EPA
United States
Environmental Protection
Agency
Environmental Research
Laboratory
Duluth MN 55804
National Effluent Toxicity
Assessment Center
Research and Development
EPA/600/3-88/035 Feb. 1 989
Methods for Aquatic
Toxicity Identification
Evaluations
Phase II Toxicity
Identification
Procedures
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EPA/600/3-88/035
February 1989
Methods for Aquatic
Toxicity Identification Evaluations
Phase II Toxicity Identification 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 Effluent Toxicity
Assessment Center
Technical Report 02-88
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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
Phase II is the second in a series of three guidance documents for use in
characterizing and identifying the cause of toxicity in effluent Toxicity Identification
Evaluations (TIEs). Phase I is intended to provide some knowledge of the
physical/chemical characteristics of the toxicant(s). Phase II builds on that information
and is intended to identify suspect toxicants. These are suspect toxicants because
evidence has implicated them. When evidence exists to implicate one or several
toxicant(s), Phase III confirmation should begin.
Phase II is incomplete and does not provide methods for many constituents, such as
anionic metals and polar organics. By using a loose-leaf binding, we hope to expedite
frequent expansions or revisions.
The sections of Phase I which address Health and Safety, Quality Assurance/Quality
Control (QA/QC), Facilities and Equipment, Dilution Water, Testing, and Sampling are
applicable to Phase II. Parts of the Introduction to Phase ! are also relevant to this
manual. These sections are not repeated in this report. We would especially note that
these methods are not intended for chronic toxicity studies. We welcome any
suggestions from users for use in future revisions.
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Abstract
This manual describes test methods that can be used to identify the specific
chemicals(s) responsible for effluent toxicity when the source of toxicity is related to
non-polar organics, ammonia, or cationic metals. These methods are not intended to
be irrefutable evidence but only to be providers of enough evidence to suggest that
Phase III, the toxicity confirmation, should be started. Phase III uses available methods
to provide adequate evidence that the true cause of toxicity is consistently due to the
identified toxicants.
IV
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Contents
Page
Foreword jjj
Abstract .. .1 ............... jv
Contents . . . v
Figures '.'.'.'.'.'.'.'.'.'.'.'.'. vi
Tables ; ; ; ' vjj
Acknowledgments vjjj
1. Introduction ^^
1.0 General Overview 1_1
2. Non-Polar Organic Compounds 2-1
2.1 General Overview 2-1
2.2 Sample Volume '.'.'.'.'.'.'. 2-3
2.3 Filtration '.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'.' 2-3
2.4 Column Size '.'.'.'.'.'"'' 2-3
2.5 C-|8 SPE Column Conditioning 24
2.6 Elution Blanks .'.'.".'.'.'.'.'.'.'.'.'.' 2-4
2.7 Column Loading with Effluent . . . 2-5
2.8 Cia SPE Column Elution 2-5
2.9 Blank & Effluent Fraction Toxicity Tests '.'.'.'.'.'.'.'.'.'.'.'.'. 2-5
2.10 Concentration of Toxic Fractions 26
2.11 HPLC Separation . ' '.' 2-7
2.12 HPLC Fraction Toxicity Tests '.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'. 2-8
2.13 HPLC Fraction Concentration and Toxicity Testing 2-8
2.14 GC/MS Analyses .'.".'."'.'.'.".' 2-9
2.15 Identifying Suspected Toxicants '.'.'.'.'.'.'.'.'.'.'.'.'.'.'.'. 2-9
3. Ammonia g «
3.1 General Overview - ..'.'.'.'.'.'.'.'. 3-1
3.2 Equitoxic Solution Test '.'.'.'.'. 3-1
3.3 Results/Subsequent Tests 3-3
3.4 Zeolite Test 3.4
3.5 Procedure '.'.'.'.'.'.'.'"' 3-4
3.6 Interpretation of Results '.'.'.'.'.'.'.'.'.'.'.'.'.'.'' 3-4
4. Cationic Metals 4_-l
4.1 General Overview 4-1
4.2 Procedure 4-2
5. References
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Figures
Number Pa9e
2-1. Schematic for Phase II identification of Cis SPE removed toxicants .............. 2-1
2-2. Step 1 for concentrating the whole effluent chemicals
on the Cis SPE column ....... ! ................................... 2-4
2-3. Procedures for eluting the column with' a gradient of methanol/water solutions ....... 2-6
2-4. Procedure for combining and concentrating toxic fractions. Concentration
factors are approximate based on 2 L of effluent and a 6 ml_
combined eluate for each methanol/water gradient ........................ 2-7
2-5. Procedure to fractionate concentrate on the HPLC .......................... 2-8
2-6. Procedure to concentrate toxic HPLC fractions
(combined and individually) ..... ................................ • • - 2-8
VI
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Tables
Number
Page
2-1. Comparison of Toxic Units in Each Toxic Fraction to Toxic Units
of All Fractions Combined and Whole Effluent 2-3
3-1. Percent Un-ionized Ammonia in Aqueous Ammonia Solutions
for Selected Temperatures and pH Values 3-2
3-2. Calculated Un-ionized Ammonia LC50 Values at Different
pH Values for Ceriodaphnia Using an LC50 at pH 8.0 and 25°C of 3.0 mg/L .... 3-2
VII
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Acknowledgments
This report is a product of the effort of many individuals. Methods for Phase II have
been under development over the last few years. As with Phase I, throughout the text
the reference to "experience" in the approach or technique is the collective experience
of the Environmental Research Laboratory (ERL-Duluth) effluent group. That group
consists of: Elizabeth Durhan, Gary Ankley, 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.
The assistance of Dorette Gueldner (AScI) in the typing, organization, and revisions is
greatly appreciated. Larry, Teresa, Liz and Gary reviewed the various drafts to improve
the document. Final reviews were willingly performed by Evelyn Hunt (ERL-Duluth)
and Teresa. In addition, Teresa prepared the figures and made changes on drafts and
final versions.
Through the support of Rick Brandes (EPA, Permits Division) and Nelson Thomas,
(ERL-Duluth) the National Effluent Toxicity Assessment Center (NETAC) continues to
refine these techniques. This report will be updated as methods are revised or
developed, and we welcome suggestions.
VIII
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Section 1
Introduction
1.0 General Overview
The major objective of Phase II is to identify the
suspected toxicant(s). Some general guidance may
be furnished by the outcome of Phase I tests (Mount
and Anderson-Carnahan, 1988; hereafter referred to
as Phase I),,but for many effluents, both separation
and concentration steps will be needed in order to
achieve the objective. For metals, Atomic Absorption
spectrometry (AA) may be sensitive enough to
measure toxic concentrations directly in the sample,
the number of metals is small enough that toxicity can
be attributed without separating one from another,
and ammonia measurements are made without
separation or concentration. However, for organic
chemicals, the number is so large that separation is
usually necessary, both for analytical, as well as
lexicological reasons.
Because there are often so many constituents within
the class of chemicals identified in Phase I, initial
efforts are most productively directed towards
separating the toxic from the non-toxic constituents.
