). Phase II (toxicant identification)
and Phase III confirmation will usually follow Phase I.
Two other EPA manuals (EPA, 1988A; 1988B) can be
consulted for more information on bench scale and
pilot plant effluent toxicity treatability studies and
source control options.
Phase I characterizes physical/chemical properties of
the effluent toxicant(s) using effluent manipulations
and accompanying toxicity tests. Each
characterization test in the Phase I series is designed
to alter or render biologically unavailable a group of
toxicants such as oxidants, cationic metals, volatiles,
non-polar organics or metal chelates. Aquatic
toxicity tests, performed on the effluent before and
after the individual characterization treatment, indicate
the effect of the treatment and provide information on
the nature of the toxicant(s). By repeating the toxicity
characterization tests using samples of a particular
effluent collected over time, these screening tests will
provide information on whether the compounds
causing toxicity remain consistent. These tests will
not provide information on the variability of toxicants
within a characterization group. Knowing that the
toxicants have similar physical/chemical properties
means that they can be identified in Phase II using
similar techniques. With successful completion of
Phase I, the toxicants can be tentatively categorized
as certain cationic metals, non-polar organics,
oxidants, substances whose toxicity is pH dependent
and others. Information on physical/chemical
characteristics of the toxicants will indicate filterability,
degradability, volatility, and solubility. Either of two
choices is available in the second phase of testing:
toxicant treatability or toxicant identification studies.
The toxicant identification option is described in
Phase II (Mount and Anderson-Carnahan, 1988).
Phase II involves several steps, all of which rely on
tracking the toxicity of the effluent throughout the
analytical procedure. Although effluent toxicants are
partially isolated in the first phase of the study, further
separation from other compounds present in the
effluent is usually necessary. Techniques are
available to reduce the number of compounds
associated with the toxicants. Unlike Phase I
procedures, Phase II methods will be toxicant
specific. Currently available techniques in Phase II are
for identifying non-polar organics, EDTA chelatable
metals, chlorine, and ammonia. Additional procedures
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Figure 1-2. Flow chart for toxicity reduction evaluations.
Phase I
Toxicant Characterization Tests
Treatability
Approach
Treatability Approach
or Identify Toxicant
Identify Toxicant(s)
Phase II
Toxicant Identification Analyses
Phase III
Toxicant Confirmation Procedures
Based on Site Specific
Considerations
Toxicity Treatability
Evaluations
~- —
^
r
Source
Investigation
Control Method Selection
and Implementation
Post Control Monitoring
for other toxicants will be added as they are
developed. Once the toxicants have been adequately
isolated from other compounds in the effluent and
tentatively identified as the causative agents, final
confirmation (Phase III) can begin.
Like Phase I, Phase III (Mount, 1988) contains
methods generic to all toxicants. No single test
provides irrefutable proof that a certain chemical is
causing effluent toxicity. Rather, the combined results
of the confirmation tests are used to provide the
"weight of evidence" that the toxicant has been
identified.
Once the toxicant has been identified, it can be
tracked through the process collection system using
chemical analyses. Toxicity cannot be used to find
the source on untreated wastes because toxicity from
other constituents that are toxic in untreated waste
but removed by treatment, will confuse the results. Of
course, using bench- or pilot-scale systems and
measuring toxicity on treated waste, is feasible.
TIEs require that toxicity be present frequently
enough so that repeated testing can characterize and
subsequently identify and confirm the toxicants in
Phases II and III. Therefore, enough testing should be
done to assure consistent presence of toxicity before
TIEs are initiated. This is done not to validate a given
test but to establish consistent presence of toxicity.
The methods described herein are applicable only to
acute toxicity. Much work remains to be done before
chronic toxicity methods are developed and proven.
In some special cases in which toxicity can be
concentrated (as in the non-polar organic section of
Phase II) one may be able to "convert" chronic
toxicity to acute toxicity by concentration and
successfully identify chronic toxicity.
To be successful, TIEs must be conducted by
multidisciplinary teams whose team members must
interact daily so that toxicologists and chemists are
aware of the many concerns that affect test results.
Speed is usually important because effluents often
decay during storage. Often subsequent tests cannot
be designed until the results of the previous ones are
known. Obviously then, waiting a week for analytical
or toxicological results may preclude more work when
the effluent sample undergoes changes during the
waiting period. If this happens, one must begin again
on a new sample in which case resources are not
then effectively being used.
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Section 2
Health and Safety
Working with effluents of unknown composition is the
nature of toxicity identification evaluations. Therefore
safety measures must be adequate for a wide
spectrum of chemicals as well as biological agents.
From the type of treatment used one may be able to
judge probable concerns. For example, extended
aeration is likely to minimize volatiles and chlorinated
effluents are less likely to contain viable pathogens.
Exposure to the wastewater during collection and its
use in the laboratory should be minimized. Inhalation
and dermal adsorption can be reduced by using
plastic gloves, laboratory aprons or coats, safety
glasses, respirators, and laboratory hoods. Further
guidance on health and safety for toxicity testing is
described in Walters and Jameson (1984).
In addition to taking precautions with effluent
samples, a number of the reagents that might be
used during Phase II toxicant identification and Phase
III toxicant confirmation studies are known or
suspected to be very toxic to humans. Analysts
should familiarize themselves with safe handling
procedures for these chemicals (DEHW, 1977;
OSHA, 1976). Use of these compounds may also
necessitate specific waste disposal practices.
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Section 3
Quality Assurance
3.1 General
Quality assurance is composed of two aspects,
quality verification and quality control. Quality
verification entails a demonstration that the proposed
study plan was followed as detailed and that work
carried out was properly documented. Some of the
aspects of quality verification include chain of custody
procedures, statements on the objective of the study
and what is known about the problem at its outset,
instrumental log books, and work assignments. This
aspect of quality assurance ensures that a "paper
trail" is created to prove that the work plan has been
covered completely. The quality control aspect of
quality assurance involves the procedures taking
place such as the number of samples to be taken and
the mode of collection, standard operating procedures
for analyses, and spiking protocols.
No set quality assurance program can be dictated for
a TIE; the formula to a successful study will be
unique to each situation. However, adherence to
some general guidelines in formulating a Quality
Assurance Plan (QAP) may increase the probability of
success.
In preparing a QAP, enough detail should be included
so that any investigator with an appropriate
background could take over the study at any time.
Cross checking of results and procedures should be
built into the program to the extent possible. Records
should be of a quality that can be offered as evidence
in court. Generally, the QAP should be provided in a
narrative form that encourages users to think about
quality assurance. To be effective, the QAP must be
more than a paper exercise simply restating standard
operating procedures. It must increase
communication between clients, program planners,
field and laboratory personnel and data analysts. The
QAP must make clear the specific responsibilities of
each individual. The larger the staff, the more
important this becomes. While QAPs may seem to be
an inconvenience, the amount of effort they require is
commensurate with the benefits derived.
3.2 TIE Quality Control Plans
A successful TIE is dependent upon a strong quality
control program. Obtaining quality TIE data is more
difficult because the constituents are unknown in
contrast to quality control procedures for a standard
analytical method for a specific chemical. In such an
analysis, one knows the characteristics of the analyte
and the implications of the analytical procedure being
utilized. Without knowledge of the physical/chemical
characteristics of the analyte, however, the impact of
various analytical procedures on the compound in
question is not known. Further, quality control
procedures are specific to each compound; quality
control procedures appropriate to one analyte may be
completely inappropriate to another.
The problem of quality control is further exaggerated
because quality control procedures for aquatic toxicity
tests may be radically different from those required
for individual chemical analyses. This additional
dimension to quality control requires a unique
framework of checks and controls to be successful.
The impacts of chemical analytical procedures on
sample toxicity must be included. Likewise,
procedures used to insure quality toxicity test results
should not impact chemical analyses. For example, in
performing standard aquatic toxicity tests, samples
with low dissolved oxygen are usually aerated. This
practice may, however, result in a loss of toxicity if
the toxicant is volatile or subject to oxidation.
3.3 Cost Considerations/Concessions
The quality control practices required in any given
experiment must be weighed against the importance
of the data and decisions to be based upon it. The
crucial nature of certain data will demand stringent
controls, while quality control can be lessened in
other experiments having less impact on the overall
outcome.
Effluent toxicant identification evaluations require a
large number of aquatic toxicity tests. The decision to
use the standard toxicity test methods described in
Peltier and Weber (1985) (involving a relatively high
degree of quality control) must be weighed against
the degree of complexity involved, the time required
and the number of tests performed; all of these affect
the cost of testing. For this reason, toxicity tests used
in the early phases of the evaluation generally do not
follow this protocol, nor do they require exacting
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quality controls because the data are only preliminary.
Phase I, and to a lesser extent, Phase II results are
more tentative in nature as compared to the tests
performed for the confirmation of the effluent
toxicant(s) in Phase III.
The progression towards increasingly definitive results
is also reflected in the use of a single species in the
initial evaluation studies and multiple species in the
later stages. The use of several species of aquatic
organisms to assure that effluent toxicity has been
reduced to acceptable levels is necessary because
species have different sensitivities to the same
pollutant. Quality control must relate to the ultimate
goal of attaining and maintaining the designated uses
of the receiving water. For this reason, final effluent
test results must be of sufficient quality to ensure
ecosystem protection. The use of dilution water in
toxicity tests which mimics receiving water
characteristics will help to ensure that the effluent will
remain non-toxic after being discharged into the
environment. In addition, it is essential that the
variability in the cause of effluent toxicity be defined
during the course of the TIE so that appropriate
control actions provide a final effluent safe for
discharge.
3.4 Variability
The opportunities to retest any effluent to confirm the
quality of initial results will be limited at best. In
addition to the shifting chemical and toxicologic
nature of the discharge over time, individual effluent
samples stored in the laboratory change. Effluent
constituents degrade at unknown rates, as each
compound has its own rate of change. The change in
a sample's toxicity over time represents the
cumulative change in all of the constituents, plus that
variation resulting from experimental error. Some
guidelines for assessing and minimizing changes in
sample chemistry and toxicity are discussed in
Sections 6 and 8. Regardless of the precautions
taken to minimize sample changes, a sample cannot
be retested with certainty that it has not changed.
3.5 Intra-Laboratory Communication
Quality control procedures in chemistry and biology
can be quite different. For example, phthalates are a
frequent analytical contaminant requiring special
precautions that are not of toxicological concern. The
toxicological problem presented by the zinc levels
typically associated with new glassware are of no
concern to those performing organic analyses. The
difference in glassware cleanup procedures is an
example of many differences that must be resolved.
Cleaning procedures must be established to cover the
requirements of both. Time schedules for analyses
must be detailed in advance. One cannot assume
compound stability; therefore, time delays between
the biological and chemical analysis of a sample
cannot be tolerated.
3.6 Record Keeping
Throughout the TIE, record keeping is an important
aspect of quality verification. All observations,
including organism symptoms, should be
documented. Details that may seem unimportant
during testing may be crucial in later stages of the
evaluation. Investigators must record test results in a
manner such that preconceived notions about the
effluent toxicants are not unintentionally reflected in
the data. TIEs required by state or federal pollution
control agencies may require that some or all records
be reviewed.
3.7 Phase I Considerations
Effluent toxicity is "tracked" through Phases I, II and
III using aquatic organisms. Such tracking is the only
way to detect where the toxicants are until their
identity is known. The organism's response must be
considered as ground truth. Therefore, the toxicity
test results must be dependable. System blanks
(blank samples carried through procedures and
analyses identical to those performed on the effluent
sample) are used extensively throughout the TIE in
order to detect toxic artifacts added during the
effluent characterization manipulations. With the
exception of tests intended to make the effluent more
toxic, or situations in which a known amount of
artifactual toxicity has been intentionally added,
sample manipulation should not cause the effluent
toxicity to change.
There are many sources of toxicity artifacts in Phase
I. These include: excessive ionic strength resulting
from the addition of acid and base during pH
adjustment, formation of toxic products by acids and
bases, contaminated air or nitrogen sources, poor
mixing of test solutions, contaminants leached from
filters, pH probes and SPE columns, the reagents
added and their contaminants. LC50 data for several
reagent chemicals and common aquatic test
organisms are provided as needed in subsequent
sections of this document.
Frequently toxic artifacts are unknowingly introduced.
For example, pH meters with refillable electrodes can
act as a source of silver which can reach toxic levels
in the solutions being measured for pH. This is
especially a problem where there is a need to
carefully maintain or track solution pH. Using pH
electrodes without membranes avoids the silver
problem which can only be detected by profuse use
of blanks.
Oil in air lines or from compressors is a source of
contamination. Simple aeration devices, such as
those sold for use with aquaria are better as long as
3-2
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caution is taken to prevent contamination of the
laboratory air which is taken in by the pump.
Worst case blanks should be used to better ensure
that toxicity artifacts will be recognized. Test
chambers should be covered to prevent
contamination by dust and to minimize evaporation.
Since small volumes are often used, evaporation
must be controlled. Plastic disposable test chambers
are recommended to avoid problems related to the
reuse of test chambers. Cups from the same lot
should be spot-checked for toxicity.
Glassware used in various tests and analyses must
be cleaned not only for the chemical analyses but so
that toxicity is not caused either by other
contaminants or by residues of cleaning agents.
Since the organisms are sensitive to all chemicals at
some concentrations, all toxic concentrations must be
removed and not just those for which analyses are
being made.
Randomization techniques, careful observance of
organism exposure times and the use of organisms of
approximately the same age ensure quality data.
Standard reference toxicant tests should be
performed with the aquatic test species on a regular
basis and control charts should be developed (Peltier
and Weber, 1985). During Phase I it will not be known
how much the toxicity of the reference toxicant varies
over time compared to the toxicant(s). When the
toxicants are known, they should be used as the
reference toxicant. Reference toxicant tests should be
performed to coincide with the TIE testing schedule.
3.8 Phase // Considerations
In Phase II, a more detailed quality control program is
required. Interferences in toxicant analysis are for the
most part unknown initially but as toxicant
identifications are made, interferences can be
determined. Likewise instrumental response, degree
of toxicant separation, and detector sensitivity can be
determined as identifications proceed.
3.9 Phase III Considerations
In Phase III of a TIE, the detail paid to quality control
and verification is at the maximum. This phase of the
study responds to the compromises made to data
quality in Phases I and II. For this reason, confidence
intervals for toxicity and chemical measurements
must be calculated. These measurements allow the
correlation between the concentration of the toxicants
and effluent toxicity to be checked for significance
based on test variability. Effluent manipulations prior
to chemical analysis and toxicity testing are minimized
in this phase in an effort to decrease the chance for
production of artifacts. Field replicates to validate the
precision of the sampling techniques and laboratory
replicates to validate the precision of analyses must
be included in the Phase III quality control program.
System blanks must be provided. Calibration
standards and spiked samples must also be included
in the laboratory quality control program. Because an
attempt will be made to correlate effluent toxicity to
toxicant concentration, spiking experiments are
important in determining recovery for the toxicant(s).
These procedures are feasible in this phase of the
study because the identities of the substances being
measured are known.
The toxicants being analyzed can be tested using
pure compounds, alleviating the need for a general
reference toxicant. Because the test organism also
acts as an analytical detector in the correlation of
effluent toxicity with toxicant(s) concentration,
changes in the sensitivity of the test organisms must
be known. This is best achieved by using the same
chemicals identified for the reference toxicants.
3-3
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Section 4
Facilities and Equipment
The facilities, equipment and reagents needed to
perform an effluent TIE will depend on the phase of
the study and the characteristics of the toxicant(s).
The equipment required for Phase I characterization
tests can be found throughout Section 8. The facility
and equipment needs in Phase II of the TIE will be
site-specific and will depend both on the
physical/chemical characteristics of the toxicants and
on the choice of the Phase II approach.
Phase I requires only basic analytical and toxicity
testing equipment which would be available in most
laboratories doing toxicity tests and performing the
usual water chemistry analyses. Phase III
requirements are largely limited to equipment found in
a typical toxicity testing lab and equipment necessary
for the analyses of the toxicant(s).
Because of the equipment needs and time required to
conduct the evaluations, complete on-site effluent
TIEs using a mobile laboratory are generally not
feasible. Measurement of the loss of toxicity over time
in several effluent samples will provide information
upon which to base acceptable storage times.
Usually, with modern rapid sample shipment methods,
off-site work is practical. The cost of shipment is
usually far less than the cost of on-site work.
Large numbers of organisms and many tests are
needed for TIE tests. Ready availability of test
organisms is important because often needed tests
are not predictable. Only after the results of one
experiment are known, can the next test be planned.
It is probably more economical to culture many of the
test species that might be used in TIEs than it is to
purchase them. A delay in testing caused by
shipment time or lack of availability of test organisms
could cost far more in work loss than it would cost to
maintain cultures for many weeks.
4- 1
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Section 5
Dilution Water
The choice of dilution water will change with the
purpose of the tests and therefore the choice will
often be more varied in Phases I and II than in Phase
III. Particularly for some toxicant groups in Phase II,
some very unusual dilution water is recommended in
order to achieve the desired chemical conditions.
Sometimes the water may in itself be toxic! Such
conditions are foreign to conventional toxicology and
rightly so, but this is not conventional toxicology.
Much of Phase I and parts of Phase II utilize organism
tolerance and relative toxicity to accomplish the
objectives of the study. Methanol, hydrogen ion
concentration and osmotic pressure may sometimes
be near lethal levels in order to test necessary
conditions. In some cases, the dilution medium may
cause complete mortality in 48 hours, but the point of
interest is whether treatment causes more rapid
mortality. If so, one can say that one condition is
more toxic than another and obtain important
information from the test. The key is to run sufficient
numbers of system blanks so that the relative
contribution to mortality is known and toxicity is not
attributed to an incorrect cause. These are examples
of the previous statement that these methods "utilize
tolerance and relative toxicity." In reality this approach
is very much like comparison of the toxicity of two
chemicals, A and B. If one determines LC50s for A
and B and concludes that A is twice as toxic as B,
lethal conditions are being compared in order to say
this. Controls are not involved in the LC50 calculation
and high control survival does not change the data
interpretation. The same concept of relative toxicity is
used here. Chemical "A" is the blank and chemical
"B" is the treated sample and the question is, "which
is more toxic?".
Where these methods are built on tolerance, chronic
toxicity endpoints cannot be used and that is why
these methods are intended only for acute toxicity.
Obviously, if one wants to measure chronic effects,
the test organisms must be able to live long enough
to display chronic effects. Many of the pH changes
and other manipulations used in these methods would
not allow sufficient survival time or health for
reproduction. Attention would also have to be given to
acclimation, feeding and general living conditions.
Because of these factors, choice of dilution water in
Phase I is of much less concern both because these
are acute tests and because of the many additives
used which change the mixture much more than the
dilution water changes it. In general for Phase I, any
water which is of consistent quality and will support
growth and reproduction of the test species is
suitable. In Peltier and Weber (1985), a variety of
dilution water choices is provided and any of these
may be used for TIEs.
In Phase III, where the objective is to confirm the true
cause of toxicity, where artifacts are to be excluded
to the extent possible and where absolute toxicity is
more important than relative toxicity, practices,
including choice of dilution water, need to follow
conventional toxicological methodology. Tolerance
must not be necessary in order to provide the desired
response. Attention must be given to simulation of the
dilution water into which the effluent is discharged.
Some toxicant dose response relationships may be
totally different as the water quality characteristics
changes. These factors must be incorporated into
Phase III where absolute toxicity is of the utmost
concern. In Phases I and II, only relative differences
are being considered.
Perhaps a cautionary note is warranted regarding the
effects of dilution water on effluent toxicity. If high
concentrations of effluent are being tested (e.g.,
80%) the physical/chemical characteristics will
resemble those of the effluent. If low concentrations
are tested (e.g., 5%) then characteristics will
resemble those of the dilution water.