The need to identify the toxicant(s) quickly is a
temptation to analyze too soon. Some methods, such
as Gas Chromatography/Mass Spectrometry (GC/MS)
for non-polar; organics, allows one to identify many
chemicals in a mixture. Identification must be followed
by association with toxicity, and this is very difficult to
do on mixtures with many constituents, for several
reasons:
• Toxicity data for many of the chemicals identified
are usually not available.
• Constituents are often not available in pure form
to measure their toxicity.
• Interactions (additivity, synergism, antagonism)
are not known for the given mixture, and one
must know interactions to apportion toxicity.
If there is a single suspect toxicant such as ammonia,
then separation needs are limited largely by the
analytical requirements. If the toxicity is caused by
one constituent, the number of other non-toxic
constituents js irrelevant when attributing toxicity.
However, Phase I results do not usually lead to a
single suspect toxicant and, therefore, separation may
be necessary.
When a method for separating the toxicant(s) is
found, concentration may be an inherent part of the
procedure (as in solvent extraction) which will simplify
the problem of finding a method to concentrate the
sample. Through the stages of separation and
concentration, measurement of toxicity will be the
only way to evaluate the success or failure of the
methods.
The interpretation of results will often be different than
we usually encounter. In the classical research
approach, experiments are designed to either accept
or reject a hypothesis. In Toxicity Identification
Evaluation (TIE) work, an experiment frequently will
allow us only to accept but not reject the hypothesis.
For example, if ammonia is the suspect toxicant, and
you want to know whether there are additional
toxicants, the ammonia can be removed using zeolite.
If the effluent is still toxic, you can conclude that there
are additional toxicants present. If the effluent is not
toxic, you cannot conclude that there are no
additional toxicants because the zeolite may have
removed other toxicants in addition to the ammonia.
The always-present question of more than one
toxicant immensely complicates data interpretation.
Phase I results are not likely to give even a clue to
this question unless the toxicant class changes over
time. Phase II results are often such that one cannot
tell whether the situation is one of partial removal of a
single toxicant or toxicity resulting from multiple
toxicants. Frequently the issue will not be resolved
until at least one toxicant is identified and measured
analytically. Experience shows that the best choice is
to try to identify the toxicant that appears easiest to
identify. Usually that will be a toxicant that can be
separated from the sample (e.g., extracted or
recovered from a column that sorbs at least some of
the toxicity) and for which there is a broad spectrum
analytical method. Above all, data should always be
interpreted under all probable scenarios, i.e., one
toxicant, multiple toxicants, and even different
toxicants from sample to sample.
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As effluent constituents are identified, a sorting
process begins in which a decision must be made as
to the probability that each one identified is a cause
of toxicity to the test organism being used. Usually,
this is based on the estimated concentration and the
constituents' toxicity. Analytical error in quantitation
may be large (10X or more) because recoveries and
instrument response factors probably will not yet have
been determined. A toxicological concern is the
uncertainty caused by differences in species
sensitivity and water quality effects. Error of the LC50
value will vary depending on the quality of the test,
the number of times it was repeated, and the
completeness by which the results and conditions
were described. Species sensitivity frequently varies
from 100X to 1.00QX. We have found 1,OOOX to be a
minimum difference in measured concentrations
versus literature LCSOs in order to reject a chemical
as a suspect when the uncertainty of the LC50 data
is high. If one has good data for the test species
being used, then this difference may be reduced
(e.g., to 10X). Since these decisions are always
subjective they will sometimes be wrong no matter
how carefully they are made. Perhaps most important
is to make these decisions in an interactive process,
First evaluate candidates that have concentrations
higher than or closest to their LC50 values and if
these prove to be negative, then examine those that
have concentrations farther below the LC50 values.
The suspected toxicant concentrations at the dilution
equal to the LC50 are the important concentrations to
compare. At some point, a decision must be made
that the toxicants have not yet been measured and
different sample preparation or analyses must be
used.
For some effluents, Phase I results will not provide
any real help in Phase II. In these cases, more drastic
characterization steps may be helpful. Boiling, ashing,
and vacuum distillation, among others, may be tried.
We have little experience upon which to recommend
procedures in these cases. The most important
concern is to realize that the more severe the effluent
treatment, the more likely it is that artifacts will be
created.
Phase II efforts should grade into Phase III
Confirmation (Mount, 1988; hereafter referred to as
Phase III) as soon as good evidence is obtained that
one or more candidates are probable toxicants. The
primary product of Phase II is the chemical identity of
the suspected toxicants to furnish the basis upon
which Phase III testing will be conducted.
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Section 2
Non-polar Organic Compounds
2.1 General Overview
A schematic of the general procedures followed in
identifying non-polar organic toxicants is shown in
Figure 2-1. In this procedure, the C-\Q Solid Phase
Extraction (SPE) column is used to extract non-polar
organic compounds from effluent samples. These
compounds are then selectively stripped off the
column by eluting the GIS sorbant with solvent/water
mixtures that are increasingly non-polar. As a series,
the "fractions" resulting from column elution contain
analytes that are decreasingly polar and decreasingly
water soluble. Each fraction is tested for toxicity. The
fractions that exhibit toxicity are then diluted in water
and a smaller Cis SPE column is used to con-
centrate the toxicants. The column is dried and eluted
with a small volume of 100% methanol. The resulting
concentrate is chromatographed using reversed
phase High Pressure Liquid Chromatography (HPLC).
HPLC fractions are collected and tested for toxicity.
The toxic fractions are again concentrated in 100%
methanol and tested as before and analyzed using
GC/MS. Those constituents identified are roughly
quantitated, by assuming that the identified con-
stituents and !the internal standard have the same
response factor, and the estimated concentrations are
compared to LC50 values for each chemical. If good
suspects are identified, Phase III steps begin. In
addition, mass balance testing (described later)
should be started to determine whether more
toxicants are present. If no candidate toxicants are
identifiable, more separation on the HPLC may help if
there are many constituents present. Additional
constituents can be identified by increasing the
concentration factor by using larger effluent samples.
At some point, the probability that the toxicants are
not chromatographing or the mass spectrometer is
not detecting the toxicants, must be considered. Use
of other types of mass spectrometry, such as liquid
chromatography/mass spectrometry (LC/MS) or direct
probe mass spectrometry should be explored.
It is likely that only a small percentage of effluent
samples will require just one sequence as shown in
Figure 2-1. Many effluent samples require several
fractionation sequences before good suspects are
found. Once the toxicants are separated into HPLC
Effluent Sample
I
Toxicity Test
Toxicity Tests
Concentration of Toxic Fractions* Toxicity Tests
Methanol Fractions
I
HPLC Fractionation
Toxicity Tests
I
Concentration of Toxic HPLC Fractions Toxicity Tests
I
GC/MS Identification
I
Compare Concentrations to LC50 Values
*ln rare cases, GC/MS can be useful here.
Figure 2-1. Schematic for Phase II identification of C18
SPE removed toxicants.
fractions, the effort is largely an analytical one until
some candidates are identified.
The Cjs SPE sorption, rather than non-polar solvent
extraction, is used because there is not a good way
to determine whether the toxicant was extracted from
the effluent. Since the non-polar solvent is not
miscible with water, getting extracted chemicals back
into water to test for toxicity is difficult, and the
solvents themselves are generally toxic. The
extracted sample will also be toxic as a result of a
small amount of the solvent dissolved in the sample.
Residual levels of solvent can be stripped from the
extracted effluent prior to toxicity testing only if the
toxicants are not stripped as well. Effluent extracts will
contain more interfering constituents, emulsions may
be a problem, and the HPLC fractions may be less
well defined. The Cia SPE column is easier when it
works. If it does not work, extracts can be used if the
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toxicants are non-volatile under the required
conditions and toxicity tracking is done.
The sorbants recommended herein for use in SPE
and HPLC columns are chemically identical. The
column packing is composed of porous silica which
has been reacted with octadecyl groups to produce a
surface GIB layer one molecule thick. The same
compounds will partition on both columns and the
order of elution of chemicals will be the same. The
major difference between the SPE and HPLC
columns is the resolution achieved. The particle size
employed in HPLC columns is smaller, providing a
greater surface area and better resolution. Despite
poorer resolution, SPE columns have the advantage
of generally possessing a higher loading capacity.
The SPE column is used as a preparatory column for
sample cleanup while the HPLC column gives far
more refined and controlled separation.
The mechanism of extraction with Cia sorbants is
relatively simple. Extraction of effluent compounds
occurs because the GIB sorbant more strongly
attracts for the non-polar compounds than the
surrounding water molecules.
Sorption of non-polar organics is also influenced by
Ionic strength, pH, and total organic carbon (TOG)
levels. The important consideration is to check for
toxicity breakthrough in the post-column effluent.
Even when the capacity of the column is exceeded,
the procedures described in this subsection may be
useful in concentrating part of the non-polar effluent
toxicants.
Just as non-polar organic compounds are separated
from other effluent components while passing the
effluent through the column, these non-polar
compounds can be further segregated according to
their polarity during column elution. To elute toxicants
extracted by the C-\Q sorbant, the eluting solvent must
have a higher affinity than octadecyl for the
compound. Elution solvent choice is complicated
because the identity of the toxicants is not known.
Without that knowledge, the degree of solvent
strength required to elute the toxicants is unknown. It
is known that the octadecyl group is extremely non-
polar, and very few solvents, except hexane
(E° = 0.0), come close to its strength. Unfortunately,
except for its compatibility with GC/MS, hexane does
not meet the necessary criteria of miscibility with
water and low toxicity. Methanol (E°=0.73), on the
other hand, is a far weaker solvent than may be
desirable but is still less polar than water (E° >0.73).
Methanol is also not the solvent of choice for GC
analysis, but can be used even though column life is
shortened. Since methanol has a very low toxicity apd
it will elute chemicals from Cis, it is an acceptable
solvent for our purposes.
Because of the limited number of solvents meeting
the criteria for acceptability, different concentrations
of methanol in water are used rather than different
solvents to selectively elute compounds. Using a
step-wise gradient elution entailing sequential
column rinses with successively increasing methanol
concentrations, the relatively hydrophilic, polar
compounds are eluted first, while the more
hydrophobic non-polar compounds are eluted last.
Given the relatively weak strength of methanol as a
solvent for non-polar compounds, it is possible that
effluent compounds, sparingly soluble in water, will
not be eluted from the C^Q sorbant. If toxicity is
extracted by the column but not eluted by methanol,
less polar solvents must be used. These will be
discussed later. Recovery of 100% of each effluent
toxicant in the C-\Q column fractions may not be
crucial, because at this stage, the concentration of
toxicant in the fraction and the toxicity of the fraction
are compared. Assumptions about the concentration
of toxicant in the whole effluent are not made at this
point, nor is any statement made regarding recovery
of whole effluent toxicity by C^Q column fractions. In
later stages of Phase II, column recovery must be
defined in order to make meaningful inferences
regarding the relationship between the concentration
of the suspected toxicant(s) and the observed toxicity.
The efficiency of C-\Q column recovery consists of
extraction efficiency (i.e., how well the column
sorbant removes the effluent components) and elution
efficiency (i.e., how well sorbed effluent compounds
are removed from the column by the solvent gradient
used). For purposes of this study, "efficiency" applies
only to recovery of those compounds causing or
affecting effluent toxicity. The question of extraction
efficiency can be determined by measuring the
toxicity of the effluent after it passes through the
column. By comparing the toxicities of aliquots
collected after different volumes of effluent have
passed through the column, a determination of
toxicant extraction efficiency can be made separate
from column selectivity. If there is toxicity, but it is
independent of the volume of effluent previously
passed through the column, then the post-column
effluent toxicity is probably caused by toxicants that
are not extracted by the column. If the toxicity of the
post-column effluent increases with the volume of
effluent passed through the column, the capacity of
the column to sorb the toxicants was exceeded.
Elution efficiency can be approximated by summing
the amount of toxicity in the toxic fractions. This will
usually be somewhat imprecise for several reasons. A
single toxicant may occur in more than one
contiguous fraction, in which case a small amount of
the toxicant in one fraction may not be detectable
because it is present below the LC50 concentration.
(This problem can be solved by combining all
fractions and measuring their total toxicity.) If more
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than one toxicant is present, they may not be strictly
additive in their toxicities, and when separated into
different fractions the sum of the fraction toxicities will
be low even if extraction and elution were 100%.
Table 2-1 illustrates a hypothetical example.
(Section 7 of Phase I defines toxic units.) Frequently
the toxicity of the combined fractions will be
somewhat greater than whole effluent. This may be
caused by enhanced toxicity due to the methanol in
the fractions. To compensate for toxicity
enhancement by methanol, methanol has been added
to the whole effluent, but this may stimulate biological
growth. If this happens, the test is negated. Fractions
should be tested in water with low TOC and
suspended solids (SS) to lessen effects of the
characteristics on toxicity.
Once significant toxicity is found in the C-\Q SPE
fractions, HPLC separation must be done to reduce
the number of chemicals associated with the
toxicant(s). In the procedure described below, the
HPLC fractions will contain methanol. Aliquots must
therefore be diluted until the methanol is below a toxic
level before the fractions can be tested for toxicity.
One must calculate the necessary dilution to reach a
non-toxic level of methanol and then determine how
much effluent must be concentrated on the SPE
column (including the pre-HPLC concentration step)
in order to have enough toxicant in the HPLC fraction
following dilution, to detect toxicity. This, of course,
depends on the toxicity of the effluent. In the sections
to follow, a typical set of dilution and concentration
factors are given, but these must be altered to fit
each situation.
The toxic HPLC fractions are concentrated using a
small SPE column and then analyzed by GC/MS. The
estimated concentrations of constituents in the final
concentrate (based on an internal standard) are then
compared to their toxicity to decide which ones may
be sufficiently high in concentration to cause toxicity.
If no candidates are found, more concentrations,
other analytical methods (e.g., LC/MS), and better
separation are possible further steps.
Table 2-1. Comparison of Toxic Units in Each
Toxic Fraction to Toxic Units of All
Fractions Combined and Whole
Effluent
Source Toxic Units
Toxic Fraction (% Methanol)
75% 0.5
80% 1.2
85% 0.6
SUM 2.3
Combined Fractions 2.7
Whole Effluent 2.5
2,2 Sample Volume
The volume of effluent needed depends on the
toxicity of the effluent, the toxicity of the chemicals
causing effluent toxicity, and the sensitivity of the
analytical method. Since only the first of these will
usually be known when Phase II begins, trial and error
will dictate needed volumes. For effluents with LC50
values from 25-100%, 2000 mL have usually been
adequate.