Little specific information can be given about the
selection of dilution water in Phase I and II except that
the desired tested conditions will often dictate its
characteristics. For example, in Section 8.6, the same
column used for the blank may not be usable for the
effluent sample if receiving water is used as the
dilution water. Secondly, sufficient numbers of blanks
must be included to interpret the results. In Phase III,
the choice should be based on the receiving water
where the discharge occurs.
5- 1
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Section 6
Effluent Sampling and Handling
A wastewater sample may be representative only of
the discharge at the time of sampling. In effect, each
sample is a "snapshot" of the effluent's toxicological
and chemical quality over time. To determine whether
any effluent sample is typical of the wastewater may
require the collection of a large population of
samples. Further, what constitutes a "representative"
sample is a function of the parameter of concern.
Because effluents vary in composition, sampling must
be extensive enough that one is confident that the
groups of samples are representative of the discharge
over time. Guidelines for determining the number and
frequency of samples required to represent effluent
quality are contained in the "Handbook for Sampling
and Sample Preservation of Water and Wastewater"
(Berg, 1982). However, since this guidance is not
based on toxicity, it should be used with caution.
Both quantitative (change in .concentration) and
qualitative (change in toxicants) variability commonly
occur in effluents and both may affect toxicity.
Changes in effluent toxicity are the result of varying
concentrations of individual toxicants, different
toxicants, changing water quality characteristics
(affecting compound toxicity) and analytical and
toxicological error. Even if the toxicity of an effluent to
an aquatic organism is relatively stable, this does not
mean that there is only a single toxicant causing
toxicity in any given sample or among several
samples.
Determining whether a sample is typically toxic is not
as simple as comparing the conventional pollutants of
the sample to long-term effluent averages. Effluent
toxicants often do not follow the same trends as
BOD, TOG and TSS. The toxicant(s) may be present
at such a low level that it does not significantly affect
the quantity of the conventional pollutant, even
though it is present in toxic concentrations.
Conventional parameters, BOD, TSS, and other
pollutants limited in the facility's NPDES permit, will
provide an indication of the operational status of the
treatment system on the day of sampling. For
industrial discharges, information on production levels
and types of operating processes may be helpful. The
condition of the facility's treatment system at the time
of sampling should be determined by the individual
collecting the sample. The type of sample, time of
collection, and other general information on the facility
should be recorded. An example of a page of a log
book is given in Figure 6-1.
Upon the arrival of the sample in the laboratory,
temperature, pH, toxicity, hardness and conductivity
should be measured. Total residual chlorine, total
ammonia, alkalinity, dissolved oxygen (DO), and
organic carbon measurements may also be
appropriate. Toxicity should be measured periodically
during storage to document any changes.
Investigators should not be surprised to find that well
operated municipal and industrial treatment systems
discharge unacceptably toxic wastewaters. Effluent
guideline-based limits reflecting best achievable
technology, do not prescribe limits for more than a
few chemicals. Many compounds present in effluents
are not regulated because the discharger is not
required to report their presence in permit
applications or they cannot be detected using typical
methods for wastewater analysis.
For chlorinated effluents, whether sampling should be
done before chlorination depends on the question to
be answered. Sometimes the question may be
whether there are toxicants other than chlorine
present. Dechlorination prior to toxicity character-
ization may be needed in order to distinguish toxicity
from causes other than chlorine. Usual methods of
dechlorination may remove more than toxicity from
chlorine alone and careful data interpretation is
needed to understand the results. Toxicity from more
than one cause is often not additive in effluents, so
relative contributions from two or more causes can be
very hard to decipher.
The choice of grab or composite samples will depend
on the specific discharge situation, (e.g., plant
retention time) questions to be answered by the TIE
and the stage of the TIE. In Phase I testing, samples
that are very different from one another give results
that are difficult to interpret; therefore composite
samples are more similar and are easier to use. In
Phase III, effluent variability is used to advantage;
therefore, grab samples are often best. If toxicity is
low or intermittently present, grab samples may be
6- 1
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Figure 6-1. Example data sheet for logging in samples.
Sample Log #:
Date of Arrival:
Facility: _
NPDES #:
Location:
Contact:
Phone Number:
Sampler.^
Sample Type:
Grab
Collected,
Date
AM/PM
AM/PM
Composite
Collected From_
Date
To
AM/PM
AM/PM
Date
Sample Conditions Upon Arrival:
Temperature
PH
Total Alkalinity_
Total Hardness
Conductivity/Salinity _
Chlorine
Total Ammonia
Total Organic Carbon_
Condition of treatment system at time of sampling:
Status of process operations/production (if applicable):
Comments:
6-2
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best during all phases. The additional difficulty of
getting flow proportional samples should be balanced
against their advantage in each situation. While grab
sampling may provide maximum effluent toxicity, it is
more difficult to catch peaks in toxicity and Phase I
sampling may require more time.
Peltier and Weber (1985) have discussed the
advantages and disadvantages of grab and composite
sampling and have also detailed methods for
sampling intermittent discharges.
If the TIE analyses are not conducted on-site,
samples must be shipped on ice to the testing
location. Effluent samples should not be filtered prior
to testing unless it is necessary to remove other
organisms. Sample filtration could affect the results of
the characterization tests, one of which entails
filtering the effluent. Sample aeration should also be
minimized during collection and transfer. Initial sample
analysis should begin as soon as practical after
effluent sampling. Phase II and especially Phase III
may require specific types of sample containers or
the addition of preservative to aliquots of sample
designated for chemical analyses. For a single Phase
I test series, 3.5 liters of effluent are needed for
analysis if test organisms such as daphnids or newly
hatched fathead minnows are used. The exact
volume required depends on the toxicity of the
effluent and to a lesser extent, the test options
chosen (cf., Section 8). For other species different
volumes may be necessary. Volumes used in each
characterization test are supplied in Table 6-1.
The extent of the analyses carried out on any
individual sample must be weighed against the cost of
additional sampling, the stability of the sample,
sample representativeness and the need to have
samples of different toxicity. Clearly, the resources
required for such TIEs are too great to expend on a
single sample or on a few samples which do not
represent the effluent. Likewise, there is not a set
number of samples which should be analyzed in
Phase I, II or III before going on to subsequent
phases of the study or taking final measures to
control effluent toxicity. The number of samples
analyzed in each phase will be a function of the
apparent variability in the effluent, the number of
toxicants, how persuasive the data are, the cost of
the remedial action, regulatory deadlines and finally,
the success of each study phase.
Table 6-1. Volumes Needed for Phase I Tests
Characterization Step
Volume for
Each Step
Total
Volumes1 (ml_)
Chemical analyses -2 <1000
pH 3 Adjustment 30 - 300
filtration 235
solid phase extraction 200
aeration 35
pH 11 Adjustment 30 —300
filtration 235
solid phase extraction 3 200
aeration 35
Unadjusted pH effluent (pH|)
initial test
baseline
filtration
solid phase extraction
aeration
EDTA chelation
oxidant reduction
Gradual pH changes
pH6
pH7
pH 8
40
80
235
200
35
100
100
40-500
40-500
40-500
-590
-120-1500
1 Total volume is — 3.5 L; this is maximum needed, does not
include subsequent testing.
2 Amount for this step is dependent on effluent
characteristics.
3 The pH is readjusted to pH 9 before it is put through the
column.
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Section 7
Toxicity Tests
7.1 Principles
Acute lethality tests with aquatic organisms are
utilized throughout the toxicity characterization
procedures described in this manual as well as in
Phases II and III. Using toxicity for such evaluations is
logical since toxicity triggers the TIE requirement. In
these tests the organism acts as the "detector" for
chemicals causing effluent toxicity. As such, they
provide the true response regardless of the outcome
of other analyses. The toxicity test is the only
analytical tool that can be used to measure toxicity.
Until the cause of toxicity is known, chemical
methods cannot be used to identify and quantify the
toxicants.
There are a number of consequences associated with
this reliance on toxicity. The organism responds to
every constituent, provided that it is present above a
threshold level either individually or collectively if the
constituents are additive. While this general response
to any compound presents an advantage as a broad
spectrum test for toxicants, it requires considerable
effort to determine the primary cause of toxicity
because it is not specific. This non-specific
response necessitates a generic chemical/physical
characterization of toxicants during Phase I testing
before specific identification is begun in Phase II.
A further repercussion of this universal response is
the probability of artifactual toxicity. Because the
analyst is reliant upon the organism's ability to track
toxicity throughout the effluent characterization steps,
sample manipulations are constrained. While
characterizing the effluent, no manipulation can
change the toxicity of the sample in an unpredictable
manner. "Toxicity-blanks and controls" are helpful
but the difficulties associated with them are far
greater than those connected with chemical analyses
because of their non-specificity. As a result many
more blanks and controls are employed in TIE testing
than in chemical analyses or standard toxicity testing.
Negative blank toxicity cannot be assumed regardless
of past results. Quite unexpected sources of
artifactual toxicity will occur in the course of
conducting an evaluation.
7.2 Test Species
Just as different analytical methods have different
detection levels for the same chemical, different
species have different sensitivities to the same
toxicants. The major difference is that the toxicity
measurement is non-specific to chemicals and so in
an unknown mixture (an effluent) one must determine
whether a different toxicity value for the effluent is
caused by different sensitivity to the same toxicant or
to different toxicants.
The choice of species to use for the toxicity test can
change the conclusion reached. In addition to the
obvious need to use species of an appropriate size,
age and adaptability to test conditions, there are other
important considerations. An effluent toxic to two
species, having equal or different LC50s may be toxic
because of different toxicants. Differences of 1.000X
in sensitivity are common and differences of 10,OOOX
occur among species exposed to a single chemical.
Anyone involved in identifying the cause of toxicity of
an effluent will be concerned when someone has
found the effluent toxic to some organism. If that is
not the case, before a TIE is begun, one should
determine to which organisms the toxicity concern is
directed.
Many effluents will be received for TIEs because they
have been found toxic to Ceriodaphnia or Daphnia,
species well suited to TIE methods. TIE test species
selection is obvious in these instances. Where toxicity
concern is based on species (trout or mysids), that
are not going to be the TIE test species, one must
demonstrate that the toxicity of concern has the same
cause as the toxicity manifested by the species to be
used in the TIE. The difficulty depends on the effluent
characteristics (especially toxicity variability), the
number of TIE steps which affect toxicity and the
difference in sensitivity between the species being
compared. Since this problem has not been one we
have experienced frequently, our suggestions are
certainly not all inclusive. The final confirmation
(Phase III) methods are designed to show whether the
wrong toxicant was identified. However, many
resources may be consumed before reaching that
7 - 1
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stage and earlier assurances should be obtained if
reasonable, to save time and cost.
One approach is to compare the LC50 values of
whole unaltered effluent samples for the species
originally raising the toxicity concern and the selected
species for the TIE. If the acute toxicity varies
similarly for each species among samples then there
is evidence that the two species are responding to
the same toxicant(s). If the LC50 values vary
differently for the two species, there is evidence that
the toxicants are different. If the LC50 values among
samples do not vary more than the precision of the
test method this approach is useless for that effluent.
If in Phase I, several steps (e.g., pH decrease,
aeration, solid phase extraction) all changed toxicity,
and if the direction and relative magnitude of change
was the same for both test species, then there is
evidence that both are sensitive to the same toxicant.
If one or more parameters are different, the evidence
is strong that the toxicants are different. This is not to
say that if a Phase I technique completely removes
toxicity to one species, it will remove it to the same
extent for the other species as well. Because different
species have dissimilar sensitivities to the same
chemical, removal of 90% of a compound in an
effluent sample may lead to a non-toxic
concentration to one species while only reducing the
toxicity to another species. If the Phase I procedures
that successfully remove or reduce effluent toxicity
differ by test species, it is unlikely that toxicity is
caused by the same chemical(s).
Symptoms are useful, especially if one is comparing
like organisms. Comparing fish symptoms to Daphnia
symptoms could be very misleading but comparing
Daphnia magna symptoms to those of Ceriodaphnia
dubia symptoms should be relatively safe. If one finds
like symptoms, the evidence is not convincing
because many toxicants cause specific symptoms but
if symptoms are distinctly different, the evidence is
strong that the toxicants are different. This is true
only when symptoms are compared at effluent
concentrations that are the same multiple of the LC50
for each species. For example, if two species have
LC50 values of 10% and 90%, comparing symptoms
at 100% concentrations could be misleading. At
100% effluent, the species with an LC50 of 10%
might go through the symptoms so fast, or skip some
symptoms, that they would appear completely
different from those of the less sensitive species.
Experience will reveal additional techniques that can
be used.
Freshwater discharges to saline receiving water
require separate considerations. Sea salts can be
added to raise the salinity of the effluent enough so
that marine species can be used in the TIE. However,
the tolerances of marine organisms to the additives
and effluent manipulations have not been determined.
To do so is costly and time consuming and a more
efficient method may be to use a freshwater species
in Phase I and II. If this is done, data must be
gathered to show that the freshwater species chosen
is sufficiently sensitive and is responding to the same
toxicant(s) as the marine species. The principles of
doing this are the same as described above for
different freshwater species. When Phase III is
reached, marine species should be used, but in that
phase, manipulations and additives are minimal and
little ancillary data are needed in order to use marine
species.
For discharges, with conductivities comparable to
brackish or marine water, caution is in order. Most
methods for measuring "salinity" (conductivity or
refraction) are non-specific for NaCMhe principal
component of sea water. Marine organisms
accomplish osmotic regulation by regulating sodium
and chloride. If salinity of an effluent is not caused by
NaCI, marine species may be stressed as much as
freshwater species by high concentrations of other
dissolved salts. Unless the "salinity" of an effluent is
known to be caused by NaCI, marine species cannot
be used to avoid the salinity effects.
7.3 Toxicity Test Procedures
The purpose of the toxicity test in Phase I is the
same as that of any analytical method~to measure
(detect) the presence of the toxicants. This use is
quite different than conventional toxicity testing where
the objective is to accurately and quantitatively
measure the sensitivity of the organism to known
concentrations of a chemical or effluent. For this latter
purpose, removing stress (due to low dissolved
oxygen (DO), other contaminants, and lack of space)
is important because such stresses may change the
sensitivity of the organism to the contaminant of
concern. In Phase I, relative sensitivity is used, that
is, we compare whether one condition is more or less
toxic than another but both may be toxic. Therefore,
concern of documenting and/or removing other
stresses is not very important. It is important to be
sure that these other stresses are similar for each
condition being compared.
The reason for this discussion under test methods is
that effort must be made to make the tests used in
Phase I as cheap as possible because for some
effluents, large numbers of tests may be needed. For
example, we have used more than 100 tests on some
effluents in Phase I. If the effort usually expended
measuring water chemistries had been done on these
tests, the cost would have been prohibitive. The
reader may wonder whether data collected from such
tests can be trusted. Confidence in the data hinges
on careful assurance that the stresses are similar
among comparisons. For example, it does not matter
if the test organisms are acclimated to a pH change.
7-2
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It does matter that stress from lack of acclimation to
pH change occurs in each treatment compared.
Sometimes, in order to achieve desired chemical
conditions, the stress from pH change cannot be
made uniform. In these situations, only gross
differences in response may be dependable. In some
cases, erroneous conclusions will be reached. While
these may cause wasted effort, the error should be
found in Phase III. That is why, in Phase III, careful
quality control must be exercised and cost saving
shortcuts are not acceptable because one of the
several purposes of Phase III is to catch errors or
artifacts that may creep into Phases I and II.
One need not use standard acute methods in Phase I
for these reasons. The following mechanics of
performing an acute test with cladocerans and newly
hatched fathead minnows has been found by
experience to be very cost effective and is offered as
an aid to those doing Phase I testing. Specific
volumes and sizes are used in this example for
simplicity but, of course, these are varied depending
on each test purpose.
For example, arrange a set of 12 plastic cups into six
pairs. Fill 10 cups with 10 mL of dilution water using a
disposable pipette. Add 10 ml of effluent to the two
empty cups to make the high concentration, (e.g.,
100%). Add 10 ml of effluent to the next pair of test
cups already containing 10 ml each of the dilution
water (Figure 7-1). The resulting concentration is
50%. From each cup of the 50% solution, transfer 10
mL to the third pair of test cups to produce the 25%
concentration. Continue this process until sufficient
exposure concentrations have been prepared. One
cup in the series contains only dilution water and
serves as the control. Mixing the solutions prior to
aliquot transfer is very important. This can be
accomplished by drawing the solution into the pipette
and discharging it back into the cup several times
prior to transfer. Additional mixing of test solutions
should be done for experiments in which reagents
such as Na2S203 and EDTA (Phase I), or effluent
fraction solvent concentrates (Phase II) are added to
effluent or dilution water.
The need for duplicates will depend on the accuracy
and precision required of the test results. Tests
requiring a measure of accuracy in the form of
confidence intervals (CIs) should be run in duplicate.
Tests designed to provide only an indication of
positive or negative toxicity need not be run in
duplicate. Beyond the Initial and Baseline effluent
toxicity tests (Sections 8.1 and 8.2), designed to
define effluent toxicity upon arrival in the laboratory
and periodically during testing, respectively, Phase I
toxicity tests usually do not require preparation in
duplicate.
The test organisms of uniform age should be
randomly placed in each test cup to better insure
valid results. Because the volume of test solution may
be small, care must be taken to minimize the volume
added during test organism transfer. If the volume of
water transferred with the organism is reduced to a
drop (50 pL), only five organisms are added to the
test chamber and a 10 mL test volume is used, the
resulting change in test solution volume will be 2.5%.
Minimizing the change in volume is more critical as
test solution volume is reduced. Such situations may
be encountered in Phase II studies, when limited
volumes of effluent fraction concentrates may
necessitate the testing of cladocerans in volumes of
even 1 mL or less.
We have stressed a relaxation of the usual water
chemistry requirements in these Phase I tests
because they are not as necessary here as they are
in Phase III. However, sometimes, in order to maintain
the desired conditions in the test (such as maintaining
a specific pH) frequent specific measurements will be
necessary. The distinction drawn here is to avoid
measurements you don't need (e.g., sample
hardness) and concentrate on those that are
important (e.g., pH). Effluents are often well buffered
and pH will change sometimes quickly if equilibrium is
not already established. POTW effluents are not in air
equilibrium when discharged and as soon as they are
exposed to air, the pH will rise. A typical POTW
effluent pH is 7.2-7.4 when discharged but it will
equilibrate after contact with air and hold at 8.2-8.5.
If pH is important to test interpretation, pH must be
monitored throughout the test.
7.4 Test Endpoints
Little effort should be expended in calculating LC50
values for Phase I toxicity tests. There is no use in
applying sophisticated and complex programs to test
results. A number of methods of estimating the LC50
from the acute toxicity data are described in Peltier
and Weber (1985). A method which is most easily
and quickly applied to the data should be used. In
many cases, the graphical method entailing
interpolation may prove to be the most convenient.
Differences resulting from the choice of data analysis
method should not impair the outcome of Phase I
studies. Phase III tests may require more
sophisticated analyses.
Toxic units (TU) have a special utility in some parts of
a TIE. For specific chemicals the TU is equal to the
concentration of the compound present in the effluent
divided by the LC50 of the compound. The TU of
whole effluent is 100% divided by the LC50 of the
effluent. For example, if the 48 hour LC50 of
compound A is 3 mg/L, a solution of 1 mg/L of this
compound contains 0.33 TU. If the LC50 of an
effluent is 25%, the effluent contains 4 TU (100/25).
By normalizing the concentration term to a unit of
7-3
-------
Figure 7-1. Schematic for preparing effluent test concentrations using simple dilution techniques.
Effluent
• Add 10 mL to each replicate
for the high concentration.
• Add 10ml_to next
concentration
Dilution
Water
• Add 10 mLto each
replicate except in
the high concentration
High
Cone
Waste
toxicity (such as the LC50), the TU allows the toxicity
of chemicals and/or effluents to be "summed,"
provided that the test length and species used are the
same in every test. This cannot be done using LCSOs
because chemicals and effluents each have different
toxicity, and different concentrations each equal one
LC50. Phase III contains more discussion about
adding toxic units; however one must be cautious in
summing toxic units. Unless toxicants are strictly
additive, simple summation of toxic units will be
incorrect.