2.3 Filtration
A 1 pm glass filter should be prepared as described
in Section 8 of Phase I. The filter should be rinsed
with 200 mL of dilution water, rather than pure
distilled water and a sample collected after most of
the volume has been filtered for the filter toxicity
blank. In subsequent steps, a dilution water blank will
be collected after passing through the SPE column.
This water must not have sorbable organics in it or
toxicity in the fraction blanks may occur. The same
type of water should be used for the filter blank as for
the column blank. Therefore, a reconstituted water
should be used for these procedures. Formulas for
reconstituted water can be found in Peltier and
Weber, 1985.
The volume of effluent that can be passed through a
single filter is sample specific. If more than one filter
is needed, a separate blank should be prepared for
each filter. Whether a vacuum can be used for
filtering or whether a pressure system must be used
will be ascertained in the volatility tests of Phase I.
When uncertainty of which method to use exists, use
pressure filtration, and a toxicity test on the filtered
effluent sample is prudent.
2.4 Column Size
Various sizes of C-\Q SPE columns are available. We
routinely have used BakerRl 6 mL high capacity (HC)
columns, putting 1,000 mL through one column
(Figure 2-2). So long as the column does not
become clogged and toxicity break-through does not
occur, the column capacity has not been reached. In
the following description, volumes for a 6 mL HC GIB
SPE column are used.
Vacuum manifolds are available for drawing the
sample and solvents through the column. The use of
vacuum is much less controllable than pumping the
sample and therefore is discouraged because
extraction efficiencies may vary. It is. adequate in the
hands of a careful worker, but in routine use, flow
rates are likely to vary greatly, and there is a danger
that the column will go dry when it should not. A
pump is more convenient for the large volume
1 J.T. Baker Chemical Company, Philipsburg, NJ
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Step 1
1000 ml.
Effluent
Concentration Factor
1X
6 mL High Capacity
dsSPE column
Test Post G!8
1X
Figure 2-2. Step 1 for concentrating the whole effluent chemicals on the Cia SPE column.
effluent samples where flow rate can be better
controlled, and therefore a pump is recommended.
Whichever system is used, it must be made of
materials that dilute acid and solvents do not destroy,
or from which chemicals are not leached that are
toxic or interfere with analytical measurements.
Teflon, glass, and stainless steel are acceptable.
2.5 Cis SPE Column Conditioning
The 6 mL Cis SPE columns are conditioned by
pumping 25 mL of 100% methanol through the
column. Before the packing goes dry, 25 mL of high
purity distilled water must be added. As the last of
that water is passing through, 25 mL of dilution water
is added, and the last 10 mL is collected for a column
blank toxicity test. If the pH of the sample must be
altered in order for the toxicants to sorb on Cis. then
the 25 mL aliquot of dilution water should be adjusted
to the same pH. Pumping is continued until no water
emerges from the column.
2.6 Elution Blanks
A three mL volume of 25% methanol in water is
pumped through the dry column and collected in an
analytically clean, labeled vial (since glass also
contains metals, the vials should also be acid
leached). This procedure is repeated with 3 mL
volumes of the 50%, 75%, 80%, 85%, 90%, 95%
and 100% methanol in water. The column should be
allowed to dry between each elution with the different
3 mL volumes of methanol/water mixtures. These
methanol solutions may also have to be pH-adjusted
for better elution efficiency, and, if so, the blanks
must also be adjusted.
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2.7 Column Loading with Effluent
The same column is again conditioned with 25 ml_ of
100% methanol and 25 ml_ of high purity water, as
described above. Without allowing the column to dry,
1,000 ml_ of filtered effluent sample is pumped
through the column at a rate of 5 mL/min (Figure 2-
2). Three 10 ml_ samples of the post-Cis column
effluent are collected after 25 ml, 500 ml_ and 950
mL of the sample passes through the column. Each
10 mL aliquot is used in a toxicity test to determine
the presence of toxicity in the effluent after it has
passed through the column. This information can be
used to determine whether toxicant breakthrough
occurred. As Phase II progresses, it can be very
helpful to catch 20-30 mL and make dilutions to
obtain LC50 values. Pumping is continued until no
effluent emerges from the column.
2.8 Ci 8 SPE Column Elution
Next, a 3 mL volume of the 25% methanol/75% water
mixture is pumped through the column and collected
in a labeled, analytically clean vial. For better
separation, two 1.5 mL volumes to elute the column
are recommended (for both blanks and sample
fractions). Subsequently, 3 mL volumes of each of
the 50%, 75%, 80%, 85%, 90%, 95% and 100%
methanol are pumped through the column and
collected in separate vials (Figure 2-3). The next
elution volume should not be added until no more of
the precedinig one is emerging from the column. In
general, if the toxicants are removed by the column
best at pH 3, then the methanol fractions should be
eluted at pH 1 or pH 11 and vice versa.
This entire procedure is repeated using a second
column for the second 1,000 mL of filtered effluent.
The dilution water column blank samples should be
kept separate. The corresponding fractions from
either the blanks or the sample from each 1,000 mL
fractionation can be combined. For example, the 3 mL
100% methanol sample fraction from the first column
and the 3 mL 100% methanol sample fraction from
the second column are combined for a total of 6 mL.
The vials containing the methanol/water fractions are
sealed with perfluorocarbon or foil-lined caps and
stored under refrigeration. These fractions represent a
"first cut" separation of effluent components.
Volumes will vary if columns of different sizes are
used or if the particular effluent under study or the
research question being posed dictates method
modification.
2.9 Blank and Effluent Fraction Toxicity
Tests
In the initial stages of Phase II, toxicity tests are
conducted on GIB SPE column fractions and blanks
to detect the presence of toxicants, rather than to
quantify the magnitude of the toxicity in each. As in
the toxicity tests conducted during Phase I, there is
no need for careful measurement and maintenance of
test solution chemistry, nor is there a real need for
duplicate exposures. The major purpose behind this
tier of tests is strictly qualitative, i.e., to assess
whether or not acute toxicity is present or absent in
the fractions and blanks. As suspects are identified,
quantitative toxicity measurements are needed to
compare with the analytical measurements.
The next step is to test the blank and sample
fractions for toxicity. The methanol in the fractions
limits the concentration that can be tested, and the
amount of methanol in the 100% fractions dictates
the necessary dilution. We usually inject 150 piL of
each blank and sample fraction into 10 mL of dilution
water. This step will give a 1.5% methanol
concentration that is below the LC50 for both
Ceriodaphnia and fathead minnows in the 100%
fraction. The resulting methanol concentration must
be adjusted for the species used (cf., Section 8 of
Phase I for methanol LC50 values). Usually five
animals in each 10 mL aliquot are used without
duplicates. Using the above volumes, the tested
solution has a greater concentration than whole
effluent, assuming 100% extraction and 100% elution
in one fraction. These test solutions can be diluted as
described in Phase I to provide an LC50 for each
sample fraction. Usually blank fractions do not need
to be diluted. If dilutions are done, the initial volumes
are best increased to 300 jiL/20 mL to provide 10 mL
with which to make serial dilutions.
Individual chemicals in the fractions could be toxic
even when they are not toxic in the whole effluent,
since the concentration tested may be as high as 5X
whole effluent. Therefore, to be toxic at whole effluent
concentrations, an individual fraction must have an
LC50 of 20% or less. Since there is no way to know
whether the toxicant(s) eluted in more than one
fraction or what the percent extraction and elution
was, fraction toxicity up to 100% (5X whole effluent)
should not be disregarded.
If toxicity occurs in any of the fraction blank tests and
it is small relative to the toxicity in the corresponding
sample fraction (e.g., 20% mortality versus 80%), the
sample fraction results are not negated. If all
organisms die, dilutions should be tested to make
sure the sample fraction is substantially more toxic
than the blank. Blanks should not, in general, have
measurable toxicity.
In addition to concentrating column artifacts to toxic
levels, effluent constituents present at non-lethal
levels may be concentrated to toxic levels in this test
if they have a relatively high recovery value. On the
other hand, actual effluent toxicants with poor
recovery may not be present in these test solutions at
2-5
-------
Step 2
6 mL High Capacity
CiaSPE column
Y
Concentration Factor
333X
Each
J
J
Conduct Toxicity Test on Each
(150jut_in 10mL Water)
5X
Figure 2-3. Procedures for eluting the column with a gradient of methanot/water solutions.
toxic levels. Spurious results of this nature will be
identified in the later stages of Phase II and/or in
Phase III.
2.10 Concentration of Toxic Fractions
As explained in the General Overview, this initial
fractionation provides only a general separation of
non-polar organics. Except in relatively uncom-
plicated effluents, GC/MS analysis of toxic CIQ SPE
fractions will result in extremely complicated
chromatographs from which the causative agents
cannot be distinguished from other effluent
components. Rarely should GC/MS analyses be tried
on these fractions. In most cases, a secondary
fractionation using HPLC is needed to further simplify
toxic effluent fractions prior to component
identification.
Because fractionation by HPLC will further dilute the
concentration of effluent components in the C^Q SPE
fractions, these fractions must be concentrated prior
to injection into the HPLC. This concentration step
will increase the concentrations of constituents in the
HPLC fractions and rid the fractions of water.
The toxic effluent SPE fractions are combined (Figure
2-4) and diluted 1:10 with high purity water. The
corresponding blank fractions are similarly treated.
The total volume should not exceed 100 mi. for the 1
mL GIB column. If the 25% or 50% fractions are
toxic, then greater than a 1:10 dilution may be
needed. This must be determined by testing for
toxicity loss after the concentration step.
For this step, a 1 mL Cis column is conditioned with
10 mL of methanol and 5 mL of water similar to the
procedures described in the SPE Column
Conditioning Section. Column blanks are advisable,
and if done, the water volume must be increased to
at least 20 mL. The diluted blank fractions are then
drawn through the 1 mL Cis SPE column under a
pressure of 15 in Hg using a vacuum manifold. The
solution passing through the column cannot be tested
for toxicity because of its methanol concentration.
2-6
-------
The column is then dried for 5 min using a nitrogen
flow rate of 13 mL/sec.
After drying, the luer tip of the column is fitted with a
luer-lock needle and 100 pL of 100% methanol is
placed into the column using a microliter syringe;
nitrogen is applied to the column at a rate of 4
mL/sec to force the methanol through the sorbent.
The first 100 pL aliquot of methanol applied to the
column will yield approximately 25 pL of eluate. Two
more 100 pL aliquots of 100% methanol are also
forced through the column. The final volume of eluate
collected will* be approximately 200 pL. Measure the
exact volume collected (using a pL syringe) for
calculation of recoveries. For a given total volume of
methanol, three separate smaller elutions are more
efficient than one large one.
The same 1 ml_ C-\Q SPE column is reconditioned
following the; directions given above in the Column
Conditioning -Section. It is then used to concentrate
the diluted toxic SPE column fractions, using the
same sequence used for the blank fractions (Figure
2-4).
Step 3
Concentration Factor
Dilute Toxic Fraction(s) 1:10
: i 33X
Sorb on i mL C18 SPE Column
(discard
post-C18)
Dry Gig Column with
Nitrogen (optional)
i
Elute Column with 3-100nL
volumes of 100% Methanol
Collect Eluate Concentrate
I
Conduct Toxicity Test
in 20 mL water)
10.000X
5X
Figure 2-4.
Procedure for combining and con-
centrating toxic fractions. Concen-
tration factors are approximate based
on 2L of effluent and a 6 mL combined
eluate for each methanol/water
gradient.
The original effluent volume of 2,000 ml is now
represented by 200 pL or is nominally 10.000X more
concentrated, ignoring the amount used for testing.
As work progresses and more quantitative results are
needed, the eluate volume must be measured to
know the concentration factor. If 10 pL of the
concentrate is diluted to 20 mL in dilution water, the
resulting concentration will be 5X whole effluent.
Dilutions of this can then be made to measure an
LC50 and toxicity recovery can be calculated by
comparing this LC50 to the LC50 of the whole
effluent. Remember that an LC50 of 20% in the
concentrate test would be equivalent to an LC50 of
100% in whole effluent. One cannot compare the
concentrate toxicity to that of the individual toxic
fractions because some of the toxicant may have
been in adjacent fractions but not detectable by the
toxicity test. The concentrate toxicity may be lower
than the whole effluent toxicity because of extraction
and elution inefficiencies.
The important concern here is not 100% recovery of
toxicants but enough recovery for analyses to be
done and to measure toxicity in the HPLC fractions. If
recovery is too low, changing the column drying time
may help. Sometimes recovery appears to increase
with drying time while other compounds are volatilized
from the column during the drying process. For
concentrates analyzed using GC, removal of water is
critical to instrument performance. Where greater
concentration factors are desirable, SPE fractionation
can be repeated with additional 1000 mL volumes of
effluent, followed by combining the 3 mL toxic
fractions before concentration. The size of the column
used for concentrating may have to be increased with
appropriate changes in dilution and elution volumes.
2.11 HPLC Separation
The same column packing should be used in the
HPLC column as is used in the SPE column except
for particle size. At later stages, when more is known
about the toxicants, other columns may be used.
The HPLC conditions presented in this section are
general. As more information on the effluent is
gained, HPLC conditions should be modified to
achieve better separation, and higher concentration
factors. We use an instrument equipped with a 5 p
GIB reverse phase column (250 mm x 4.6 mm) and
UV detector. An elution gradient of 30%
methanol/water to 100% methanol over 20 minutes
with a five minute hold period at 100% methanol is
suggested. Generally, the 278 and 230 nm
wavelengths are monitored continuously while
spectral scans (210 nm-600 nm) are acquired only
for chromatograph peaks. First, 100 pL of the blank
concentrate is injected and 25-1 mL fractions are
collected in analytically clean glass vials (Figure 2-
5). The same procedure is followed using 100 pL of
the effluent sample concentrate. The vials must be
sealed (for example, with perfluorocarbon lined caps)
and stored under refrigeration.