7.5 Feeding
Most species used in acute testing are not fed during
the test. In our use of Ceriodaphnia, especially in
Phase II, it was necessary to perform tests in water
with very low dissolved solids. In such water, very
small concentrations of contaminants (e.g., metals
from glassware) can be toxic. Therefore, we routinely
feed all animals in test exposures at the beginning of
the tests. This includes the 100% effluent. This
practice has become standard in all three phases.
The decision will be species specific and dependent
on the characteristics of the effluent. Consistency
throughout the three phases is most important. All of
the data for Ceriodaphnia given in tables in Section 8,
are based on tests in which animals were fed.
7-4
-------
Section 8
Phase I Toxicity Characterization Tests
The first phase of a TIE involves a characterization of
the toxic effluent. The characterization information
gathered in Phase I forms the basis and direction for
Phase II identification of the specific toxicants or may
be useful for treatability evaluations. In Phase I,
simple toxicity removal or alteration is performed on
the whole effluent. Acute toxicity tests utilizing aquatic
organisms are used to determine whether the toxic
chemicals have certain physical or chemical
characteristics. Two objectives are accomplished
during the toxicity characterization phase: a) the
physical and chemical characteristics of the
toxicant(s) are broadly defined and b) some
information is gathered to indicate whether the
toxicants are similar in effluent samples taken over
time. For ammonia and chlorine, Phase I results may
be rather convincing that they are the cause of
toxicity but otherwise Phase I only provides evidence
of groups of chemicals that may be the toxicants.
This information can subsequently be used in the
second phase of the study, either in the development
of bench-scale wastewater treatment processes
(EPA, 1988A; 1988B) or in choosing separation and
analytical procedures for toxicant identification as
described in Phase II.
The tests described in this section are designed for
acutely toxic effluents. Methods for chronic toxicity
have not yet been developed. The methods in this
section are based on the use of small test organisms.
If larger species are used modifications to these
methods will have to be made.
Analysis of samples should begin as soon as practical
following collection. Until experience is gained with
the effluent, there is no way to predict how long
samples can be stored before substantial changes in
toxicity occur. In the laboratory as well as in transit,
the bulk effluent should be held near 4°C and kept
headspace free. Those characterization solutions
(Sections 8.3-8.6) held prior to serial dilution and
toxicity testing should also be kept at 4°C and
covered to minimize loss by evaporation. If the test
organism to be used is sensitive to supersaturation,
then supersaturation must be removed. Ceriodaphnia
are not very "sensitive" to such situations but newly
hatched fathead minnows are. Once in the laboratory,
testing on individual samples may continue indefinitely
provided that whole effluent toxicity stabilizes. The
degree of toxicity can remain similar, but the cause of
toxicity can change with age. Especially in the early
stages, fresh samples should be used regardless of
toxicant stability.
The degree to which any single sample is analyzed
should be weighed against the cost of the analyses
and the probability that the sample is an adequate
representation of typical effluent. Obviously, when
several samples show that a single class of
compounds is responsible for effluent toxicity, Phase
II procedures should be initiated.
Each of the characterization tests described in
Section 8 is designed to change toxicity of groups of
constituents (Figure 8-1). Toxicity before and after
the characterization treatment will indicate for which
groups the toxicity was changed. All but one of these
tests is performed at the same time in order to
minimize confounding effects resulting from sample
degradation over time. While it is not critical that each
characterization treatment be performed at exactly the
same time, the toxicity tests should be initiated at
approximately the same time. One species of test
organism should be used throughout the initial stages
of Phase I. Other species may be useful in the later
stages of Phase I.
For each day following receipt of the effluent sample
the various steps are given in Table 8-1. Day 1 is
when the sample arrives in the laboratory. On day 1
initial physical/chemical measurements are taken for
the effluent sample and an initial toxicity test is
conducted on an aliquot of the sample. This LC50 is
used to set the desired exposure conditions for
subsequent toxicity tests and is termed "initial"
toxicity to distinguish it from the "baseline" toxicity
described below. Other aliquots of the sample are
adjusted to pH 3 and 11, filtered, aerated and/or
chromatographed using a C-\Q solid phase extraction
(SPE) column. Following these manipulations, each
effluent aliquot is readjusted to the initial pH (pH|) of
the effluent. These aliquots and the remainder of the
effluent are then held at 4°C overnight. Delaying the
majority of the toxicity testing until the next day (day
2) allows the test exposures to be set at
concentrations bracketing the 24 hour LC50 of the
8- 1
-------
Figure 8-1. Overview of Phase I effluent characterization tests. (Note: pH( stands for initial pH.)
Baseline Toxicity
Test (Day 2)
Toxic Effluent Sample
Initial Toxicity Test
(Dayl)
Acid
PH|
Base
C-, 8 Solid Phase
Extraction Tests
(Day 2)
Acid
pH,
Acid
pH|
pH6
pH 7
pH8
day 1 initial toxicity test. This procedure also allows
pH adjusted effluent aliquots more time to stabilize,
and additional pH adjustments can be made as
necessary.
On the second day the aliquots of effluent prepared
on day 1 are diluted to 4X-, 2X-, 1X-, and 0.5X-
LC50 (24 hour) of the effluent and subsequently
tested for toxicity. This dilution series is used so that
for highly toxic effluents, smaller changes in toxicity
can be detected than would be the case if 100%
effluent was used. (See Section 9 regarding multiple
toxicants and this dilution series.) Other toxicity tests
involving the addition of chelating and reducing
agents and less severe pH adjustments are also
conducted. A second toxicity test is begun using
unaltered effluent, now 24 hours old. The results of
this and subsequent whole effluent tests are referred
to as the "Baseline" effluent LC50.
Toxicity test results are read on subsequent testing
days and depending on the outcome of the Phase I
test series, additional toxicity tests designed to further
define or confirm the nature of the toxicants are
conducted.
For an experienced analyst the amount of time
required to conduct the tasks scheduled for each day
is approximately one full day. If at 24 hours, less than
50% lethality of test organisms exposed to 100% day
1 effluent has occurred, the sample can be discarded
and a new sample collected with relatively little loss of
resources or time. Alternatively, the test can be
continued to 48 or 96 hours which may produce an
LC50. In such cases, the baseline toxicity tests
prepared on the second day (day 2) following sample
arrival are set up at exposure levels of 100%, 50%,
25%, 12.5%, 6.25% effluent.
Several Phase I characterization tests are relatively
broad in scope, intended to include more than one
class of toxicants. Therefore, if a significant change in
effluent toxicity is seen following these
characterization procedures, additional tests are
needed to further delineate the nature of the toxicity.
The amount of testing beyond the initial
characterization of the sample will depend on the
stability of effluent toxicity, the nature of the toxicity,
and previous Phase I results for the effluent (i.e.,
observed trends in the nature of the toxicity). A
"significant reduction" in toxicity between aliquots of
the day 2 whole effluent (Baseline LC50) and treated
effluent must be decided based upon the laboratory's
test precision. Usually a change in the LC50 equal to
one concentration interval can be considered
significant but when precision is good smaller
differences can be used. This suggestion is arbitrary
and should not replace good judgement and
experience. None of these tests by themselves are
conclusive, so the danger of type I or type II errors is
not great. Experience has shown that for many
effluents, at least one Phase I characterization test
will be successful in substantially altering effluent
toxicity. If not all toxicity is removed, other groups of
toxicants (not addressed by Phase I procedures) may
be present in the effluent or a single toxicant may be
8-2
-------
Table 8-1. Outline of Phase I Effluent Manipulations
Description Section
DAY 1 SAMPLE ARRIVAL
Chemical analyses 6.0
• temperature
• pH
• chlorine
• hardness
• alkalinity
• conductivity
• ammonia
• TOC
• DO
"Initial" toxicity test 8.1
Sample manipulation:
• pH adjustment (pH 3, pH(, pH 11) 8.3
• pH adjustment/filtration 8.4
• pH adjustment/aeration 8.5
• pH adjustment/Gig solid phase extraction 8.6
DAY 2 TOXICITY TESTING:
Warm effluent samples from Day 1 and set-up
toxicity tests
• baseline toxicity 8.2
• pH adjustment samples 8 3
• aeration samples 8.4
• filtration samples 8.5
• Gig solid phase extraction samples 8.6
• oxidant reduction samples 8.7
• EDTA chelation samples 8.8
• graduated pH samples 8.9
Read mortality on "initial" toxicity test 8.1
DAYS 3 AND 4 MONITORING TESTS:
Read 48 h mortality initial toxicity test 8.1
Read 24 h and 48 h mortality on all tests from Day 2 8.2-8.9
present in the effluent at such high concentrations
that only partial toxicity removal is achieved.
Additional testing to resolve these findings involves
applying the successful Phase I test at a higher level
(i.e., increased degradation time, increased aeration,
larger C\Q SPE column volume, increased reagent
concentrations).
Another outcome of the Phase I characterization test
series may be that several tests succeed in partially
removing effluent toxicity. In this situation, one may
be dealing with several toxicants, each with different
physical/chemical characteristics, or a single toxicant
of such a nature as to be removed by more than one
Phase I test. These results may be resolved by
treating a single aliquot of the sample with all of the
characterization tests that significantly reduced the
Baseline toxicity of the effluent. If effluent toxicity
removal is enhanced as compared to the reduction
provided by individual characterization tests, the
sample may contain more than one type of toxicant. If
the final toxicity removal at the end of the series of
characterization tests is approximately the same as
that provided by the most efficient single Phase I test,
then it is likely that all of the test methods involved
are successful in reducing the same toxicant to
varying extents. This outcome is also suggested
when one or more Phase I tests completely remove
toxicity while some number of other tests partially
reduce toxicity. Phase I tests overlap somewhat in
their abilities to remove groups of toxicants. For
example, increasing pH may cause a metal to
precipitate and EDTA may also remove its toxicity. In
any case, results of this nature are useful in selecting
Phase II options.
When several treatments are applied to the same
sample, tests must be designed to ensure that toxicity
does not result from the additives used (acid, base,
EDTA) rather than from the effluent's toxicants. The
assumption must not be made that toxicants are
either additive or synergistic. Our experience shows
that independent action (one or more of multiple
toxicants act independently of the rest, as though the
others were not present) is not uncommon in
effluents. Experience also shows that one should not
use selected tests to confirm a suspicion that a
certain toxicant is the cause of toxicity. Time and
again, this leads to wasted effort. There are so many
possible causes of toxicity that such guesses are
rarely the helpful and more often channel thinking and
delay final solution. On the other hand, if one wants
only to know whether a certain chemical is the
toxicant, these tests can be selected to accomplish
that goal. Frequently one needs to know whether the
toxicity is due to ammonia or whether there are
toxicants present other than salt. These questions are
quite different from the former case where one is
playing "I'll bet you the toxicant is..." game.
No Phase I characterization test should be dropped
from use on the basis that the toxicants it is designed
to address are not likely to be present in the effluent.
In excluding any Phase I test, the analyst may be
limiting the information that can be gained on effluent
toxicants. The investigator should approach effluent
characterization without a preconceived notion as to
the cause of toxicity.
There are two types of checks that can be used to
detect artifact toxicity. A "toxicity blank" consists of
performing the same (Phase I) test on dilution water
and measuring to determine whether any toxicity is
added by the test procedure. A toxicity blank,
however, does poorly in identifying artifact toxicity if
toxicity is affected by the wastewater matrix. For
example, the toxicity of a Phase I reagent, EDTA,
may be completely different in dilution water and in
effluent. If so, a toxicity blank is inappropriate for the
chelation test. A "toxicity control", on the other hand,
involves a comparison of the test solution and the
8-3
-------
Baseline effluent. In this case, the comparison must
demonstrate that the effluent test solution has not
become more toxic than the unaltered effluent. If it
has, the test procedure has produced artifactual
toxicity. For some treatments, valid blanks or toxicity
controls cannot be made.
No procedure should be assumed to be free of
artifactual toxicity. Many of the Phase I toxicity tests
involve relatively severe or unorthodox effluent
manipulations. Blanks and controls must be
consistently and conscientiously used to detect the
introduction of toxic artifacts or other changes to the
effluent that increase sample toxicity.
8.1 Initial Effluent Toxicity Test
Principles/General Discussion:
The major purpose of the Initial effluent test is to
provide an estimate of the 24 hour LC50 for purposes
of setting exposure concentrations in Phase I tests.
Volume Required:
Initial toxicity test is performed in duplicate exposure,
and 40 ml of effluent is needed.
Apparatus:
Disposable one ounce test chambers, automatic
pipette (10 mL), disposable pipette tips (10 ml_), eye
dropper or wide bore pipette, light box and/or
microscope (optional).
Test Organisms:
Test organisms, 60 or more, of the same age and
species.
Procedure (Day 1):
A concentration series using 10 mL in duplicate of
100%, 50%, 25%, 12.5%, 6.25% effluent, and a
control will suffice for most effluents. Obviously more
toxic effluents will require a lower range. If nothing is
known about the toxicity, more concentrations should
be included. A sample data sheet for the Initial test is
shown in Figure 8-2.
8.2 Baseline Effluent Toxicity Test
Principles/General Discussion:
In order to determine the effects that the various
Phase I characterization tests have on effluent
toxicity, the toxicity of the effluent sample, prior to any
treatment in the laboratory, must be determined. The
portion of the effluent sample, tested for toxicity the
day after it arrives in the laboratory (day 2), will be
referred to as the "Baseline effluent". The Baseline
effluent LC50 will be compared to results of toxicity
tests initiated on day 2 on aliquots of the effluent
carried through characterization tests. Such a
comparison will demonstrate whether the removal or
alteration of various groups of toxicants changes
effluent toxicity. Thus, by comparing these results, an
indication of the physical/chemical nature of the
toxicants can be obtained. If the Baseline effluent
LC50 is substantially different from the toxicity of the
effluent when it arrived in the laboratory (Initial
toxicity), one must decide whether the schedule
suggested in these methods should be revised to
reduce delay in testing. If Phase I testing is extended
to additional days, Baseline tests must be done each
time on succeeding days, and used for comparison to
these later tests.
Volume Required:
The Baseline toxicity test is performed in duplicate
using 10 mL per replicate. The total volume
necessary will depend on the 24 hour LC50 of the
day 1 initial effluent test, but 80 mL will be adequate
for most species.
Apparatus:
Disposable one ounce test chambers or glass
beakers, automatic pipette (10 mL), disposable
pipette tips (10 mL), eye dropper or wide bore
pipette, light box and/or microscope (optional).
Test Organisms:
Test organisms, 60 or more, of the same age and
species.
Procedure (Day 2):
Two concentration series will be used in duplicate for
the static acute toxicity test. One test series will
provide exposures at effluent dilutions of 100%, 50%,
25%, 12.5% and 6.25% (or as appropriate if the
effluent is more toxic). This series will enable a
comparison of the results of the Baseline (day 2)
effluent test to the Initial effluent LC50 (cf., Section
8.1). In testing^ the day 2 effluent, any obvious
physical changes, (e.g., formation of precipitates,
odors) which occurred during storage, should be
noted.
The exposure levels in the other test series will be
based on the 24 hour LC50 of the Initial (day 1)
toxicity and will include day 2 effluent concentrations
at 4X-, 2X-, 1X-,and 0.5X-LC50. Most of the
toxicity tests with the characterization solutions will
also be performed using these same exposure
concentrations. If the 24 hour LC50 of the initial
effluent is greater than 25%, the series should begin
at 100%, include four exposure concentrations and
the lowest concentration should equal one-half the
8-4
-------
Figure 8-2. Example of data sheet for initial effluent toxicity test.
Test Type: INITIAL
Test Initiation (Date
Investigator:
EFFLUENT
& Time):
Sample Loq #, Name:
Date of Collection:
Species/Age: _
No. Animals/No. Reps:_
Source of Animals:
Dilution Water/Control:,
Test Volume:
Other Info:
Cone. (% effluent)
100
50
25
12.5
6.25
Control
24 h
A B
LC50 B
c> I
Survival Readings'
48 h
A B
ILC50 H
c> 1
72 h
A B
ILC50 1
ci §
96 h
A B
HLCSO
Hci
Comments:
8-5
-------
LC50. In this case, the method for making dilutions
described earlier must be changed. If the 24 hour
LC50 of the day 1 initial effluent is equal to 25%, the
second exposure series will be unnecessary because
the first series described fulfills the requirements for
comparison to the initial effluent test and character-
ization solution toxicity test results. Throughout this
section, the alternate series will be assumed when
4X-LC50 exceeds 100%.
A sample data sheet is shown in Figure 8-3. In order
to compare the Baseline effluent toxicity and the
toxicity of the effluent aliquots subjected to
characterization tests, all of the day 2 toxicity tests
should be initiated at approximately the same time.
The Baseline toxicity test should be repeated each
time additional characterization tests are performed
on the sample after the initial Phase I battery. The
Baseline test will serve as the basis for determining
the effects produced by the additional characterization
tests, and will also provide information on degradation
of sample toxicity. If sample toxicity is greatly
changed compared to the toxicity of the sample on
the day of its arrival (e.g., greater than 50% change)
it is advisable to discard the remaining sample and
collect a fresh one.
/nterferences/Contro/s and Blanks:
The control treatment in this test is used for
comparison to several subsequent tests and provides
an important reference for diluent water acceptability.
Mortality in these controls will negate other work.
Results/Subsequent Tests:
Baseline LC50's should be generated for as long as
the effluent sample is being used and a Baseline test
should be started every time the effluent sample is
put through any characterization steps. (Note: similar
procedures should be followed in Phases II and III.)
8.3 pH Adjustment Test
Principles/General Discussion:
The pH has a substantial effect on the toxicity of
many compounds found in effluents. Therefore pH
adjustment is used throughout Phase I to provide
more information on the nature of the toxicants.
Changes in pH can affect the solubility, polarity,
volatility, stability and speciation of a compound,
thereby affecting its bioavailability as well as its
toxicity. Before describing the pH adjustment test,
some discussion on the effect of pH on various
groups of compounds is warranted.
Two major groups of compounds significantly
impacted by solution pH are acids and bases. To
understand how organic and inorganic compounds of
this type are affected by pH changes, one must have
a basic understanding of the thermodynamic
equilibrium acidity constant, Ka, for the proton transfer
reaction.
HA + H.O = H 0+ + A~
£
-------
Figure 8-3. Example of data sheet for definitive baseline effluent toxicity data.
Test Type: BASELINE EFFLUENT
Test Initiation (Date & Time):
Investigator:
Sample Log #,
Date of Collect
Name:
ion:
Species/Age:_
No. Animals/No. Reps:.
Source of Animals:
Dilution Water/Control:_
Test Volume:
Other Info:
Cone. (% effluent)
100
50
25
12.5
6.25
4X-LC50/
2X-LC50/
1X-LC50/
0.5X-LC50/
Control
• L(
c
24 h
A B
350 Hll
1 •
Survival Readings:
48 h
A B
LC50 Si
ci •
72 h
A B
LC50 H
ci •
96 h
A B
ILC50
C,
Comments:
8-7
-------
B: unprotonated base
Kb: thermodynamic equilibrium constant for the base.
e.g., C6H5NH2 + H20 =
+ OH'
In the above reaction, BH+ can be considered the
"conjugate acid of the base", that is, the protonated
form of the base. Thus, the same reaction can be
expressed as follows:
[BH+]
Note:
e.g., C6H5NH3-1- + H2O = H30+ + C6H5NH2
conjugate acid
K =
a
=2.34xlo-6
[C6H5NH3+]
This convention can be used to simplify dealing with
equilibrium constants for acids and bases.
As with acids, when the solution pH is equal to the
pKa of the conjugate acid of a base, equal amounts of
the base will exist in the ionized and un-ionized
forms. For example, ammonia in an aqueous solution
at pH 9.25 (the pKa of ammonia) will be found as
50% NH4+ and 50% NHs. At one pH unit above the
pKa (i.e., 10.25) roughly 90% of the ammonia will be
in the un-ionized form (NHs) and the remainder will
be in the NH44- form. At pH 8.25, one unit below the
pKa of ammonia, approximately 90% of the ammonia
will be in the NH4+ form, and approximately 10% will
be in the NHa form.
The above can be summarized by the following:
Predominant Species
Organic
Inorganic
pH>pK,
acid RCOO", RCO" A'
base RNH2 B
pH < pK,
acid RCOOH, RCOH HA
base RNH3* BH +
R = aliphatic or aromatic group
The effect of pH on the ratio of the ionized and un-
ionized forms of acids and bases has a number of
impacts on Phase I results. First, compounds may be
more toxic in the un-ionized form as compared to
the ionized form. For example, NH3 is generally
recognized as the toxic form of ammonia while NH4 +
is of far less concern. A second implication of this
effect relates to toxicant solubility. Un-ionized forms
of acids and bases can be considered less polar than
their ionized forms, which interact to a greater extent
with water molecules. Consequently, un-ionized
forms of acids and bases can be more easily stripped
from water using aeration (Section 8.5) or extraction
with non-polar solvents or solid phase column
techniques (Section 8.6). Likewise, changes in
compound solubility with pH change many mediate
removal through filtration (Section 8.4).
Another implication of the pH effect involves metal ion
complexes. An example of how pH can alter the form
of a metal in a natural water system is shown in
Figure 8-4. Given a pe, the equilibrium electron
activity, (in a simple sense, whether the system is
aerobic or anaerobic), one can see how various forms
of manganese are created and eliminated as pH
shifts.
Figure 8-4. pt-pH diagrams for the CO2, H2O, and Mn-CO2
systems (25°C). Solid phases considered:
Mn(OH)2(s) (pyrochroite), MnCO3(s) (rhodo-
chronsite), Mn3O<(s) (hausmannite), r-MnOOH
(manganite), r-MnOz (nsutite). (Reprinted with
permission from Stumm & Morgan, 1981.)
c
CD
- 1.5
Each of the different forms of the metal will be
manifested differently in aquatic organism effects.
Some forms of the metal will be relatively insoluble;
these forms may not affect toxicity. Likewise, as with
acids and bases, the toxicity of the soluble forms of
the metal will be a function of the actual species
present (e.g., the LC50 of Mn2+ as compared to the
LC50 of MnO42'). The actual species formed will
depend, in addition to pH and pe, on the other
8 - 8
-------
chemical constituents present in the water. The
hydrolysis rate of organics is greatly affected by pH,
and pH changes may alter organic toxicity as well.
Regardless of the speciation effect on toxicity,
changes in solution pH may affect the toxicity of any
given compound. The pH of the test solution may
affect membrane permeability at the respiratory
surface as well as the chemistry of the toxicant. One
might expect that changing the pH, only to return it to
its original pH in a short time, would not alter toxicity.
Experience shows that this is not the case and that
this adjustment sometimes results in reduction or loss
of toxicity. If the kinetics of the pH driven reaction (on
return to the original effluent pH) are slow or
irreversible, pH adjustment alone may be effective in
evidencing toxicants affected by pH change. Some
organics may also degrade due to pH change.
Another purpose of the "pH Adjustment Test" is to
provide a blank for subsequent Phase I pH
adjustment tests performed in combination with other
operations. This test will demonstrate whether toxic
concentrations of ions have been reached as a result
of the addition of acid and base.
Toxicity blanks for sample ionic strength increases,
which are based on adding the same volumes and
strengths of the acid and base *o dilution water, do
not give comparable results when added to the
effluent. Effluents already contain substantial
concentrations of major anions and cations, which are
not found in dilution water. Further, the volumes and
strengths of the acid and base necessary, for
example, to lower an effluent to pH 3 and raise it
back to pH 7.6 are not likely to result in the same
final pH when added to dilution water.
Volume Required:
600 mL are needed to make pH adjustments. A 300
ml_ aliquot of the day 1 effluent sample is lowered to
pH 3 with four exposure concentrations, and the
second 300 mL sample is raised to pH 11.
Approximately 30 mL will be needed for the test but
the actual amount depends on the 24 hour LC50 of
the initial effluent test. The remaining 270 mL of each
of these solutions is reserved for the pH
Adjustment/Filtration, pH Adjustment/Aeration and pH
Adjustment/Cia SPE Phase I tests.
Apparatus:
Burettes for acid and base titrations, pH meter and
probe, 2-500 mL beakers, 2-500 mL graduated
cylinders, 12-30 mL beakers, stir plate, and stir bars
(perfluorocarbon), automatic pipette, disposable
pipette tips, eye dropper or wide bore pipette, light
box and/or microscope (optional), pH meter and
probe.
Reagents:
1.0, 0.1 and 0.01 N NaOH, 1.2, 0.12 and 0.012 N
HCL (ACS grade in high purity water) and buffers for
pH meter calibration.
Test Organisms:
Test organisms, 40 or more, of the same age and
species.
Procedure (Day 1):
The general procedure for the pH Adjustment test is
shown in Figure 8-5. Using a burette, and stirring
constantly, 1.0 N NaOH is added dropwise to a 300
mL aliquot day 1 effluent until the solution pH nears
11.0. (Note: overshooting results in the addition of
more salts and volume may cause toxicant
decomposition and should be avoided.) In order to
minimize any over-adjustment of pH, 0.1 N NaOH is
added dropwise in the latter stages to bring the
effluent aliquot to pH 11. The solution should be
allowed to equilibrate after each incremental addition
of base. The amount of time necessary for pH
equilibration will depend on the buffering capacity of
the effluent. Caution should be taken to prevent any
solution pH of greater than 11.0. If pH 11 is
exceeded, 0.12 N HCI must be used to lower the pH
to 11.0. The goal of the pH adjustment step is to
reach pH 11, while minimizing both the change in
aliquot volume and the increase in ionic strength.
Volumes and strengths of base (and any acid added)
should be recorded. A 30 mL volume is held for the
same length of time it takes to complete other Phase
I manipulations (performed on day 1) with pH 11
effluent. Once other work has been completed with
the total volume of the pH 11 effluent, the 30 mL
volume at pH 11 is returned to the initial pH (pH|) of
the day 1 effluent. (The other aliquots of pH 11
effluent are also returned to pH| at this time.) This is
accomplished by the slow, dropwise addition of 0.12
N HCI first and later 0.012 N HCL as the pH of the
stirred solution nears pH|. If pH| is exceeded, the pH
must be appropriately increased with 0.01 N NaOH.
Again, the volumes and strengths of acid and any
base added should be recorded.
The pH of the solution should be checked periodically
throughout the remainder of the work day and
readjusted as necessary. Changes in the total volume
of acids and bases added should be recorded.
This entire procedure is repeated with a second 300
mL aliquot of effluent, using 1.2 N and 0.12 N HCI to
lower the pH to 3. As with the pH 11 effluent, 270 mL
of the pH 3 effluent is used for the pH
8-9
-------
Figure 8-5. Flow chart for pH adjustment tests.
Effluent Sample 600 mL at pH|
300 ml, adjusted to pH 3
270 mL at pH 3
30 mL at pH 3
Day 1
4
Use for other
Phase I
tests:
HOLD
Use for other
Phase I
tests:
i
•Filtration
•Aeration
•C18SPE
'Adjust to pH|
•Filtration
•Aeration
•C18 SPE
•Adjust to pH|
Day 2
Set up Toxicity
Tests
Adjustment/Aeration, pH Adjustment/Filtration, and pH
Adjustment/Cia SPE tests. The remaining 30 mL of
the pH 3 effluent is held until the first day's work on
all of the pH 3 effluent has been completed. At this
point, the pH of the 30 mL volume of pH 3 effluent is
readjusted to pH| by the dropwise addition of 0.1 N
and 0.01 N NaOH. Maintenance of pH| must be
assured through periodically checking and readjusting
the sample throughout the work day. All volumes and
strengths of acid and base added should be recorded.
(Day 2): At the beginning of the work day (on the day
after the arrival of the effluent in the laboratory), the
pH of both of the 30 mL volumes is again checked to
ensure that pH| has been maintained. Any additional
pH adjustments are made and recorded. The acute
toxicity of each pH-adjusted solution is tested at
4X-, 2X-, 1X-, 0.5X-LC50 (the 24 hour Initial
LC50) as described in Section 7. Test solution pH
should be measured and recorded every 24 hours. A
sample data sheet is shown in Figure 8-6.
Set up Toxicity
Tests
Interferences/Controls and Blanks:
Controls prepared for the Baseline toxicity test act as
a check on the organisms, dilution water, and test
chambers for this test as well.
The Baseline effluent test acts as a control for the pH
Adjustment test, indicating whether the addition of
NaCI in the form of the acid and base has increased
effluent toxicity. The pH Adjustment test acts as the
control for other Phase I tests entailing pH
adjustment. In addition to serving as a control for
other pH adjustment tests, increased toxicity following
pH adjustment, not as a result of NaCI concentration,
indicates a pH effect on toxicity (see below).
Results/Subsequent Tests:
If either the pH 3 or pH 11 adjusted effluent tests
have significantly greater toxicity than the Baseline
effluent test, two possible sources of toxicity exist: 1)
the ions (Na + , Cl") added by the acid and base
have resulted in a solution with an ionic strength
intolerable to the test organism; or 2) chemical
8-1 0
-------
Figure 8-6. Example of data sheet for pH adjustment test.
Test Type: pH ADJUSTMENT
Test Initiation (Date & Time):
Investigator
Sample Log #, Name:_
Date of Collection:
Species/Age:
No. Animals/No. Reps:_
Source of Animals:
Dilution Water/Control:_
Test Volume:
Other Info:
pH/Concentration
(% pH adjusted
effluent)
3/
3/
3/
3/
11/
117
11/
IV
•
24 h
A pH
9 LC50 1
Survival Readings:
48 h
A pH
H LC5°
72 h
A pH
• LC50
96 h
A pH
• LC50
11
300 mL pH 3
300 mL pH 11
30 mL pH 11
30 mL pH 3
HC1
NaOH
Comments:
8-1 1
-------
reactions driven by the pH change have not reversed
upon readjusting to pH|. Neither of these phenomena
would be detected through the use of a dilution
water-based blank. To help resolve this situation a
listing of NaCI LC50 values for common test
organisms is provided in Table 8-2. The minimum
concentration of NaCI in the test solution (i.e., not
including the concentration of NaCI originally present
in the effluent) can be calculated from the volumes
and strengths of the acid and base added and final
solution volume. The data in the table can be used
only as a rough guide, however, because the toxicity
of sodium chloride depends on the other anions and
cations as well as the total osmotic pressure exerted
by the dissolved substances. The toxicity of the
added NaCI is best determined by adding that amount
directly to the effluent and see if the addition
increased effluent toxicity.
If pH 3 and/or pH 11 adjustment results in a
significant decrease in effluent toxicity, it could result
from volume changes by acid and base addition or
chemical reactions driven by pH change may not
have been re-established or are irreversible. These
two possibilities can be checked by adding a volume
of dilution water equivalent to the total volume of acid
and base added to the 30 mL effluent volume. If a
similar loss in toxicity of the diluted wastewater
occurs, the pH Adjustment test should be repeated
using more concentrated acid and base.
A reduction or loss of toxicity may also be the result
of the degradation of toxicant at the altered pH
values. Both organics and inorganics can be so
changed with a probable loss in toxicity. In some
cases, the toxicity could also be increased if the
degradation product is more toxic than the original
compound.
For most of the Phase I combination pH adjustment
tests, the pH Adjustment test will act as an equivalent
or "worst case" control for changes in test solution
ionic strength and volume. In effect, most of the
operations applied to the pH adjusted effluent in
Sections 8.4-8.6 will not affect pH or will serve to
drive it closer to pH|. This may not be the case for
the pH Adjustment/Aeration test, however. Because
pH 3 and 11 must be maintained throughout the
aeration process and because the loss of volatiles
may result in pH shifts towards pH|, more acid and/or
base may be added to these test solutions as
compared to the pH adjustment only solutions.
8.4 pH Adjustment/Filtration Test
Principles/General Discussion:
The filtration experiment provides information on
effluent toxicants associated with filterable material.
Toxic pollutants associated with particles may be less
biologically available. Aquatic organisms can I
exposed to these pollutants through ingestion of tl
particles, however. This route of exposure may I
significant for cladocerans and other filter feede
ingesting bacterial cells and other solids with sorb<
toxicants. The degree to which any compound exis
sorbed or in solution depends on a number of facto
including particle surface charge (or lack thereo
surface area, compound polarity and charge, solubili
and the effluent matrix. By filtering particles from tr
effluent, both a source and a sink of toxic chemica
may be removed.
In addition to determining the effect of filtration on th
toxicity of the whole effluent, the effects of p
adjustment in combination with filtration are als
assessed. As discussed in Section 8.3, changes i
solution pH can result in the formation of insolubl
complexes of metals (Figure 8-4). Similarly, organi
acids and bases existing in ionic form can b
transformed into the non-ionic form by pi
adjustment. Shifts in effluent pH can also act to driv<
dissolved toxicants onto particles in the effluent (e.g.
shifting the dissolved/adsorbed equilibrium away fron
the free form). Changes in toxicant polarity resultini
from solution pH change can make sorru
particle/toxicant interactions stronger. In other cases
the increase in effluent ionic strength resulting frorr
the shift in pH may force non-polar organic
compounds onto uncharged surfaces to a greatei
extent.
By filtering pH adjusted aliquots of effluents, those
compounds typically in solution at unadjusted pH bu1
insoluble or associated with particles to a greater
extent at more extreme pHs, are removed. By
removing the toxicant contaminated particles or
precipitated compounds prior to readjustment of the
sample to pH|, these toxicants are no longer available
for dissolution in the effluent. The pH change may
also destroy or dissolve the particles thereby
removing the sorption surfaces or drive the
dissolved/sorbed equilibrium in the opposite direction.
Positive pressure filtration is recommended. Use of a
vacuum to draw the effluent sample through the filter
may result in a loss of volatile compounds by
degassing the solution during filtration. This problem
is potentially worsened in pH adjusted effluents if
toxicants become more volatile as a result of pH
changes. If vacuum filtration is used and effluent
toxicity is reduced, subsequent tests must be
performed to define the nature of the toxicity loss.
The solid phase extraction characterization test
(Section 8.6) requires the use of filtered effluent.
Without knowledge of the effect of filtering on the
effluent toxicity, it is not possible to tell whether or not
the column or the filtration removed the toxicity
Filtering may also be useful in connection with other
Phase I tests.
8-12
-------
Table 8-2. Acute Toxicity of Sodium Chloride to Selected Aquatic Organisms
LC50 (g/L) (95% Cl)
Species Water Type
Ceriodaphnia dubia DMWa.b
Daphnia magna \_\fjti.c
RW
Pimephales promelas DMWb
Sottb.d
Lepomis macrochirus Soft RWe
Life-stage
<24h
<24 h
<24 h
<24 h
NR
NR
<24h
11 wk
1-9g
24 h
4.2
(")
3.3
3.0
2.3
(2.0-2.6)
3.3
(NR)
6.4
(NR)
7.9
(7.0-9.0)
7.9
(NR)
48 h
2.3
(2.0-2.6)
2.7
2.1
2.3
(2.0-2.6)
3.1
(NR)
5.9
(NR)
7.9
(7.0-9.0)
-
-
72 h 96 h
6.9 4.6
(5.5-8.7) (7.4-7.9)
7.7
(7.4-7.9)
12.9
(NR)
a Data for C. dubia and fathead minnows was generated at ERL-Duluth.
Both species were < 24 h old at test initiation and C. dubia were fed. Dilution water used was
diluted mineral water.
b Static, unmeasured test.
0 Dowden and Bennett, 1965.
d Adelman et al., 1976.
e Patrick et al., 1968.
LW = lakewater; RW = reconstituted water; NR = not reported
(--) Confidence interval cannot be calculated as no partial mortality occurred.
Volume Required:
A 235 mL aliquot of pH|, day 1, effluent is filtered.
Also, 235 mL each of pH 3 and pH 11 effluent
aliquots (Section 8.3) are filtered. The remaining 35
mL of each is reserved for the pH
Adjustment/Aeration Tests. A maximum volume of 30
mL of each of these three solutions is needed to
perform the filtration toxicity tests. Each test requires
four exposure concentrations (10 mL each). The
exact effluent volume required for the toxicity test will
be a function of the effluent toxicity (Section 7). The
remaining filtered effluent volumes (200 + mL) of pH
3, pH 11, and the pH| solution are each reserved for
the Cis SPE tests (Section 8.6). Excess volume has
been included to cover losses occurring during the
filtration operation.
Apparatus:
Six-250 mL graduate cylinders, 6-250 mL beakers,
6-50 mL beakers, pump with sample reservoir,
teflon tubing, in-line filter housing, ring stands,
clamps. Alternatively, vacuum flask, filter stand,
clamp, vacuum tubing, water aspirator or vacuum
pump. Glass-fiber filters (nominal size 1.0 pm,
without organic binder), stainless steel forceps,
burettes for acid and base titrations, pH meter and
probe, stir plate, perfluorocarbon stir bars, automatic
pipette (10 mL), disposable pipette tips (10 mL), eye
dropper or wide bore pipette, light box and/or
microscope (optional).
Reagents:
Solvents and high purity water for cleaning pump
reservoir and filter, 0.1 N and 0.01 N NaOH, 0.12 N
and 0.012 N HCI (ACS grade in high purity water),
buffers for pH meter calibration.
Test Organisms:
Test organisms, 75 or more, of the same age and
species.
Procedure (Day 1):
The first step for the Filtration Test is filter
preparation, shown in Figure 8-7. First the filters are
prepared, then the blanks, and finally effluent
samples at three pH's are filtered (cf., Figure 8-8).
Use of glass-fiber rather than cellulose-based
filters should minimize the adsorption and loss of
dissolved non-polar organic compounds from the
effluent sample. Adsorption of toxic dissolved organic
compounds onto the filter can lead to spurious
results.
8-13
-------
Figure 8-7. Overview of filter preparation and dilution water blanks for filtration test and Ctg solid phase extraction column
test samples.
Da
=
Da
Preparing
the Filter
/1
Dilution
Water
Blanks
r
y2
L
300 mL High
I
HCI — ^r
T
Purity Water
r
1 + NaOH
100mLofpH3 100 mL of pH, (unadj.) 100mLofpH1l
4^
Filer
4-
•^ -^
Filter
Filter
4- 4
Discard water Discard water
Discard water
Dilution Water
I
pH3
4
1
pH
0
•i-
prepared filter prepared filter
4
200 mL 20 mL 200 mL
•*• 4 — NaOH +
4
pH 11
Jr
prepared filter
' *•
20 mL 200 mL 20 mL
NaOH Thru C18 Thru C18
| SPE Column SPE Column
L— + I.
1 r i
Tox. Tox. Tox.
Test Test Test
1
HCI — >4 4- HCI
Thru Gig
SPE Column
HCI — »
4- IT
Tox. Tox. Tox.
Test Test Test
Filter Preparation
To prepare the 1.0 iim glass-fiber filter for use,
wash two 50 mL volumes of high purity water through
the filter. For pH 3 effluent filtration test, the filter
should be washed with high purity water adjusted to
pH 3 using HCI. Likewise, the filter used with the pH
11 effluent samples should first be washed with high
purity water adjusted to pH 11 using a concentrated
NaOH solution. Washing the filters with water
adjusted to the same pH as the effluent should
preclude sample contamination with water soluble
toxicants contained on the filters.
Blank Preparation
The next step is to prepare filter blanks using dilution
water (Figure 8-7). These blanks are used to detect
the presence of any water soluble toxicants which
may remain on the filter following the washing
process. The pH| filtration blank is simply prepared by
passing 220 mL of pH unadjusted dilution water
through a prepared filter. The filtered dilution water is
collected and 200 mL of this volume is reserved for
the GIB SPE test blank (Section 8.6). The remaining
20 mL is used on day 2 as a blank in a toxicity test.