2- 7
-------
2.12 HPLC Fraction Toxicity Tests
Toxicity tests on the HPLC blank and sample
fractions using non-replicated exposures of five
animals each, without dilution, are normally used. The
methanol in the HPLC fraction limits the concentration
that can be tested. The percent methanol varies with
the particular fraction so those that are 100%
methanol determine the necessary dilution. Again, a
1.5% methanol concentration, an acceptable
methanol concentration for many species, is sug-
gested.
If 100pLof the ~200 pL concentrate is injected on
the HPLC (Figure 2-5), then each 1 mL HPLC
fraction equals 1,000 mL of effluent (assuming no
loss and elution in one fraction) or a 1.000X
concentration. If each HPLC fraction is then diluted to
1.5% for testing (150 pL to 10 mL) the resultant
concentration is 15X whole effluent concentration. Of
course, some loss of toxicant will occur in each step
and the toxicity will usually be less.
Step 4
Concentration Factor
Inject 100 nL of the 10.OOOX
Concentrate on the
HPLC C18 Column
1
Collect 25-1 mL
HPLC Fractions
I
Conduct Toxicity
Test on Each (150
liL/10 mL water)
1.000X
15X
Figure 2-5.
Procedure to fractionate concentrate
on the HPLC. Concentration factors
are approximate.
The HPLC fractionation is precise enough that more
concentrate can be fractionated without the need to
do toxicity tests, in order to increase sample quantity
for analyses.
The blank fractions should not be toxic. If they are,
then dilutions will have to be made on both blanks
and toxic fractions to tell if there is enough additional
toxicity in the sample fractions to warrant analyses.
2.13 HPLC Fraction Concentration and
Toxicity Testing
The toxic HPLC fractions and corresponding blanks
must be concentrated in a solvent suitable for GC/MS
or other analytical techniques. The procedure is
identical to Section 2.10 Concentration of Toxic
Fractions, and is depicted in Figure 2-6. Judgment
must be used to decide whether to concentrate each
toxic fraction separately or to combine various toxic
fractions prior to concentration. If, for example, three
successive fractions are toxic, there is a good
probability that the same toxicant is in all three. If
there are three other toxic fractions separated from
the first set by several non-toxic fractions, there is a
high probability that the second three contain a
different toxicant from the first three. There is also a
good probability that at least one non-toxic fraction
on either side of the toxic fractions contains some of
the toxicant. The virtue of combining fractions is to
reduce the work load and increase the concentration
in the concentrate. The disadvantage is that more
constituents that are not the toxicant will be included.
The decision has to be based on trial and error and
experience. As soon as an identification is obtained,
then HPLC conditions (gradient and fraction size and
number) can be optimized. Blanks corresponding to
the toxic fractions are concentrated the same way.
Steps
Concentration Factor
Dilute Fraction(s) 1:10 10OX
i
Extract on 1 mL
C18 SPE Column
(discard post-Ci8)
Figure 2-6.
Dry C-jg SPE Column
with Nitrogen
(Optional)
1
Elute Column with
3-100 pL Volumes
of 100% Methanol
i
Collect Eluate
Concentrate 5.000X
1
Conduct Toxicity
Test(20nL/lOmL 10X
water)
I
Analyze
Concentrate on 5.000X
GC/MS
Procedure to concentrate toxic HPLC
fractions (combined or individually).
Volume of eluate should be measured.
The concentrate must be finally checked for toxicity
before analysis, as described in Section 2.10. This is
the last opportunity to assure that the analytical
instrument receives the toxicant. Whether the toxicant
is detected by the analytical detector is always a
question. Since GC/MS detects only about 20% of
2-8
-------
organic chemicals (cf., Phase I), even such a broad
spectrum analytical instrument is not a certain way to
measure the toxicant.
2.14 GC/MS Analyses
Procedures and methods provided in this section are
based upon our experience in performing GC/MS
analyses on fractions from numerous effluents. In
general, these procedures are suggested and should
be used.
The typical instrument conditions are as follows: A
GC/MS system equipped for doing standard residue
chemistry analyses is suggested; i.e., a 30 m capillary
column, electron impact ionization, scan range of
50-500 amu, scan rate of 1 or 2 scans/sec, a GC
temperature program of 50 to 300°C at 5°C/min, and
data system with library searching ability.
For the GC/MS analysis, the prepared blank and toxic
fractions (in ~200 pL of methanol) should be tested
for toxicity (Figure 2-6). After verification of the
toxicity in the methanol concentrate, inject 1 or 2 uL of
the concentrate and collect the mass spectral data.
Note, methanol is not a typical solvent for GC
analysis. However, since only methanol fractions can
be tested for toxicity verification, methanol solvents
must be used. Injection of methanol on a capillary
column will shorten its life. Thus, your routine GC/MS
QA/QC procedures should closely monitor the
performance of your column.
After collection of the mass spectral data, peak
detection, and integration, algorithms should be used
on the data. All detected peaks should be library
searched; reverse search is preferred. The EPA/NIH/
NBS library (approximately 45,000 chemicals) for
performing the library searching has been adequate in
our laboratory.
Once the library search results are available, the
search report for each peak must be examined. By
this examination one must decide if the identification
by the search is valid and reasonable. In general, a
trained GC/MS chemist is required to do this
validation process. Factors considered in our
laboratory for this process include: A) are all major
ions present in the correct proportions, B) is this
identification consistent with other information about
the fraction, C) do forward and reverse searching
provide similar fits, and D) are the library searching
fits greater than 70%? Factor A must be met!
Consistency, factor B, considers circumstances such
as "has the identified chemical been found in vastly
different fractions," or "has the same identification
been given to numerous peaks in the same
chromatogram?" Both factors C and D are somewhat
relative and depend a lot on the sample and its
matrix. In addition, the toxicants are often very minor
components in the GC/MS total ion chromatogram
and thus, the quality of the mass spectral data even
after background subtraction will lead to poor results
for factors C and D.
After examination of the library search results, a list of
identified chemicals is assembled and evaluated using
the methods in the following section. For the
confirmation analyses we suggest EPA method 680
(Alford-Stevens et al., 1985).
2.15 Identifying Suspected Toxicants
The goal of the rest of Phase II is to determine if all
the toxicants have been identified. Two parallel lines
of investigation should be pursued to reach that goal.
One is to develop evidence that the concentrations of
the suspects are sufficient to cause toxicity. The
second is to develop data to estimate the proportion
of the whole effluent toxicity that is due to the
suspected toxicants, so a decision can be made as to
whether other toxicants need to be identified.
The first investigation should begin by comparing the
estimated concentrations of identified chemicals in
the concentrate at its LC50 to their LC50 values.
Because at this stage, the compound quantification
will have been done using one or more unknown
internal standards and recoveries , considerable error
may be involved in the estimate. Secondly, the LC50
data, if available, are likely to be for different species
than the one used in the TIE. Species differences are
usually as large as 100X and often, 1.000X. Given
these two sources of uncertainty and the chance that
they may reinforce one another, certainly if the
estimated concentration of a chemical at the fraction
LC50 is within 1,OOOX of the literature LC50, the
chemical should remain suspect. To the extent that
data for either compound quantitation or compound
LC50 values are known to be better, concentration
differences of smaller magnitude may be used to
eliminate suspects.