Again, excess is included to cover any possible loss
during rinses.
The procedures to prepare the pH 3 and 11 filtration
blanks are somewhat different. To prepare the pH 3
filtration blank, 255 mL of dilution water is adjusted to
pH 3 with 0.12 and 0.012 N HCI, caution being taken
to minimize the increase in dilution water ionic
strength. Of the pH 3 dilution water, 35 mL is
reserved for use as a blank in the Aeration test
(Section 8.5). The remaining 220 mL of pH 3 dilution
water is passed through a filter previously washed
with pH 3 rinse water. The pH 3 dilution water is
collected and 200 mL of this volume is reserved for
the pH 3 CIB SPE test blank. The remaining 20 mL is
readjusted to the initial pH of the dilution water (pH0,
using 0.01 N NaOH, again taking care not to exceed
pH0 (pH0 symbolizes the equilibrium pH of the
8-14
-------
Figure 8-8. Overview of steps needed with the effluent for the filtration and C-| g SPE column tests.
Day 1
NaOH
200 mL
+•
35 mL
Thru C}8
SPE Column*
U- NaOH
4
i
«—
- NaOH
200 mL
+•
35 mL
Thru Cig
SPE Column*
r ^
r *
HCI
HCI
200
mL
~H-
35 mL
Thru C18
SPE Column*
~>
r ^
r ^
* HCI
W
Day 2
"See Figure 8-13 and Section 8 6 for details.
dilution water) during the readjustment process. This
solution is used on day 2 in a single exposure toxicity
test.
The pH 11 blank is prepared in a similar fashion using
255 mL of dilution water adjusted to pH 11 with 0.1 N
and 0.01 N NaOH. Of the pH 11 dilution water, 35 mL
is reserved for use in the Aeration test. The remaining
volume is filtered using the filter previously washed
with pH 11 rinse water and 200 mL of the filtered pH
11 dilution water is collected for use as the basic C-\Q
SPE blank. The remaining 20 mL is readjusted to pH0
with 0.012 N HCI and used on day 2 in a single
exposure toxicity test.
Sample Preparation
The same filter used to prepare the pH0 filtration
blank is now used to filter a 235 mL aliquot of the pH|
effluent. Effluent passed through the filter is collected
and 200 mL is reserved for the GIB SPE test. The
remaining volume is held overnight for toxicity tests
initiated on day 2.
Using the same filter used to prepare the pH 3
filtration blank, 235 mL of pH 3 effluent (see Section
8.3) is filtered and collected. The filtered pH 3 effluent
is split into 200 mL and 35 mL aliquots. The 200 mL
aliquot is used in the pH 3 GIB SPE test. The 35 mL
aliquot is readjusted to pH| using 0.1 and 0.01 N
NaOH. Care must be taken to minimize both an
increase in aliquot volume and ionic strength. The pH
readjusted 35 mL aliquot is held for the day 2 toxicity
testing.
Finally, the filtration step is repeated using 235 mL of
the pH 11 effluent (Section 8.3) and the filter
originally used to filter pH 11 dilution water. Again,
200 mL of the pH 11 filtered effluent is used in the
basic Ci8 SPE test; 30 mL is readjusted to the pH| of
the effluent with 0.12 N and 0.012 N HCI and used to
conduct a toxicity test on day 2.
In filtering effluent samples with high solids content, it
may be necessary to change filters in order to obtain
235 mL of filtered effluent. If so, the filter preparation
step must be repeated to provide additional filtration
blanks. Alternatively, it may be possible to centrifuge
samples high in suspended solids and filter the
supernatant through a single filter. If this option is
taken, the toxicity of the supernatant must be tested
on day 2 and compared to that of the Baseline
effluent.
In the above procedures, the effluent filtration system
must be cleaned between pH adjusted aliquots to
prevent any carry-over. This means all equipment
should be thoroughly rinsed with 10% HNOa, acetone
and high purity water between aliquots of effluent.
The pH of the pH adjusted blanks and effluent
aliquots, designated for day 2 toxicity tests, should be
checked periodically throughout the work day.
Adjustments should be made as necessary in order to
8-15
-------
maintain pH0 and pH|, respectively, of these
solutions.
(Day 2): Prior to initiating the toxicity tests, the pH of
the pH 3 and 11 blanks and filtered effluent aliquots
should be measured and readjusted to the pH0 and
pH|, respectively. Toxicity tests performed on all three
(pH 3, pH|, and pH 11) filtration blanks involve testing
without dilution. Based on the 24 hour Initial LC50 of
the day 1 effluent, toxicity tests performed on the
effluent aliquots filtered at pH 3, pH 11, and pH| are
set up at 4X-, 2X-, 1X-, and 0.5X-LC50, or as
described in Section 8.2. Measurement of exposure
pH should be made daily on the highest tested
concentration, concurrently with survival readings. A
sample data sheet for the Filtration tests is shown in
Figure 8-9.
Interferences/Controls and Blanks:
Controls prepared for the Baseline toxicity test serve
as a check on the quality of organisms, dilution water
and test conditions.
Results of the pH Adjustment test (Section 8.3) will
indicate whether or not toxic levels of NaCI have been
produced through pH adjustment. Results of the
effluent filtration tests at each pH should be
compared with the filtration blank performed at the
corresponding pH to determine the validity of the
toxicity test outcome. No significant mortality should
occur in any of the filtration blanks. If unacceptable
mortality of control organisms occurs in either or both
of the pH adjusted filtration blanks, further
investigation will be necessary to determine whether
lethality resulted from toxicants leached from the filter
at pH 3 and/or 11, or whether the increase in dilution
water ionic strength (via acid and base addition) is
responsible for the problem. This can be
accomplished by repeating the filtration step on pH
adjusted dilution water providing a pH adjustment
blank (i.e., pH adjusted unfiltered dilution water).
Additionally, if the pH 3 and/or 11 filtration, aeration
(Section 8.5) and C-\Q SPE (Section 8.6) dilution
water blanks have approximately the same final
concentration of acid and base, any ionic strength
related toxicity should be detected in them.
If a filtration blank shows unacceptable acute toxicity
but the corresponding filtered effluent is equally or
less toxic than the Baseline effluent, it is possible that
the dilution water blank removed the final traces of
toxic filter artifacts. In some cases, the effluent matrix
may have also prevented the artifacts from leaving
the filter or masked their presence. Alternatively the
observed filtered effluent toxicity may represent the
net effect of toxicant removal via filtration plus
contamination by filter artifacts.
Results/Subsequent Tests:
The LC50s for the aliquots of pH 3, pH| and pH 11
filtered effluent are compared to the Baseline effluent
LC50 to determine whether any of these processes
resulted in a significant change in effluent toxicity.
If any or all of these pH/filtration combinations result
in less effluent toxicity (which cannot be attributed to
the effects of pH adjustment alone), it may be
possible to confirm the findings of the test. This can
be attempted through a transfer of the solids
contained on the filter back into the filtrate at pH|.
This can be done by reversing the flow of the filtrate
through the filter or rinsing the solids off the filter with
filtrate. The toxicity exhibited by this solution should
be similar to that of the original effluent, provided that
the final concentration of solids in the test solution
approximates the solids level in the sample that was
filtered. For precipitates formed as a result of pH
changes or for contamination of suspended solids
facilitated by pH adjustment, time must be allowed for
the precipitate to redissolve in the pH| filtrate or for a
new equilibrium to be set up between the
contaminants on the solids and in solution. The
results of this test are not likely to be quantitative due
to the recovery problems inherent in the process.
In order to determine whether the effluent matrix
affects the toxicity of filterable particles (e.g., its ionic
strength, dissolved organic carbon content), the
filtered material can also be added to a volume of pH|
dilution water equal to the volume of effluent that was
passed through the filter. The toxicity of this dilution
water, spiked with effluent solids, can be compared to
the toxicity of the unfiltered (Baseline) effluent and the
filtrate spiked with its own solids.
We have had limited experience with effluents in
which acute toxicity could be removed by filtration at
a normal pH (7.0-8.0). The additional tests
suggested herein may or may not provide the
intended information.
If toxicity can be removed by filtration, either with or
without pH change, one has a method for removing
the toxicants from other material in the effluent. This
knowledge itself provides an important advance
because further characterization and analyses will be
less confused by those constituents separated from
the toxicants. Usually further characterization will be
the next step. Tests must be designed to determine
whether the mechanisms causing removal are
precipitation, sorption, change in equilibrium or
volatilization. One usually necessary step is to learn
how to recover the toxicity from the filter. If this
cannot be done and the loss is not by volatilization,
then the whole experiment may have little utility. Use
of pressure and vacuum filtration may reveal if
8-1 6
-------
Figure 8-9. Example of data sheet for filtration tests.
Test Type: FILTRATION
Test Initiation (Date & Time):
Investigator:
Sample Log #, Name:_
Date of Collection:
Species/Age:
No. Animals/No. Reps:.
Source of Animals:
Dilution Water/Control:_
Test Volume:
Other Info:
pH/Conc.
(% effluent)
3/
3/
3/
3/
3/ blank
PH|/
PH|/
PH|/
PH,/
pH| / blank
11
11
11
11
1 1/ blank
Survival Readings:
24 h
A pH
• 1
48h
A pH
72h
A pH
96h
A pH
1 LC50 ^BLC5° ffll-C50
• HI " Hi
8-1 7
-------
volatilization is involved. If one gets an idea of the
toxicant through other tests, filtration can be used to
remove the toxicants. Then if the suspected toxicant
is the true one, its concentration should be lower or
zero after toxicity is removed by filtration.
8.5 pH Adjustment/Aeration Test
Principles/General Discussion:
The Aeration test is designed to determine how much
effluent toxicity can be attributed to volatile or
oxidizable compounds. The test is performed with
pH-adjusted and unadjusted effluent. By comparing
the toxicity test results for aerated acidic, pH| and
basic samples, toxicity may be changed and this
knowledge can be used for further characterization.
Some compounds can be removed or oxidized most
easily at one pH, whereas others are most easily
removed or oxidized at a different pH. Thus, the
aeration is performed at several pH values.
Whether a constituent is completely removed, or
sufficiently removed to reduce toxicity, depends on
many chemical/physical conditions. At a minimum,
one must be certain that the geometry of the sparging
process is always the same and that the duration is
constant. Otherwise, the test is of little value. The pH
of many effluents will change, sometimes rapidly,
during sparging and so pH must be frequently
checked and maintained during the entire aeration
period.
Air is used for sparging so that oxidation is included.
Subsequent tests with nitrogen may be used to
separate sparging from oxidation. We have grouped
them first, to avoid many tests initially, and because
many treatment processes remove such constituents
well. Oxidation can change many constituents in
many ways and one must determine if oxidation or
sparging is the mechanism before additional tests can
be designed. Water soluble constituents such as
ammonia and cyanide are not readily stripped by this
test and one should not assume that they will be
removed.
Volume Required:
Thirty-five mL volumes of pH 3, pH 11 (see Section
8.3) and pH| effluent are needed for this test. A
maximum volume of 30 mL of each of these solutions
is required for the toxicity tests on aerated solutions.
An excess volume has been provided to allow for
losses without removing it from the aeration vessel.
Each toxicity test utilizes four exposure
concentrations (10 mL each). The exact volume
required for the toxicity test on each pH adjusted or
unadjusted aerated solution will depend on the toxicity
of the effluent (the 24 hour Initial LC50).
Apparatus:
Aeration device or compressed air system with a
molecular sieve, six glass diffusers, six-50 mL wide
graduated cylinders, burettes for acid and base
titrations, pH meter and probe, stir plate(s),
perfluorocarbon stir bars, automatic pipette (10 mL),
disposable pipette tips (10 mL), eye dropper or wide
bore pipette, light box and/or a microscope (optional).
Reagents;
0.01 N NaOH, 0.012 N HCI (ACS grade in high purity
water), buffers for pH meter calibration.
Test Organisms:
Test organisms, 75 or more, of the same age and
species.
Procedure (Day 1):
Six different solutions are aerated in this test; pH 3,
pH|, and pH 11 effluent, and pH 3, pH0, and pH 11
dilution water, (cf., Section 8.3 and 8.4, respectively
for preparation of pH adjusted effluent and dilution
water.) A flow chart for the effluent samples of the
Aeration test is shown in Figure 8-10. Each sample
is transferred to a 50 mL cylinder containing a small
perfluorocarbon stir bar. The diameter and length of
the pH probe must be such that it can be placed into
the solution during aeration. The taller the water
column and the smaller the bubbles, the better the
stripping will be. Each solution should be moderately
aerated (~10 mL air/minute) for a standard time such
as 60 minutes. Formation of precipitates should be
noted.
The pH of the acidic and basic effluent and dilution
water aliquots is checked every five minutes during
the first 30 minutes of aeration and every 10 minutes
thereafter. If the pH of any solution had drifted more
than 0.2 pH units, it must be readjusted back to the
nominal. The volume and concentration of additional
acid and/or base added to the solutions should be
recorded so that the final concentration of Na+ and
CI" in each solution can be calculated following final
pH readjustment. Solutions should be stirred during
any pH readjustment. Again, precautions must be
taken in order to minimize the amount of acid and
base added. Aeration time does not include the time
intervals during which aeration is temporarily
discontinued to readjust pH. A constant pH is not
maintained in the "pH|" effluent because this solution
represents the generalized effects of aeration on the
effluent without regard to pH. Only slight changes in
the pH of the pH0 dilution water are likely since such
water is usually brought to air equilibrium.
Air contaminated with oil (droplets or vapor), natural
gas or any other substance is not acceptable.
8-18
-------
Figure 8-10. Diagram for preparing aeration test samples.
HCI
Day 1
Aerate 35 mL 1 h;
Maintain pH
NaOH
NaOH
Aerate 35 mL 1 h;
Maintain pH
NaOH
Aerate 35 mL 1 h;
Maintain pH
HCI
HCI
Day 2
'All steps are conducted on dilution water to prepare blanks for testing.
Contaminated air is probable from air lines containing
oil or in cases where the source of the air is
contaminated (e.g., boiler room). Small air pumps,
sold for home aquaria are adequate, if the room air is
free of chemicals. Chemistry laboratories where
concentrated chemicals are used often do not have
suitable air quality. Following aeration, the pH of each
solution (including the 35 mL portions of pH
unadjusted effluent and dilution water) is returned to
the pH of the initial effluent or dilution water using the
necessary volumes of 0.01 N NaOH and 0.012 N
HCI. Returning all effluent solutions to the initial pH of
the wastewater will ensure that a valid comparison
can be made with the Baseline LC50. The pH of each
sample must be periodically checked throughout the
remaining work day and readjusted as necessary. If
stable pH can be attained prior to toxicity test
initiation, the pH during the test is likely to change
less.
(Day 2): Prior to initiating the toxicity tests, the pH of
all of the aerated effluent and blank solutions should
be checked and adjusted to pH| or pH0. Toxicity tests
are performed on a single 100% concentration of all
three dilution water blanks (pH 3, pH0 and pH 11).
These blanks will provide information on toxic artifacts
resulting from aeration.
Based on the 24 hour Initial LC50 of the day 1
effluent, toxicity tests are performed on each aerated
effluent solution at concentrations of 4X- (or 100%),
2X-, 1X-, and 0.5X-LC50 (cf., Section 8.2). The
pH of each of test concentration should be measured
and recorded daily. An example of the data sheet for
the Aeration test is given in Figure 8-11.
Interferences/Controls and Blanks:
Controls prepared for the Baseline toxicity test also
act as controls on organisms, dilution water and test
conditions for this test. Results of the pH Adjustment
test (Section 8.3) will suggest whether or not toxic
levels of NaCI may have been reached as a result of
the addition of acids and bases to the effluent.
No significant mortality should occur in any of the
three aeration blanks. If there is significant mortality,
the cause must be found and corrected before the
test can be meaningful. Blank toxicity, especially in all
three, suggests contaminated air. Other possible
causes include contaminated equipment, such as
electrodes or glassware (especially where low or high
pH solutions were in contact), or the addition of too
much acid or base. To determine which of these
factors resulted in blank toxicity, the toxicity of pH
adjusted aerated dilution water can be compared to
that in the same pH adjusted unaerated dilution water.
Approximately the same quantities and concentrations
of acid and base should be added to both these
samples of dilution water to make them comparable.
Another approach to this question involves observing
the pH Adjustment/Filtration and pH Adjustment/C^a
SPE blanks (Sections 8.4 and 8.6). Assuming the
concentration of acid and base in the final blank
8-19
-------
Figure 8-11. Example of data sheet for aeration tests.
Test Type: AERATION
Test Initiation (Date & Time):
Investigator:
Sample Log #, Name:_
Date of Collection:
pH3
pH,
PH11
Species/Age:
No. Animals/No. Reps:
Source of Animals:
Dilution Water/Control:,
Test Volume:
Other Info:
pH/Conc.
(% effluent)
3/
3/
3/
3/
3/ blank
PH|/
PH|/
PH|/
pH|/
pH| / blank
1V
11/
n/
11/
1 V blank
Survival Readings:
24 h
A pH
48h
A pH
72h
A pH
96h
A pH
HC;
NaOH
8-20
-------
solution is approximately the same in all dilution
waters for the three tests, toxicity in the aeration
blanks but not in the filtration or C^Q SPE blanks
suggests that aeration rather than pH adjustment has
led to contamination. Compare the toxicty from the
Baseline test to the toxicity of all three aerated
effluents. When the Baseline toxicity is significantly
less than that of any one of the aerated samples,
toxicity was added or created during effluent
manipulations. This check is especially important
because pH adjustment of aerated effluent may have
required larger quantities of acid and base as
compared to the pH Adjustment test (Section 8.3).
Dissolved oxygen depletion is likely to be caused by
nitrogen stripping. If a relatively large surface-to-
volume ratio is used (such as the 10 mL volume in a
1 ounce plastic cup) during the overnight holding
period, DO should not be a problem.
Results/Subsequent Tests:
The LCSOs for the aliquots of pH 3, pH|, and pH 11
aerated effluent are compared to the Baseline effluent
LC50 to determine whether any of these processes
resulted in a significant change in effluent toxicity. If a
substantial reduction in toxicity is seen for any or all
of the three aerated effluent solutions, one must next
determine whether the separation of effects was
caused by sparging or oxidation. This is done by
repeating those tests in which toxicity was reduced,
substituting nitrogen for air in the stripping process.
Use of nitrogen eliminates oxidation as a removal
process. If side-by-side effluent stripping tests with
air and nitrogen provide the same results, toxicant
removal is probably caused by the sparging process.
If only the test(s) conducted with air succeed(s) in
reducing or removing effluent toxicity, oxidation is a
probable cause. An effluent sample may contain
toxicants removed through sparging and oxidation. An
example would be where aeration at pH 3 and pH|
reduces toxicity, but nitrogen stripping removes the
toxicity only in the pH 3 effluent.
An additional removal process, which is not
volatilization, may also occur under both air and
nitrogen sparging. Materials such as surfactants will
be carried by bubbles and deposited above the liquid
level on the sides of the cylinder. If air and nitrogen
both remove toxicity, this zone should be checked for
such deposits.
Removal of toxicants by precipitation resulting from
pH change alone in this test should also be detected
by the pH Adjustment/Filtration test. Oxidation of
compounds can cause precipitation. If oxidation is the
cause, the pH Adjustment/Filtration Test will not
change toxicity. If sparging has removed the toxicant,
the "volatile toxicant transfer" experiment described
below may provide separation of the volatile toxicant
from other constituents. Our experience with this
Figure 8-12. Closed loop schematic for volatile chemicals.