Once a list of suspects is available, the
measurements for both concentration and toxicity
should be refined. This will usually require obtaining
or making pure compound to make better analytical
measurements and to establish LC50 values for the
species of concern. This is one of the steps that
pushes us to do as much separation as practical
before analysis so that the list of suspects is small.
At this stage, only the concentration of the suspected
toxicant(s) in the concentrate is known; until recovery
through all the fractionation and concentration steps
is completed, suspect compound concentrations in
whole effluent are poorly known. Since the
concentrate is virtually devoid of suspended solids
and much of the effluent TOC, both of which may
dramatically affect toxicity of non-polar organics, the
toxicity of non-polar chemicals may be quite
different in the fraction tests than in the effluent test.
2-9
-------
Therefore, the toxicity of suspects in the fraction test
should be compared to the suspect's toxicity in a
relative pure water, such as reconstituted water. In
general, one would expect the toxicity to be lower in
the effluent.
During this same stage, the steps leading to the final
concentrate should be better checked for toxicity
recovery. The objective is to place a good estimate
on how much of the whole effluent toxicity is
contained in the final concentrate. This is best done
by testing the toxicity of the concentrate at
concentrations near those of whole effluent,
correcting for volume losses due to toxicity testing
SPE column fractions, which was previously ignored.
If the toxicity of the final concentrate is similar to that
of whole effluent, allowing for losses, and if the
concentration of the suspect(s) in the test
concentration which produces an LC50 is similar
enough to the LC50 to account for the concentrate's
toxicity, it is time to begin Phase III. If multiple
toxicants occur, the toxic units of those are compared •
to the whole effluent toxic units.
If the concentrations from improved quantitation and
toxicity measurement are close, Phase III should be
started, recognizing that other toxicants may yet be;
identified. If no suspects are found, then different
analytical methods are called for, and more
concentration, more separation, and more sophis-
ticated analyses must be done. When the concentrate
is toxic, the problem is not biological but is an
analytical one. Not much more can be done with
toxicity testing at this point. In some of the effluents
we have tested, finding other candidates has taken up
to four months. Since few laboratories will have all the
needed analytical equipment, contractors may be
used or help from laboratories that have the needed
equipment may be sought.
Because artifact toxicity can be created that equals
toxicity due to lost or unidentified toxicants, the
suspected toxicant(s) may be found to be the wrong
ones as Phase III progresses. One purpose of Phase
III is to identify such errors. Should this occur, one
must go back to the beginning of Phase II, or even to
Phase I and start over. If a number of different
effluent samples were used during Phase II work,
redoing Phase I on additional samples may be time
well spent since the effluent may have changed in the
interim.
While the distinction between Phase II and III may
seem well defined, as discussed here, in practice
there is no sharp boundary. In general, as soon as a
probable suspect is identified, confirmation proce-
dures of Phase III should start. The assumption that
one of the toxicants has been identified when it has
not, can badly mislead identification of other
suspected toxicants.
A final suggestion is to look at additive toxicity of
several to many constituents, if one or a few seem
not to account for toxicity. Enhancement of toxicity by
methanol should also be checked.
2-1 0
-------
Section 3
Ammonia
3.1 General Overview
Unlike Phase II procedures for non-polar organic
compounds or metals, the toxicant identification
methods described in this section are specific for
ammonia. The procedures used in this step of the
study assume that Phase I tests have implicated
ammonia as one of the toxicant(s) (Phase I, Section
8).
These tests should separate toxicity caused by
ammonia from toxicity caused by other compounds
which also become more toxic as pH increases. Two
methods can be used to add further evidence to
implicate ammonia as the toxicant. Equitoxic solutions
of effluent can be created through pH adjustment and
dilution of effluent aliquots, and a zeolite resin can be
used to strip the effluent sample of ammonia. Toxicity
tests and ammonia measurements are subsequently
performed on pre- and post-column effluent.
Depending on the presence of other toxicants in the
effluent, additional sample manipulations may be
needed before using these tests. For example, if toxic
oxidants such as chlorine are also present in the
effluent, sodium thiosulfate must be added to the
sample before conducting the Phase II ammonia
tests. If other toxicants can be removed by C-| Q SPE,
then these tests can be done on post-Cia SPE
effluent.
3.2 Equitoxic Solution Test
Principles/General Discussion:
As described in Phase I, ammonia toxicity is relatively
unique in its behavior as pH changes. For a constant .
temperature situation, Table 3-1 shows that as pH
increases by one unit, there is a nearly 10X increase
in the percent of total ammonia present as NHs (toxic
form) in aqueous solutions at pH 6.0-8.5. Table 3-1
has been constructed from the dissociation values for
ammonia (Thurston, et al., 1979). Once the effect of
pH and temperature on the percentage of total
ammonia as NHs are accounted for, as in Table 3-1,
there is a separate effect of pH on NHs toxicity as
shown in Table 3-2, wherein NHs toxicity increases
(decreasing ;LC50) with decreasing pH. By combining
these two pieces of information, radically different
concentrations of effluent can be made to be equally
toxic by pH adjustment and dilution. Reading the text
in the ammonia criteria document (EPA, 1985A) and
understanding the effect of pH on the acute toxicity of
ammonia will aid in understanding this test. As
discussed in the document, the slope of the LC50-
pH curve is similar enough for tested species, i.e.,
that an average slope is adequate for any of the
tested species. The formula for that curve is given at
the bottom of Table 3-2. You can see from Table
3-2 that NHa is about three times less toxic at pH
8.0 than at pH 7.0. On the other hand, about 10 times
more ammonia in the toxic form is present at pH 8.0
than at pH 7.0 (Table 3-1). Therefore, the toxicity of
a given amount of total ammonia is about three times
more toxic at pH 8.0 than at pH 7.0. By making the
proper dilutions of effluent, accompanied by the
proper pH adjustments, different effluent
concentrations will be equally toxic if ammonia is the
principal cause of toxicity. The probability that any
other chemical would have both the same dissociation
curve and the same change in toxicity with pH is very
small. If other toxicants are involved, the expected
toxicity will not occur unless they are removed or
neutralized prior to this test-
In using the equitoxic approach, it is necessary to
prepare dilutions/concentrations based on the
conflicting effects of pH on ammonia toxicity. Thus,
when pH is increased, a dilution factor must be
calculated that takes into account both increased
amounts of un-ionized ammonia present in the
solution, and the decreased toxicity of the un-
ionized ammonia to the organism due to the higher
pH. Conversely, when pH is lowered, a concentration
factor must be calculated that takes into account both
decreased amounts of un-ionized ammonia in the
sample, and increased toxicity of un-ionized
ammonia due to the lower pH. A descriptive example
of this calculation follows.