PUMP
!5__
•100 mL
Dilution water
at 25 °C
•1000 mL
Effluent
at 25 "C
technique is limited to a few effluents. To perform the
"volatile toxicant transfer" experiment, a closed loop
stripping apparatus is used (Figure 8-12). This
apparatus consists of a pump which can pump
nitrogen gas, two airtight fluid reservoirs (cut
graduated cylinders work well), perfluorocarbon tubing
and diffusers. The arrangement should be that air or
nitrogen can be circulated through one reservoir and
then through the second before returning to the first
reservoir.
Numerous operating conditions can be selected, each
telling something different. This system should not be
operated as a conventional purge and trap system.
The reason is that since one does not know, as yet,
the identity of the toxicant, conditions for trapping are
not known. The objective initially should be to get
measurable toxicity moved into the trap. This will
establish that there are at least some volatile
toxicants present. At this stage the goal is not to
move all the toxicant to the trap. If the same
concentration of the toxicant can be transferred to the
trap as exists in the unaltered effluent, the data are
easiest to interpret. For this purpose the volume of
sparged effluent should be large and the trap volume
small. The nitrogen sparging gas is recirculated so
that if the trap is inefficient in removing the toxicant
from the nitrogen, the toxicant will not be lost from
the sample. Because conditions cannot be selected
to optimize transfer, longer sparging times should be
chosen.
8-21
-------
The first experiments should involve no pH changes if
any measurable change in toxicity occurred in the
earlier tests without pH change. The reason for this
selection is that drastic changes in pH can cause so
many unknown effluent changes, and artifacts are
likely to occur. Of course if only pH changes caused
toxicity changes, then pH will have to be altered.
When pH is altered, then equilibrium objectives,
mentioned above, are not possible and the entire
process takes on characteristics of more conventional
purge and trap experiments. The usual resin traps
described in EPA methods are not suitable because
the trap cannot be tested for toxicity. The trapping
medium must be, or be able to be, made into a
toxicity testable water.
If sparging only affects toxicity with pH change, then
the system should be operated in a conventional
manner. That is, the trap volume should be small
relative to the sample volume and the trap pH should
be opposite the sample pH (e.g., if the sample pH is
3, then the trap pH should be 10). One can no longer
conclude anything about the original effluent
equilibrium and the procedure is one of separation.
Toxicity in the trap may or may not be the same
substance as the one causing original effluent toxicity.
Obviously, all the precautions mentioned above
regarding NaCI addition and other adjustments must
be tracked with blanks just as in any other
experiment. We have not found many effluents where
the transfer technique is useful but for those effluents
where it works, it is a powerful tool.
8.6 pH Adjustment/CIS Solid Phase
Extraction Test
Principles/General Discussion:
The solid phase extraction (SPE) test is designed to
determine the extent of effluent toxicity caused by
those organic compounds and metal chelates that are
relatively non-polar. The effluent is passed through a
small column packed with an octadecyl (Cis) sorbent.
Compounds in the effluent interact through solubility
and polarity with the sorbent and are extracted from
the effluent onto the sorbent. This type of
chromatography in which the mobile phase (the
effluent) is polar and the solid phase (C18 sorbent) is
non-polar, is referred to as reversed phase, SPE.
Any organic compound present in water can be
considered "soluble" by virtue of its presence in the
water. Obviously relative degrees of water solubility
exist. Many highly toxic pollutants found in effluents at
very low concentrations are not considered water
soluble despite the fact they are present at toxic
concentrations.
Compounds extracted by the C18 sorbent from a
neutral aqueous solution are usually soluble in hexane
or chloroform. The C18 sorbent can also be used to
extract organic acids and bases as organic acids and
bases can be made less polar by shifting the
equilibrium to the un-ionized species. By adjusting
the effluent to a low pH and a high pH, some of these
compounds will exist predominately in the un-ionized
form and will sorb to the C18 column. Because of
C18 column degradation, the use of pHs above 10
and below 2 are not used. To ensure column
integrity, pH of the effluent will be lowered only to 3
and raised only to 9 (not 11) in this test.
Manufacturer's data should be consulted for tolerable
column pH ranges and for exact column conditioning
proceedures which must be done to get proper
performance.
Volume Required:
A maximum volume of 35 ml_ of solution at each pH
is required for the pH adjustment/filtration toxicity
testing, and the additional 200 ml_ volume is pumped
through the C\Q column. The remaining 200 mL
volumes of filtered pH 3, pH 11, and pH| effluent from
Section 8.4 are used in this test. Each toxicity test is
conducted on four exposure concentrations (10 mL
each). The exact volume required for the toxicity test
on each pH adjusted or pH| post-column effluent will
depend on the toxicity of the effluent (the 24 hour
Initial LC50).
Apparatus:
Six-250 ml graduated cylinders, 8-25 mL
graduated cylinders, burettes for acid and base
titrations, pH meter and probe, stir plate,
perfluorocarbon stir bars, pump with sample reservoir,
perfluorocarbon tubing, ring stands, clamps, 3-3 mL
Cis SPE columns (200 mg sorbent), automatic
pipette (10 mL), disposable pipette tips (10 mL), eye
dropper or wide bore pipette, light box and/or
microscope (optional).
Reagents:
HPLC grade methanol, high purity water, 0.01 N
NaOH, 0.012 N HCI (ACS grade in high purity water),
buffers for pH meter calibration, and solvents for
cleaning the pump and reservoir.
Test Organisms:
Test organisms, 135 or more, of the same age and
species.
Procedure (Day 1):
The first step (Figure 8-13) in the test involves
conditioning the solid phase extraction column.
Column conditioning procedures may vary with the
manufacturer of the column. The procedures
described in this document are modifications of the
8-22
-------
Figure 8-13. Step-wise diagram for C18 solid phase extraction column test samples.
Step
1
Step
2
Step
3
HCI ->,£
Prepare C18 SPE Columns (3)
• 25 mL methanol
• 25 mL high purity water
DO NOT LET SORBENT GO DRY
200 mL pH 3
Filtered Water
+
Prepared
Column
-*•
Collect 1 0 mL Sample
L — NaOH
_ _J. .
Toxicity
Test
£~ HCI
200 mL pH 3
Filtered Water
•*•
Prepared Column
from Step 2*
±
Collect 30 mL Samples
After 25 mL & 150 mL
>
L DISCS
r Wate
Dilution Water
I I
4r
I
200 mL pH0
Filtered Water
+
Prepared Column
•*•
Collect 1 0 mL Sample
I
*
Toxicity Test
Effluent Sample
|
^
I
200 mL pH,
Filtered Water
4-
rd Methanol &
r After Rinses
•J
^ — NaOH
200 mL pH 9
Filtered Water
+
Prepared Column
•*•
Collect
10 mL Sample
i
4 — HCI
r
Toxicity Test
NaOH ^l
200 mL pH 9
Filtered Water
•*•
Prepared Column
from Step 2"
.^
Collect 30 mL Samples
After 25 mL& 150 mL
Prepared Column
from Step 2*
^
Collect 30 mL Samples
After 25 mL & 150 mL
4 — NaOH _M HCI
Toxicity Test
Toxicity Test
Toxicity Test
Day
1
Day
2 .
Day
1
Day
2
"Use same column used with the dilution water.
conditioning steps used with Baker® 1 C-\Q SPE
columns.
Using a flow-rate of 10 mL/min, 25 mL of HPLC
grade methanol is pumped through the column and
discarded. Next 25 mL of high purity water, adjusted
to pH 3 with HCI, is placed in the sample reservoir.
Care must be taken in timing the addition of solutions
after the methanol has passed through the column.
While the mixing of methanol with subsequent
solutions must be minimized, the column must also
1 J.T. Baker Chemical Company, Phillipsburg, NJ.
be prevented from going dry following the methanol'
wash. The amount of time needed between
introduction of solutions to prevent any column drying
will be unique to each investigator's apparatus. This
timing should be determined before performing this
procedure with actual effluent samples. If the column
dries at any time after introduction of the methanol,
the column must be reconditioned (with methanol).
As the last volume of pH 3 high purity water is
entering the column, the filtered pH 3 dilution water is
placed into the reservoir. Again, the column must not
be allowed to dry before the pH 3 dilution water
enters the column. The pH 3 high purity water
8-23
-------
passing from the column should be measured to
determine the point at which the dilution water begins
to leave the column. The pH 3 high purity water used
to condition the column is discarded. The full 200 ml_
of the filtered pH 3 dilution water is collected, and a
10 mL aliquot is taken for the toxicity blank to detect
toxicity leached from the column. This 10 mL aliquot
is readjusted to the initial pH of the dilution water
using 0.01 N NaOH and reserved for day 2 toxicity
testing. Care should be taken to minimize changes in
sample volume and ionic strength during pH
readjustment.
As the last several mL of filtered pH 3 dilution water
are entering the column, the 200 mL volume of
filtered pH 3 effluent is placed in the sample
reservoir. Again, the column sorbent must not be
allowed to dry between the dilution water blank and
the effluent. Collect a 30 mL aliquot of column
effluent after 25 mL of the wastewater passes through
the system. A second 30 mL aliquot is collected after
a total of 150 mL of the effluent passes through the
column. Collection of the first post-column effluent
sample after 25 mL of effluent has passed the column
ensures that any dilution water left in the system will
not be present in the post-column effluent sample.
The second sample of post-column effluent provides
information on column overloading and toxicant
breakthrough. Both of these 30 mL aliquots are
readjusted to the initial pH of the effluent using the
drop-wise addition of 0.01 N NaOH. The total
volume of NaOH necessary for pH adjustment should
be recorded. These aliquots are reserved for day 2
toxicity testing. Columns are not re-used but should
be saved for subsequent elution.
If a dilution water is used, for example receiving
water, in which trace organic contaminants may be
present or in which organics such as humic acid may
be present, the same column should not be used for
the effluent. Rather, a new column should be
conditioned and in place of the 200 mL dilution water
blank, 200 mL of a synthetic dilution water should be
used. It should be checked for toxicity in the same
way. The pH should be adjusted to the same value as
that of the effluent sample.
The above procedure is repeated using a new
conditioned 3 mL C-\Q SPE column and the filtered
pH| effluent (Figure 8-13). Prior to attaching a new
column to the apparatus, the reservoir and pump
must be cleaned with acetone and high purity water.
After the samples are started through the column, the
pH of the aliquots of dilution water and effluent
collected should be checked. It is unlikely however,
that pH readjustment to pH0 and pH| (respectively)
will be necessary. If pH adjustment is necessary it
should be performed using 0.012 N HCI or 0.01 N
NaOH, recording all volumes added.
In the final CIB SPE test, pH 9 (not 11) dilution water
and effluent are chromatographed as described
above. While use of pH 11 effluent offers the likely
advantage of shifting a larger number of basic organic
compounds farther towards the predominately un-
ionized form, and therefore removal, the C-\Q column
will not withstand a pH above 10. For this reason, the
pH 11 filtered dilution water and effluent aliquots
prepared in Sections 8.3 and 8.4 are readjusted to pH
9 with 0.12 N HCI and 0.012 N HCI before application
to the column. The 25 mL of high purity water used to
rinse the column following methanol conditioning must
also be adjusted to pH 9 with NaOH. The single 10
mL aliquot of post-column pH 9 dilution water and
both 30 mL aliquots of post-column pH 9 effluent
are further adjusted to pH0 and pH| respectively, prior
to toxicity testing. The total volume of acid added for
pH readjustment is recorded. The pH of all aliquots of
the chromatographed dilution water and effluent
should be checked and readjusted as appropriate
throughout the remainder of the work day.
(Day 2): The pH of all of the post-column dilution
water and effluent aliquots should be checked and
readjusted if pH has drifted over night. Toxicity tests
are performed on a single 100% concentration of all
three of the dilution water blanks collected. These
blanks will provide information on the presence of
toxicity leached from the Cia column at different pHs.
The six 30 mL post-column effluent aliquots are
tested for toxicity using an exposure series based on
the 24 hour LC50 of the original effluent.
Chromatographed effluent aliquots are tested at
concentrations of 4X-, 2X-, 1X-, and 0.5X-LC50
(cf., Section 8.2 for alternate series). The pH of each
solution tested should be measured daily and
recorded along with organism survival. A sample data
sheet for the GTS SPE test is shown in Figure 8-14.
Interferences/Controls and Blanks:
Controls on test organism performance, dilution water
quality, and such are provided by the control from the
Baseline toxicity test. The pH Adjustment and
Filtration tests (Sections 8.3 and 8.4) provide
information on the effects of pH adjustment and
filtration on effluent toxicity apart from any additional
changes caused by CIQ SPE. Effluent and blank
results from these two tests must be consulted prior
to interpreting the results of the Cis SPE test, both in
terms of identifying any toxic artifacts added during
filtration and pH adjustment and in allocating toxicity
reduction to the three components potentially
impacting effluent toxicants in the Cis SPE test.
The Cis technique is, of those methods so far
discussed, the most dependent on more
manipulations. More problems are likely to be
encountered with toxic blanks because in addition to
those associated with pH adjustment and filtering, the
8-24
-------
Figure 8-14. Example of data sheet for effluent solid phase extraction test with and without pH adjustment.
CD
ro
en
Test Type: Cia SPE
Test Initiation (Date & Time):
Investiga
Sample
Date of
%Etf.
cone.
tor:
Log #, Name:
Collection:
PH
3
3
3
3
3
3
3
3
3
vor
25
25
25
25
150
150
150
150
blank
Species//*
No. Anirrtc
Source of
Dilution to
Test Volu
ae:
Jls/No. R(
Animals:
teter/Con
me:
30S.
rol:
Other Information:
Survival Readings:
24 h
A pH
481)
A pH
72 h
A pH
96 h
A pH
PH
PH|
PH,
PH,
PH|
pH,
PH|
PH|
PH|
PH|
vor
25
25
25
25
150
150
150
150
blank
24 h
A pH
48 h
A pH
72 h
A pH
96 h
A pH
PH
11
11
11
11
11
11
11
11
11
vor
25
25
25
25
150
150
150
150
blank
24 h
A pH
48 h
A pH
72 h
A pH
96 h
A pH
HCI
NaOH
HCI
NaOH
25 ml pH 3
25 mL pH|
25 mL pH 9
"Volume through column.
150mLpH3
150 mL pH(
150mLpH 9
-------
method involves use of resin and methanol as
well. Blanks for toxicity must be checked in the same
manner as before for acid and base addition, filter
toxicity, pH drift, as well as toxicity from the CIQ
column. But in addition to these, some effluents
behave in a peculiar way after passing through the
SPE column. They become toxic sometimes
apparently due to "slime" growth or precipitate and
other times for no apparent reason. Observations and
judgement must be used to detect such problems and
only through experience can one recognize these
when they occur. Failure to recognize them will result
in the conclusion that the C^Q column did not remove
toxicity when it in fact may have.
Results/Subsequent Tests:
The above unique properties of some effluents and
the potential for blank toxicity problems make
interpretation of results more subjective. If toxicity is
not reduced in post-column effluent, not too much
credence should be placed on the results. Going
back and sorting through the possible causes can be
very time consuming. If none of the other Phase I
treatments have affected toxicity, a wise choice is to
elute the columns with 100% methanol. If a 2 ml_
volume of methanol is used, and the sorption and
elution efficiency are 100%, any substances retained
by the columns will be concentrated 100 times. If 150
pL of the methanol fraction is diluted to 10 ml_ in
dilution water, the methanol concentration is 1.5%
and below the LC50 for all species given in Table 8-
3. This provides a concentration of effluent
constituents 1.5X whole effluent concentration. This
small amount of concentration over whole effluent
allows detection even if some loss occurred either in
sorption or elution. One should run a methanol blank
for comparison to the fraction test.
If toxicity is found in the fraction, proceed to Phase II
for identification of that component of toxicity.
If the post-column effluent is not toxic, then
considerable weight can be attached to the results.
The next step is to go on to Phase II CIQ procedures
for that toxicity removed by the column. Some
checking should be done to assure that the GIB
column has not just served as a smaller pore size
filter than the glass filter used to filter the sample
before passing it through the column. This caution is
especially applicable to the samples in which pH was
adjusted.
8.7 Oxidant Reduction Test
Principles/General Discussion:
This test is designed to determine to what extent
constituents reduced by sodium thiosulfate are
responsible for effluent toxicity. Chlorine, a commonly
used biocide and oxidant, is frequently found at
Table 8-3. Toxicity ol Methanol to Several Freshwater
Species.
Species
Ceriodaphnia
dubia
Daphnia
magna
Daphnia pulex
Hyalella
azteca
Sa/mo
gairdneri
Pimephales
promelas
Lepomis
macrochirus
Life-
stage
<6 ha
<24 ha
<48ha
<24 hb
<14hd
juvenile0'
juvenile'
<24 ha
28-32 df
juvenile'
LC50 (%, v/v (95% Cl))
24 h
>3.0
(--)
2.7
(2.6-2.9)
2.4
(2.2-2.6)
NR
2.56
(2.3-2.8)
2.5e
(1.9-2.8)
2.5
(2.5-2.7)
4.0
(-)
3.8
(3.7-3.9)
2.4
(2.2-2.7)
48 h
>3.0
(--)
2.7
(2.6-2.9)
2.0
(1.9-2.2)
3.2C
(2.5-3.7)
NR
NR
2.5
(2.5-2.7)
4.0
(")
3.8
(3.7-3.9)
2.4
(2.2-2.7)
72 h 96 h
NR NR
NR 2.5
(2.5-2.7)
3.7 3.7
(3.2-4.2) (3.2-4.2)
NR 3.7
(3.6-3.9)
NR 1.9
(1.8-2.3)
a Data for C. dubia and fathead minnows were generated at ERL-
Duluth. C. dubia were fed. Dilution water used was diluted mineral
water.
Randall and Knopp, 1980. (Tested in spring water; static &
unmeasured.)
0 48 h EC50.
d Bowman et al., 1981. (Tested in well-water, static &
unmeasured.)
6 18HLC50.
1 Poirier et al., 1986. (Tested in Lake Superior water, and all tests
were measured and flow-throughs.)
Note: (--) Confidence interval cannot be calculated as no partial
mortality occurred. NR = Not reported.
acutely toxic concentrations in municipal effluents.
Other chemicals used in disinfection (such as ozone,
and chlorine dioxide), formed during chlorination,
(such as mono and dichloramines), and compounds
such as bromine, iodine, manganous ions and some
electrophile organic chemicals are also neutralized in
this analysis.
This test does not simply affect chlorine toxicity.
Chlorine is unstable in aqueous solutions and
decomposition is more rapid in solutions when
chlorine is present at low concentrations. Phase I
Initial and pH 3 Aeration tests will provide information
on chlorine toxicity as will the Oxidant Reduction
Test.
In this test, varying quantities of sodium thiosulfate
(Na2S2O3), are added to aliquots of the effluent to
produce increasing ratios of reducing agent/total
thiosulfate reducible constituents. Frequently, the
reduced form of the toxicant has a much lower
toxicity.
Data available for Ceriodaphnia dubia, Daphnia
magna and fathead minnows (Table 8-4) show that
sodium thiosulfate has a low toxicity. Other data
8-26
-------
generated at ERL-D shows that for Ceriodaphnia,
both feeding and reduced hardness lowers the LC50
value. The LC50 for Ceriodaphnia ranges from 300 to
3,300 mg/L in reconstituted water of 10 to 300 mg/L
(as CaCOs) hardness in fed tests. Even at the lowest
value, 300 mg/L is nearly equal to 2 M sodium
thiosulfate. Some of the added thiosulfate will
combine with certain oxidants present in effluent, thus
lowering the concentration of reactive, toxic
thiosulfate. The LC50 values shown are therefore
lower than might be expected in effluents because in
reconstituted water, the thiosulfate is not likely to
react. More importantly, since an effluent
concentration of 4X the LC50 is tested, toxic oxidant
levels should not be excessively high, as might be the
case if 100% effluent were always tested. As a result
there should not be a need to add very large amounts
of thiosulfate to neutralize toxic oxidants in the test
solution.
Table 8-4. Toxicity of Sodium Thiosulfate to
Ceriodaphnia, Daphnia, and Fathead
Minnows.
LC50 (g/L) (95% Cl)
Species
Ceriodaphnia
dubia3
Daphnia
magnab
Pimephales
promelasa
24 h
2.5
(--)
2.2
(NR)
8.4
(7.6-9.3)
48 h
0.85
(0.72-1.0)
1.3
(NR)
8.4
(7.6-9.3)
72 h
79
(7.4-8.5)
96 h
7.3
(6.4-8.3)
a Data for C. dubia and fathead minnows were generated at
ERL-Duluth. Both species were < 24 h old at test initiation and
C. dubia were fed. Dilution water used was diluted mineral
water.
b Dowden and Bennett, 1965; dilution water was an artificial water.
Note: (--) Confidence interval cannot be calculated as no partial
mortality occurred. (NR) Not reported.
The approach is to add a concentration (at the
highest addition) that is approximately equal to the
^28203 LC50 of the test species. If the test species
is not listed in Table 8-4, an N32S203 LC50 will
have to be determined.
For cases where oxidants account for only part of the
toxicity, sodium thiosulfate may only reduce the
toxicity as opposed to completely eliminating it. Time
to mortality must be measured rather than observing
mortality at a fixed time. Time to mortality
measurements are necessitated as there are no
dilutions from which to calculate an LC50. In other
sections of Phase I, except for Section 8.7 and 8.8,
we fix the time at which mortality is measured and
vary the exposure concentrations. The endpoint is the
estimated concentration that will kill half the
organisms in a specified time. In time to mortality
tests, the concentration is fixed and the time required
to kill 50% of the organisms is measured. Obviously,
to do so requires frequent mortality observation.
Since toxicity is a function of time and concentration,
both LCSOs and LTSOs (lethal time to 50% mortality)
are measures of the same property.
This test is useful even when chlorine appears to be
absent in the effluent. Oxidants other than chlorine
occur in effluents and this test should not be omitted
just because the effluent is not chlorinated. Likewise,
removal of toxicity by thiosulfate does not prove that
chlorine was the cause of effluent sample toxicity.
Volume Required:
A maximum volume of 100 mL effluent is required for
the Oxidant Reduction Test. The exact volume
required will depend on the 24 hour Initial LC50. The
test requires 10 effluent aliquots at 4X-LC50 or
100%.
Apparatus:
Glass stirring rods, 1 mL glass pipettes, automatic
pipette (10 mL), disposable pipette tips (10 mL), 10
and 100 microliter syringes, eye dropper or wide bore
pipette, light box and/or microscope (optional).
Reagents:
The sodium thiosulfate stock concentration should be
10X the N32S2O3 LC50 concentration for the test
species being used. The stock solution should be
prepared in freshly boiled water.
Test Organisms:
Test organisms, 50 or more, of the same age and
species.
Procedure (Day 2):
To perform the test, put 10 mL aliquots of effluent
diluted to 4X-LC50 (or 100%) in 10 test chambers.
Add 1.0, 0.8, 0.6, 0.4, 0.2, 0.1, 0.05, 0.025, and
0.012 mL to nine aliquots (mix), and do not add any
to the tenth. The treatment receiving 1 mL should
contain the approximate concentration of sodium
thiosulfate equal to the LC50 of the test species.
Figure 8-15 contains an example form for recording-
the data. A suggested schedule for observing time to
mortality is shown on the data form.
Interferences/Controls and Blanks:
Controls prepared for the Baseline toxicity test act as
a check on the general health of test organisms,
dilution water quality and test conditions. By
comparing the time to mortality in the various
Na2S2Os exposure concentrations with that in the
treatment without thiosulfate, one can determine
whether the addition of N32S2O3 increased time to
mortality at some thiosulfate concentration. If, in all of
8-27
-------
Figure 8-15. Example of data sheet for the oxidant reduction test.
Test Type: OXIDANT REDUCTION
Test Initiation (Date & Time):
Investigatory
Sample Log #, Name:_
Date of Collection:
Species/Age: _
No. Animals/No. Reps:
Source of Animals:
Dilution Water/Control:,
Test Volume:
Other Info:
Stock =
Comments:
4X-LC50:_
TRC:
or 100%
mL Stock
added
1.0
0.8
0.6
0.4
0.2
0.1
0.05
0.025
0.012
0.0
Survival Readings:
2h
4 h
8h
10 h
24 h
48 h
72 h
96 h
g/L Na2S2O3
8-28
-------
the effluent exposures, time to mortality decreases as
the volume of sodium thiosulfate added increases the
test should be repeated with a 10 times weaker
Na2S2Os solution. If a significant loss in effluent
toxicity is apparent over the first 24 hour period after
sample arrival in the laboratory (i.e., Initial LC50 <
Baseline LC50), it may be necessary to conduct
future Oxidant Reduction tests immediately upon the
sample's arrival in the laboratory.
Results/Subsequent Tests:
If oxidants are causing toxicity, time to mortality
should increase somewhere in the range of tested
thiosulfate additions. No change in toxicity suggests
either no oxidant toxicity or not enough thiosulfate
was added. The experiment should be repeated,
increasing the concentration of thiosulfate added.
If oxidant toxicity is evident, a measurement of free
chlorine should be made and the concentration
compared to the chlorine toxicity value for the test
species used. Mono and dichloramine should also be
measured since they have different toxicities than free
chlorine (see Phase III for confirming mixtures as
toxicants). A comparison of Aeration and Cis SPE
Test results to the Oxidant Reduction Test results
may provide even more information on the
physical/chemical nature of the oxidants.
8.8 EDTA Chelation Test
Principles/General Discussion:
To determine the extent to which effluent toxicity is
caused by certain cationic metals, increasing amounts
of a chelating agent (EDTA) are added to aliquots ot
the effluent sample. The form of the metal (e.g., the
aquo ion, insoluble complex) has a major effect on its
toxicity to aquatic organisms (Magnuson et al., 1979)
and specific metal forms may be more important in
aquatic toxicity than the total quantity of the metal.
Addition of the ethylenediaminetetraacetate ligand
(EDTA), a strong chelating agent, will produce
relatively non-toxic complexes with many metals.
The success of EDTA in removing metal toxicity is a
function of solution pH, the type and speciation of the
metal, other ligands in the solution, and the binding
affinity of EDTA for the metal versus the affinity of the
metal for the tissues of the organism (Stumm and
Morgan, 1981). Because of its complexing strength,
EDTA-metal complexes will often displace other
soluble forms such as chlorides and oxides of many
metals. Among the cations typically chelated by
EDTA are aluminum, barium, cadmium, cobalt,
copper, iron, lead, manganese ( + 2), nickel, strontium
and zinc. EDTA will not complex anionic forms of
metals such as selenides, chromates and
hydrochromates and forms relatively weak chelates
with arsenic and mercury. For those metals with
which it forms relatively strong complexes, the toxicity
of the metal to aquatic organisms is frequently
reduced.
Since EDTA will complex relatively non-toxic metals
(e.g., calcium, magnesium) as well as more toxic
heavy metals, fairly concentrated solutions are
needed. The mass of chelating agent required should
be approximated because EDTA can become toxic
when present above a certain concentration. The
range of EDTA concentrations that will adequately
bind the metals but not be toxic appears to be smaller
than that for sodium thiosulfate and oxidants.
Table 8-5 contains LC50s for disodium EDTA at
various hardness and salinity values to Ceriodaphnia
and fathead minnows. Note that the concentration of
EDTA tolerated by organisms increases directly with
both water hardness and salinity. By measuring the
hardness and salinity of the effluent, the range of
EDTA concentrations that should not be toxic in an
effluent sample can be estimated. "Salinity" not due
strictly to NaCI will have different effects on toxicity.
This calculation, for prediction of the EDTA
concentration, is more involved than is at first
apparent. The data in Table 8-5 indicate that over
the physiological range of hardness and salinity,
hardness affects the toxicity of EDTA more than does
sodium chloride. The usual methods for measurement
of salinity (conductivity meter, salinometer or
refractometer) do not specifically measure sodium
chloride. The choice of EDTA concentrations should
always be based first on hardness and secondly on
salinity only when the salinity is known. The particular
combination of hardness and salinity present in an
effluent sample may have to be tested to get an
accurate EDTA LC50. If the salinity is composed of
ions other than sodium and chloride, the hardness of
the dilution water should be made equal to the
effluent hardness and the additional "salinity" added
in the form of other major cations and anions such as
potassium, sulfate and carbonate.
An EDTA LC50 value derived in a standard toxicity
test water is likely to be much lower than the LC50
for EDTA in effluent. For example, the values
contained in Table 8-5 represent worst case
conditions presented by EDTA in relatively pure
water. Likewise, the toxic concentration of EDTA in
one effluent will not be the same as the concentration
causing toxicity in a different effluent or even a
different sample of the same effluent. For this reason
the concentrations of EDTA added to the effluent
aliquots should bracket the expected LC50 based on
clean water with a similar salinity and hardness value
as per the above discussion.
As in the pH Adjustment and Oxidant Reduction tests,
the effluent itself is used as a control rather than a
blank based on dilution water. By adding increasing
concentrations of EDTA to each effluent aliquot, at
8-29
-------
Table 8-5. Toxicity of Disodium EDTA to Ceriodaphnia dubia and Fathead Minnows in Water of Various
Hardnesses and Salinities.
Species
Ceriodaphnia
dubia
Pimephales
promelas
Water Type
Very Hard
Hard
Mod. Hard
Soft
Very Soft
Soft
Soft
Soft
Soft
Very Hard
Hard
Mod. Hard
Son
Very Soft
Sort
Soft
Soft
Soft
Water Hardness Salmitya
(mg/L as CaCO3) (ppt)
280-320
160-180
80-100
40-48
10-13
40-48 3
40-48 2
40-48 1
40-48 0.5
280-320
160-180
80-100
40-48
10-13
40-48 3
40-48 2
40-48 1
40-48 0.5
LC50 (g/L) (95% Cl)
24 h
0.71
(.S8-.87)
0.50
(.42-.60)
0.23
(.21 -.27)
0.12
(.10-.13)
0.04
(.03-.04)
0.44
0.33
(.27-.41)
0.12
(.10-.13)
0.05
0.81
(.6S-.97)
0.54
(.43-.66)
0.29
(.23-.3S)
0.14
(.12-.18)
0.04
(.03-.04)
48 h
0.41
(.36-.47)
0.44
0.22
0.11
0.03
0.32
(.23-.4S)
0.23
(.21-27)
0.11
0.05
0.81
( 68-. 97)
0.50
(.40-. 62)
0.27
(.22- 33)
0.14
(.12-.18)
0.03
(.03-.04)
72 h 96 h
0.81 0.81
(.6S-.97) (.S2-.83)
0.47 0.44
(.36- 60) (.34:56)
0.27 0.25
C22-.33) (.20-.31)
0.11 0.08
(.08- 14) (.07-.09)
0.03 0.03
(.03-.04) ( 02-.04)
0.37
(.2S-.48)
0.23
(.17-.32)
0.17
(-13-.21)
0.11
a Brine from evaporated seawater used as source of salinity. All data generated at ERL-Duluth using synthetic waters described in
Horning and Weber, 1985. All C. dubia were <24 h old and the fathead minnows were all <36 h old at test initiation.
Ceriodaphnia were fed during exposure.
(--) Confidence interval cannot be calculated as no partial mortality occurred.
some addition the metals will be chelated but
unbound EDTA will not be present at toxic
concentrations. The goal of this test is to add enough
EDTA to reduce metal toxicity. This is most easily
achieved by adding insufficient EDTA in the lowest
EDTA additions so metal toxicity is not removed. In
the midrange of EDTA additions, the metals will be
rendered non-toxic by the EDTA, and at the high
end of the range of EDTA additions, the unreacted
EDTA is itself toxic. By using an effluent
concentration of 4X-LC50 or 100% if the LC50 is
greater than 25%, the potential for exceeding the
binding capability of the added EDTA is lessened,
especially for very toxic effluents {LC50 < 10%).
Volume Required:
A volume of 100 mL effluent usually is required for
the EDTA Chelation test. The exact volume needed
will depend on the 24 hour Initial LC50 and the
particular option chosen to determine the EDTA
concentration series required.
Apparatus;
Glass stirring rods, burettes for EDTA addition,
automatic pipette (10 mL), disposable pipette tips (10
mL), 10 and 100 pL syringe, eye dropper or wide
bore pipette, light box and/or microscope (optional).
8-30
-------
Reagents:
EDTA stock solution (see discussion under
"Procedure"), reagents for determination of effluent
hardness and salinity (see APHA, 1980; Methods 314
and 210).
Test Organisms:
Test organisms, 50 or more, of the same age and
species.
Procedure (Day 1):
There are four options to determine the concentration
of EDTA to add. The most accurate approach, when
it can be used, is to measure the hardness of the
4X-LC50 effluent concentration (or 100% when the
LC50 is >25%) using the standard method for
measuring hardness (APHA, 1980). The concentration
of EDTA that produced the endpoint for the effluent
sample is the concentration of EDTA needed at the
midrange of EDTA additions in the toxicity test. An
example will illustrate the calculation. In a 36%
effluent sample (4X-LC50), 5 ml of 0.01 M EDTA
was needed to titrate the hardness (100 ml_ sample
size). This amount corrected for a 10 ml_ sample
would be 10/100 X 5 mL = 0.5 mL. Therefore 0.5 mL
of 0.01 M EDTA added to a 10 mL sample of 36%
effluent provides the EDTA concentration desired at
the midrange. To provide this EDTA concentration at
the 0.2 mL addition, increase the 0.01 M
concentration by 0.5/0.2 or 2.5X = 0.025 M EDTA
stock. (Molecular weight (MW) of N32EDTA is 372.3
g.) When the hardness measurement endpoint cannot
be discerned because of interferences, other options,
described below, can be used.
A second option is to measure the calcium and
magnesium of the sample, and calculate the amount
of EDTA needed to chelate the calcium and
magnesium. EDTA binds with both Ca2+ and Mg2 +
on a 1:1 basis. The total number of moles of Ca2 +
(MW = 40.08) and Mg2+ (MW = 24.305) in 10 mL of
effluent at 4X-LC50 must equal the number of moles
of EDTA added to the same effluent sample. This
calculated concentration should be the one added at
the midrange of EDTA additions. The calcium and
magnesium should be measured at 100% if the LC50
is greater than 25%.
The third option is to increase the range of EDTA
concentrations tested rather than attempt to measure
the calcium and magnesium and calculate the needed
EDTA. Our experience is that setting up the toxicity
test with more EDTA concentrations may be easier
than specifically measuring the calcium and
magnesium.
A fourth option is to set the EDTA concentration at
the midrange, equal to the EDTA LC50 concentration.
Choice of the EDTA LC50 must be based on effluent
hardness (and salinity). It may be necessary to
determine the EDTA LC50 for the particular
combination of effluent salinity, and hardness and test
organism used.
(Day 2): To perform the test, 10 aliquots (or more
depending on the option chosen) of the effluent are
prepared at a concentration equal to 4X-LC50, or
100% effluent where the LC50 is greater than 25%
(where the LC50 value is calculated from the 24 hour
Initial toxicity test results). Next, 1.0 mL of the EDTA
stock is added to the first 10 mL aliquot of the
effluent. To the second 10 mL sample of effluent, 0.8
mL is added, to the third, 0.6 mL, and so on until the
fifth 10 mL effluent sample has received 0.2 mL.
Continue the procedure with four more 10 mL aliquots
of the 4X-LC50 (or 100%) effluent using 0.1, 0.05,
0.025, and 0.012 mL of EDTA stock. The tenth is a
blank used to compare treatment effects on time to
mortality. A microliter syringe will be needed for the
smaller additions. If more than 1 mL of EDTA is
required, a stronger stock concentration of EDTA
should be used. If the effluent has a low toxicity
(LC50 = 50-100%) a series of dilution blanks may
be necessary to check for the dilution effect of the
EDTA stock addition. No more than 10% dilution of
the effluent aliquots should be allowed unless a
dilution blank series is included. Special care must be
taken to mix the contents of each chamber before
introducing the test organisms. A sample data
collection sheet is shown in Figure 8-16.
Interferences/Controls and Blanks:
Controls prepared for the Baseline Toxicity test
provide quality control for test organisms, dilution
water and test conditions. The untreated aliquot acts
as a blank for use in determining the presence of
EDTA toxicity. Time to mortality at each EDTA
addition is compared to the untreated aliquot. If time
to mortality is shorter in all treatments, repeat the test
using a lower EDTA range. If time to mortality is not
reduced in any treatment, repeat using a higher range
of EDTA concentrations. Erratic patterns in mortality
cannot be used. If this occurs it suggests that this
test is not useful for the particular effluent being
studied.
Results/Subsequent Tests:
If the appropriate EDTA concentration range is
utilized, the time to mortality will not change from that
seen in the exposure 4X-LC50 of unaltered effluent
at low additions of EDTA. In the midrange, toxicity will
be reduced and at high additions of EDTA, toxicity will
be as high or higher due to unbound EDTA toxicity
and effluent toxicants other than chelatable metals if
present. Time to mortality must be used to detect
partial toxicity removal.
8-31
-------
Figure 8-16. Example of data sheet for the EDTA chelation test.
Test Type: EDTA CHELATION
Test Initiation (Date & Time):
Investigatory
Sample Log #, Name:_
Date of Collection:
Stock =
Comments:
Species/Age:_
No. Animals/No. Reps:
Source of Animals:
Dilution Water/Control:
Test Volume:
Other Info:
4X-LC50:
g/L EDTA
or 100%
mL Stock
added
1.0
0.8
0.6
0.4
0.2
0.1
0.05
0.025
0.012
0.0
Survival Readings:
2h
4 h
8h
10 h
24 h
48 h
72 h
96 h
8-32
-------
Toxicity may be removed at all exposures if the
lowest addition of EDTA removes metal toxicity and
the highest addition does not cause EDTA toxicity. If
toxicity is not reduced in any treatment, either the
effluent has no chelatable metal toxicity or not enough
EDTA was added. Increased toxicity over untreated
effluent suggests EDTA toxicity and a lower EDTA
range should be tested.
If toxicity is reduced in a systematic manner, proceed
to Phase II methods for specific identification of the
metal(s).
8.9 Graduated pH Test
Principles/General Discussion:
This test is designed to determine whether effluent
toxicity can be attributed to ammonia. The test will not
confirm ammonia as the toxicant (cf., Phases II and
III) but will indicate whether its presence in the
effluent should be further investigated.
Ammonia is specifically addressed in Phase I because
of its frequent presence in municipal and industrial
effluents. Total ammonia concentration in an effluent
sample does not relate very well to the toxicity
because the toxicity of ammonia is a function of DO,
pH, and temperature. The pH has a very large effect
on ammonia toxicity and for many effluents, especially
POTW effluents, pH rises upon contact with air such
as in a toxicity test. Literature data can be used only
as a general guide because of the large effect of very
small pH changes. These are usually not reported
fully enough to be useful.
One might expect ammonia to be removed during the
pH 11/Aeration test. Based on the authors'
experience, ammonia is not readily reduced below
concentrations toxic to aquatic organisms. Other
techniques which can be used to remove ammonia-
related toxicity may also displace other toxicants with
completely different physical and chemical
characteristics. For example, ion exchange resins
(e.g., zeolite) in addition to removing ammonia, may
also remove toxic organic compounds through
adsorption. For this reason, a specific test to address
toxicity related to this common pollutant is useful.
Ammonia acts as a basic compound in water. The
un-ionized, more toxic form, NHa, predominates
above pH 9.3 and the ionized, essentially non-toxic
form, NH4 + , is most abundant below this pH at
25°C. Through the pH range from 7.0-8.0, the
percent of total ammonia in the toxic form increases
rapidly. It is important to note that the toxicity of the
toxic form decreases with increasing pH (EPA, 1985).
Therefore, the increase in the concentration of the
toxic form is partially compensated by the decrease in
toxicity. As pH increases, the percentage in the toxic
form is greater but the toxicity of the toxic form is
less. As pH decreases, the percentage of total
ammonia as NHs decreases, but the toxicity of NH3
increases. Temperature also affects dissociation of
ammonia but since it is usually held constant in
toxicity tests, it can be ignored for purposes here.
The increase in the percent of total ammonia
occurring in the toxic form with increasing pH is
greater than the decrease in its toxicity. The net
result is an increase in toxicity, based on total
ammonia in the sample.
These opposing effects of pH change can be used to
detect the presence of ammonia toxicity. By diluting
aliquots of effluent to the 24 hour Initial LC50 and
adjusting each aliquot to a different pH value, effluent
toxicity can be greatly enhanced or completely
eliminated. For example, at pH 6 and 25°C, 0.0568%
of the total ammonia in solution is present in the toxic
form. At pH 7 and 25°C, 0.566% of the total
ammonia is present as NHs and at pH 8, 5.38% is
present in the un-ionized form. Similar changes in
percent NHs for pHs 6, 7, and 8 occur at other
temperatures, if a test temperature different from
25°C is needed. This difference in the percent of
un-ionized ammonia is enough to make the same
amount of total ammonia about 3 times more toxic at
pH 8 as at pH 6. Whether or not toxicity will be
eliminated at pH 6 and the extent to which toxicity will
increase at pH 8 will depend on the total ammonia
concentration. But if a dilution of effluent equal to the
LC50 is used for pH adjustment, this should
"normalize" the total ammonia concentration and one
should see toxicity differences between pH 6 and 8.
Either pH 8 will have a higher toxicity than the
unadjusted effluent LC50 or pH 6 will have a lower
toxicity depending on the pH of the unaltered effluent.
Perhaps the greatest challenge faced in this test is
that of maintaining a constant solution pH. This is a
necessity if the concentration of un-ionized ammonia
is to remain constant and the test results are to be
valid. In conducting toxicity tests on effluent, it is not
unusual to see the pH of the test solutions with high
effluent concentration drift 1 to 2 units greater over a
48 to 96 hour period (see Procedure for suggestions).
Volume Required:
Depending on the method chosen for pH stabilization,
120 mL to 1,500 mL of effluent is required for this
test. The volume used is split into three aliquots and
tested at the 24 hour Initial LC50 concentration,
without dilution or replication.
Apparatus:
Burettes for acid and base addition, 3-50 mL
beakers1, Parafilm®! or 3-600 mL beakers2, wire
mesh test chambers2 (described below), magnetic
stirrers, and perfluorocarbon stir bars, eye dropper or
8-33
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wide bore pipette, light box and/or microscope
(optional).
Reagents:
1.0 and 0.1 N NaOH2 or 0.1 N and 0.01 N NaOHl,
1.2 N and 0.12 N HCI2 or 0.12 N and 0.012 N HCI1.
Test Organisms:
Test organisms, 15 or more, of the same age and
species.
Procedure (Day 2):
If the dissolved oxygen content of the effluent is
adequate and BOD is not high, the following small
volume pH stabilization procedure should be tried. An
aliquot of wastewater is diluted to the 24 hour Initial
LC50 concentration using dilution water for a final
volume of 120 ml. A 40 ml_ portion of this solution is
adjusted to pH 7 using 0.12 and 0.012 N HCI or 0.1
and 0.01 N NaOH. Caution must be taken to minimize
the volume of acid or base added to the test solution.
To ensure this, as the pH nears 7.0, titration with the
stronger acid or base solution should be subrogated
with the addition of the weaker solution of acid or
base. Approximately half of this volume is transferred
into a one ounce disposable transparent test chamber
and five test organisms are added. The remaining
volume is carefully transferred into the test chamber
until the volume of pH 7 effluent rises slightly above
the edge of the container. A section of Parafilm®,
slightly larger in area than the mouth of the test
chamber, is laid on top of the effluent. The Parafilm®
is carefully pulled down around the edges of the test
chamber, caution being taken to prevent test
organism loss at this time. Care must also be used to
exclude any air bubbles during the sealing process.
This procedure is repeated with the other two 33 mL
aliquots of the effluent adjusted to pH 6 and pH 8.
Test organisms can be observed through the
transparent test chamber. The Parafilm® cover should
only be removed at the end of the test period in order
to measure test solution pH and dissolved oxygen.
If the above method does not hold pH or if oxygen is
too low, the following large volume pH stabilization
procedure may be tried.
An aliquot of wastewater is diluted to the 24 hour
initial LC50 concentration for a final volume of 1.5 L.
A 500 mL portion of this solution in a 600 ml beaker
is adjusted to pH 7 using 1.2 N and 0.12 N HCI or 1.0
N and 0.1 N NaOH. Caution must be taken to
minimize the volume of acid or base added to the test
solution. To ensure this, as the pH of the solution
1 Required for the small volume pH stabilization method.
2 Required for the large volume pH stabilization method
nears 7.0, titration with the stronger acid 01 base
should be subrogated by titration with the weaker
solutions. This procedure is repeated with a second
500 mL aliquot, adjusted to pH 6, and a third adjusted
to pH 8.
A 2 inch high stainless steel mesh cylinder (60
mesh/inch) closed at one end with a "Petri dish
type," water-tight bottom is suspended in each of
the 500 mL test solutions. Test organisms are
carefully transferred into these cages. This cylinder
allows organism exposure to the static pH solution
while providing a mechanism for maintaining the
organisms in a small area for observation purposes.
The cylinders -can be removed for test organism
observation without incurring harm to the animals.
They can also be removed as necessary for pH
readjustment. Solution pH should be checked (and
readjusted as necessary) every half hour during the
first few hours and as needed for the rest of the test.
DO should also be measured to be sure it is
adequate. Records of pH drift and DO levels should
be kept. An example data collection sheet for the
Graduated pH test is contained in Figure 8-17.
The "cage" method described above is useful
because the cages can be placed under a
microscope for observation. Remember that the DO
need is not as high as is usually required in
conventional toxicity tests. If the DO is not at or below
a lethal level for the duration of the test, the DO can
be considered adequate. More importantly, the DO
should be comparable in each test vessel.
If pH drifts from nominal more than 0.1 or at most 0.2
pH units, the results may be unusable. Short of
flow-through testing other approaches have to be
used.
An alternative is to use higher effluent concentrations,
perhaps 4X-LC50, adjusting pH initially and every 10
or 15 minutes as needed to maintain it at nominal.
Then observe the animals every 15-30 minutes and
note onset of symptoms. Ammonia does not have a
long lag period before symptoms develop, so the
observations can be done in a work day. Care will
have to be taken to avoid trauma to the animals
during pH adjustment.
Interferences/Controls and Blanks:
Blanks prepared for the Baseline test act as a check
on the general health of test organisms, dilution water
quality and test conditions. The Baseline test acts as
a control for the detection of problems (especially in
the smalt volume pH stabilization method) and
problems associated with acid or base addition. If the
Baseline effluent pH is close to the pH of any of the
pH adjusted test solutions, the toxicity should be
similar. Significantly greater toxicity suggests that an
interference from other factors (cf., Section 8.3 for
discussion on ionic strength related toxicity).
8-34
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Figure 8-17. Example of data sheet for the graduated pH test.
Test Type: GRADUATED pH TEST
Test Initiation (Date & Time):
Investigator:
Sample Log #,
Date of Collect
Name:
on:
Species/Age:_
No. Animals/No. Reps:_
Source of Animals:
Dilution Water/Control:,
Test Volume:
Other Info:
1X-LC50:
or 100%
pH
6.0
7.0
8.0
Survival Readings:
2h'
A pH DO
4h*
A pH DO
6h"
A pH DO
24 h
A pH DO
48 h
A pH DO
72 h"
A pH DO
96 h'
A pH DO
Not performed in small volume pH stabilization method.
Comments:
8-35
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Other compounds with toxicities that increase directly
with pH may lead to confounding results or may give
results similar to ammonia. Phase II contains a
suggested test to more specifically identify ammonia
as the cause of toxicity.
Results/Subsequent Tests:
When ammonia is present in the effluent at toxic
levels, the pH 6 test solution should be less toxic than
the pH 7 solution, which, in turn, should be less toxic
than the pH 8 solution. However, ammonia is not the
only possible cause. Using the pH at the Baseline
effluent LC50, the relative toxicity of each pH
adjusted solution can be predicted if ammonia is the
sole cause. For example, if, for the Baseline effluent,
the average pH in the 100% concentration in which
no organisms survived was 8.0 and the average pi-
was 7.5 for the 50% concentration in which al
organisms survived, the estimated pH at the LC5C
(71%) could be approximated as 7.7. One would
expect greater than 50% mortality in the pH 8 test
solution and significantly less lethality in the pH 7
solution. One should then proceed to Phase II for
identification. If ammonia is one of several toxicants in
an effluent this procedure may pose problems. For
this reason, if effluent total ammonia levels are
greater than 20 mg/L, it may be appropriate to include
a pH 6 effluent treatment interfaced with other Phase
I tests (cf., Section 9). Methods for further identifying
ammonia as the toxicant can be found in Phase It.
8-36
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Section 9
Time Frame and Additional Tests
9.0 Time Frame for Phase I Studies
The amount of time necessary to adequately
characterize the physical/chemical nature of, and
variability in, an effluent's toxicants will be discharge
specific. Among the factors affecting the length of
Phase I studies for a given discharge is the
appropriateness of Phase I tests to the toxicants, the
existence of long or short-term periodicity in
individual toxicants and to a lesser extent, the
variability in the magnitude of toxicity. An effluent
consistently containing 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. The decision as
to when to go beyond Phase I should be based in
part on the regulatory implications and resources
involved in subsequent actions. Where a great
amount of resources is involved, it is crucial that
Phase I results be adequate.
There are no clearly defined boundaries between
Phase I and Phase II. The "subsequent tests"
described for the characterization methods may be
thought of as intermediate studies between Phases I
and II. In terms of guidance on the TIE'S time frame,
several samples should be subjected to the Phase I
Characterization Test battery. The decision to do
subsequent tests on these samples to confirm or
further delineate initial results is a judgement call and
will depend on how clear-cut the results of Phase I
turn out to be. The time required to perform a
complete Phase I battery on a sample will depend on
many circumstances, not the least of which is how
well organized and experienced the performing lab is
at doing TIEs.
If Phase I tests needed to remove or neutralize
effluent toxicity vary with the sample, the number of
tested samples must be increased. The frequency of
testing should be sufficient to include all major
variability. While true, this statement is of no help.
Again, judgement will have to be used but the
differences seen among samples can be used to
decide when further differences are not being found.
Phase I toxicant 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 many weeks or months this may
take-each problem is unique. The LC50 of
samples can be very different but the same screening
tier tests must be successful in removing and/or
neutralizing effluent toxicity.
The individual Phase I tests previously successful in
changing toxicity should be used as a starting point
for Phase II identification. The first step in Phase II
will often be to reduce the number of constituents
accompanying the toxicants. These efforts may reveal
more toxicants than suggested by Phase I testing. In
Phase II one may discover that toxicants of a quite
different nature are also present but were not in
evidence in Phase I. More Phase I characterization
may then be needed.
Phase I results will not usually provide information on
the specific toxicants. Therefore, if effluent toxicity is
consistently reduced for example through the use of
GIB SPE, this does not prove the existence of a
single toxicant. Several non-polar organic
compounds may in fact be causing effluent toxicity
over time, but, the CIQ SPE technique only detects
the presence of these compounds as a group. This is
very important during Phase II toxicant identification.
9.1 When Phase I Tests are Inadequate:
For some effluents, the Phase I tests described above
will provide little or no clue as to the characteristics of
the toxicants. For such effluents, other approaches
must be tried. Some additional approaches are given
below with much less specificity because our
experience with them is minimal. In addition to these,
one should not hesitate to use originality and
innovation to develop other approaches. So long as
toxicity is used to track the changes, any approach
may be helpful.
Activated Carbon
Chemists are reluctant to use carbon because it is
much less selective than ion exchange or SPE
columns and extraction is less precise and more
difficult. Carbon's non-selectivity is an advantage in
9- 1
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some situations. A rather wide array of more specific
methods have failed if Phase I tests above have not
changed toxicity, and therefore a "chemical sponge"
may be useful. In order to start, one must be able to
alter toxicity somehow in order to tell what changes
are occurring. A second objective in early work is a
way to remove the toxicants from the sample (i.e., to
concentrate them). Carbon has a high capability to do
both. Furthermore, the knowledge about carbon
sorption and extraction is large and help can be found
in the literature. True, carbon may alter some
chemicals, but there are many that it does not. We
must recognize that other conventional methods such
as ion exchange are also not specific. Ion exchange
columns can sorb non-polar organics and SPE
columns can sorb metals. Carbon may be very
helpful.
Other Specific Ion Columns
Many other types of resin columns are available
through commercial sources. Many of these have
"insurmountable" blank toxicity problems but some
show promise. Mixed bed ion exchange columns
appear promising because pH is not drastically
altered as the sample passes through the resin bed
and blank problems appear tolerable. Of course with
any of these lessser used methods, the organism's
tolerance must be determined before any chemicals
can be used.
Other LJgands
EDTA reduces toxicity for only part of the cationic
metals. Other ligands such as citrate, nitrilotriacetate,
and cysteine may hold promise.
Clustering Phase I Tests
Our experience suggests independent action and less
than additivity are much more common than we
realize, at least in effluents. When these interactions
occur, interpreting Phase I data may be very difficult
and in some instances, especially with independent
action, no apparent effect on toxicity will be seen
unless Phase I tests are clustered.
The pH of effluents plays an amazingly powerful role
in affecting both form of toxicants and their toxicity.
Including pH adjustments to different values than
suggested in Phase I may be helpful. Combining
EDTA addition with post SPE column sample may
reveal additional information. We have used
aeration/filtration/pH adjustment/Ci8 SPE in various
combinations to decipher changes occurring. The
presence of more than one toxicant may often require
such combinations.
A special effect occurs when an effluent, having two
toxicants at very different concentrations, is diluted.
Suppose toxicant A would produce an LC50 at 50%
effluent and toxicant B causes an effluent LC50 at
5%. In most cases, only toxicant B will materially
affect toxicity because the effect of A will be "diluted
out" long before the LC50 of B is reached. In Phase
I, tests for such an effluent would be done near the
LC50 of B (20% and down) so that the toxicity of A
will not be noticed. If one finds toxicity at effluent
concentrations in the very low range (such as 10%)
additional Phase I testing at higher effluent
concentrations should be done subsequently. Such
cases should be caught in Phase III, but earlier
detection will be much more cost-effective.
The two objectives which usually must be achieved
before the identity of the toxicant can be made is that
the toxicant(s) must be separated and concentrated.
Anything that can be done in Phase I to achieve
these goals will speed the process.
9-2
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Sect/on 10
fleferences
Adelman, I.R., LL Smith Jr., and G.D. Siesennop.
1976. Acute Toxicity of Sodium Chloride,
Pentachlorophenol, Guthion®, and Hexavalent
Chromium to Fathead Minnows (Pimephales
promelas) and Goldfish (Carassius auratus).J.
Fish. Res. Board Can. 33:203-208.
American Public Health Association, American Water
Works Association Water Pollutin Control
Federation. 1980. Standard Methods for the
Examination of Water and Wastewater. 15th
Edition. American Public Health Association,
Washington, DC.
Berg, E. 1982. Handbook for Sampling and Sample
Presentation of Water and Wastewater. U.S. EPA,
Cincinnati, Ohio. EPA-600/4-82-019.
Bowman, M.C., W.L Oiler, T. Cairns, A.B. Gosnell
and K.H. Oliver. 1981. Stressed Bioassay Systems
for Rapid Screening of Pesticide Residues. Part I:
Evaluation of Bioassay Systems. Arch. Environ.
Contam. Toxicol. 10:9-24.
Department of Health, Education and Welfare. 1977.
Carcinogens - Working With Carcinogens. Public
Health Service, Center for Disease Control,
National Institute of Occupational Safety and
Health. Publication No. 77-206.
Dowden, B.F. and H.J. Bennett. 1965. Toxicity of
Selected Chemicals to Certain Animals. J. Water
Pollut. Control Fed. 37(9):1308-1316.
EPA. 1985. Ambient Water Quality Criteria for
Ammonia. EPA-440/5085-001. Environmental
Protection Agency, Environmental Research
Laboratory-Duluth, Duluth, MN, and the Criteria
and Standards Division, Washington, DC.
EPA. 1988A. Draft Toxicity Reduction Evaluation
Protocol for Municipal Wastewater Treatment
Plants. EPA Water Engineering Research
Laboratory, Cincinnati, Ohio. Draft EPA Research
Series Report.
EPA. 1988B. Draft Generalized Methodology for
Conducting Industrial Toxicity Reduction
Evaluations (TREs). EPA Water Engineering
Research Laboratory, Cincinnati, Ohio. Draft EPA
Research Series Report.
Federal Register. 1984. U.S. EPA: Development of
Water Quality Based Permit Limitations for Toxic
Pollutants; National Policy. U.S. EPA, Volume 49,
No. 48, Friday, March 9, 1984.
Horning, W. and C.I. Weber, (Eds.). 1985. Short-
Term Methods for Estimating the Chronic Toxicity
of Effluents and Receiving Waters to Freshwater
Organisms. U.S. EPA, Cincinnati, Ohio. EPA-
600/4-85-014.
Magnuson, V.R., D.K. Harriss, M.S. Sun, O.K. Taylor,
G.E. Glass. 1979. Relationships of Activities of
Metal-Ligand Species to Aquatic Toxicity. ACS
Symposium Series, No. 93. Chemical Modeling in
Aqueous Systems, E.A. Jenne, Editor:635-656.
Mount, D.I. 1988. Methods for Aquatic Toxicity
Identification Evaluations: Phase III Toxicity
Confirmation Procedures. Draft EPA Research
Series Report. Environmental Research Laboratory,
Duluth, MN.EPA/600/3-88/036.
Mount, D.I. and L. Anderson-Carnahan. 1988.
Methods for Aquatic Toxicity Identification
Evaluations: Phase II Toxicity Identification
Procedures. Draft EPA Research Series Report.
Environmental Research Laboratory, Duluth, MN.
EPA/600/3-88/035.
Occupational Safety and Health Administration. 1976.
OSHA Safety and Health Standards, General
Industry. 29 CFR 1910. OSHA 2206 (Revised).
Patrick, R., J. Cairns Jr., and A. Scheier. 1968. The
Relative Sensitivity of Diatoms, Snails, and Fish to
Twenty Common Constituents of Industrial Wastes.
Prog. Fish-Cult.:137-140.
Peltier, W. and C.I. Weber, (Eds). 1985. Methods for
Measuring the Acute Toxicity of Effluents to
Freshwater and Marine Organisms. 3rd Edition.
U.S. EPA, Cincinnati, Ohio. March 1985. EPA-
600/4-85-013.
Poirier, S.H., M.L Knuth, C.D. Anderson-Buchou,
L.T. Brooke, A.R. Lima, and P.J. Shubat. 1986.
Comparative Toxicity of Methanol and N,N-
Dimethylformamide to Freshwater Fish and
Invertebrates. Bull. Environ. Contam. Toxicol.
37(4):615-621.
Stumm, W. and J.J. Morgan. 1981. Aquatic
Chemistry - An Introduction Emphasizing
Chemical Equilibria in Natural Waters. John Wiley
& Sons, Inc., New York, NY. 583 p.
Randall, T,L. and P.V. Knopp. 1980. Detoxification of
Specific Organic Substances by Wet Oxidation. J.
Water Pollut. Control Fed. 52(8): 2117-2130.
Walters, C.I. and C.W. Jameson. 1984. Health and
Safety for Toxicity Testing. Butterworth Publ.,
Woburn, MA.
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* US GOVERNMENT PRINTING OFFICE 1988- 548-156/87041
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