In the following example, the effluent has an LC50 of
50% and its pH at the LC50 is 7.7. Using the above
general principle, the following calculations for raising
the pH to 8.0 are:
3- 1
-------
Table 3-1. Percent Un-ionized Ammonia in Aqueous Ammonia Solutions for Selected Temperatures and pH
Values^
Temperature ("C)
PH
6.5
6.6
6.7
6.8
6.9
7.0
7.1
7.2
7.3
7.4
7.5
7.6
7.7
7.8
7.9
8.0
15
20
0.0865 0.125
0.109
0.137
0.172
0.217
0.273
0.343
0.432
0.543
0.683
0.858
1.08
1.35
1.70
2.13
2.66
0.158
0.199
0.250
0.314
0.396
0.497
0.625
0.786
0.988
1.24
1.56
1.95
2.44
3.06
3.82
21 i
0.135 I
0.170
0.214
0.269
0.338
0.425
0.535
0.672
0.845
1.06
1.33
1.67
2.10
2.62
3.28
4.10
22
0.145
0.182
0.230
0.289
0.363
0.457
0.575
0.733
0.908
1.14
1.43
1.8
2.25
2.82
3.52
4.39
23
0.156
0.196
0.247
0.310
0.390
0.491
0.617
0.776
0.975
1.22
1.54
1.93
2.41
3.02
3.77
4.70
24
0.167
0.210
0.265
0.333
0.419
0.527
0.663
0.833
1.05
1.31
1.65
2.07
2.59
3.24
4.04
5.03
25
0.180
0.226
0.284
0.358
0.450
0.566
0.711
0.893
1.12
1.41
1.77
2.21
2.77
3.46
4.32
5.38
aThurston, et al., 1979.
Table 3-
pH
8.0
7.9
7.8
7.7
7.6
7.5
7.4
7.3
7.2
7.1
7.0
2.
Calculated Un-ionized Ammonia LC50 Values
at Different pH
an LC50 at pH
LC50 at NH3a
(mg/L)
3.00
2.84
2.68
2.50
2.30
2.10
1.88
1.67
1.45
1.25
1.07
Values for Ceriodaphnia Using
8.0 and 25° C Of 3.0 mg/L
pH
6.9
6.8
6.7
6.6
6.5
6.4
6.3
6.2
6.1
6.0
LC50 at NH3a
(mg/L)
0.90
0.75
0.62
0.51
0.42
0.38
0.30
0.24
0.19
0.15
(1) Increase
in Nhta dissociation is from 2.77% to
5.38% (Table 3-1).
Therefore,
f «i n
O.dO
=1.94 = dilution factor.
(2) Decrease
toxicity:
LC50
LC50
2.77
the dilution
at pH 7.7 =
at pH 8.0 =
The toxicity decrease factor
3.00
2.50
factor by
2.50 (Table
3.00 (Table
is:
= 1.20
the reduced
3-2)
3-2)
The LC50 value at pH 8.0 that is used is not important since
the ratios, not the absolute concentration, are used in the
calculations for dilution. The following formula from EPA
(1985A) was used to calculate values:
Therefore,
1.94(1)
= 1.6 dilution factor corrected for toxicity
Formula 1/350=
(LC50[pH=8.0]) (1.25)
1 + 10
7.4-pH
1.2(2)
(3) Effluent concentration needed at pH 8.0 is:
50%
- =3 1.25% effluent
1.6
For lowering the pH to 7.5:
(4) Decreased in NHs dissociation is from 2.77 to
1.77 (Table 3-1).
3-2
-------
Therefore, concentration factor
2.77
= 1.57
1.77
(5) Reduce the concentration factor due to increased
toxicity caused by reduced pH:
LC50 at pH 7.7 = 2.5 (Table 3-2)
LC50 at pH 7.5 = 2.1 (Table 3-2)
Toxicity correction factor
2.5
2.1
Therefore,
concentration factor 1.57(4)
toxicity correction factor 1.19 (5)
= 1.3
(6) Effluent concentration at pH 7.5 is 50% x 1.3 =
65% effluent.
Consideration must be given to the CO2 that will be
released when the pH of water is lowered one to two
pH units. Of course, the more carbonate and
bicarbonate alkalinity present, the more free CO2 will
be released. We have tested both young fathead
minnows and Ceriodaphnia and they are not bothered
by 600 mg/L of CC>2, which is the approximate
concentration occurring when 300 mg/L (as CaCOs)
hardness water is lowered from a pH of 8.5 to 6.0. If
effluent/water mixtures are used with more carbonate
and bicarbonate alkalinity, the effect of the released
CO2 must be checked with the test species. High
CO2 concentrations may also change the toxicity of
ammonia, so its effect should also be measured
where appropriate.
Since any dilution can be made, the selection of the
high pH condition can be any pH which is
physiologically acceptable. However, the change in
ammonia toxicity above pH 8.0 is smaller and the
data are less precise. Therefore, pH 8.0 may be the
upper useful pH limit.
For the lower pH, some trial and error is needed.
Obviously 100% effluent is the highest concentration
obtainable, so the necessary concentration factor
must not exceed the value 100%/LC50(%). In the
above example when the LC50 is 50%, that value is
two. It is obvious that, at most, we can only double
the LC50 concentration to 100%. In the above
example, since only 65% effluent is required at pH
7.5, a lower pH would be better so that the difference
in effluent concentrations tested would be larger. If it
were larger, dilution effects on toxicants other than
ammonia would be larger and the effect would be
easier to measure. For example, if we used a pH of
7.2, 90% effluent would be needed. ,
The key problem in doing this test is to measure and
control the pH. A 0.1 pH unit change from nominal is
too much variation. To illustrate, the change in pH
from 8.0 to 7.9 lowers the concentration of the toxic
form of ammonia 20%. Experience has shown that
just any pH meter is not good enough. The meter
used must read accurately to two decimal places. If
the meter used does not lock on the stabilized
reading after the rate of change has diminished to a
specified rate, then pH readings must be made using
a measured elapsed time. Readings should be
repeated using constant and reproducible stirring
rates that do not result in excessive loss of CO2 and
therefore change pH.
Secondly, the ammonia concentration must be high
enough so the test can be done in a work day to
monitor the pH carefully and frequently. The pH of
pH-adjusted effluents cannot be controlled accep-
tably for 48 hours. As a guide, the ammonia
concentration needs to be 2X the 48 hour LC50 to
produce mortality rapidly enough to complete the test
in eight hours. For many effluents, ammonia may
need to be added to raise the concentration to this
range. Of course there is an upper limit to the
percentage the ammonia can be increased, and
useful data obtained. If one had to increase the
concentration 8-1 OX then the error of the method is
too great to use it. An increase of 2 or 3 times is a
reasonable upper limit for spiking. A series of known
ammonia/pH combinations in laboratory water,
duplicating the effluent exposure should be run in
parallel for comparison and as a check on the
method.
If all air is excluded from the test chamber, the pH will
frequently hold very well. Since time to mortality is
used, frequent observations are necessary and,
therefore, the cover used needs to allow observations
of the test animals. If pH needs to be adjusted during
the exposure, then the cage method must be used
(Phase I). Proper stirring and measurement cannot be
done if the test animals are present. Once the test
organisms die, the pH can change appreciably and
pH measurements should be made at the time of
mortalities. As this document goes to press, we are
experimenting with the use of controlled
concentrations of CO2 in a sealed headspace over
the sample. The approach appears very promising
and simple.
3.3 Results/Subsequent Tests
No LC50 is calculated for the pH-adjusted effluent
samples used in this test. Instead, a comparison is
made based on survival and time to onset of
symptoms in the three exposures. If ammonia is not
the sole toxicant in the effluent, the expected change
3-3
-------
in toxicities will not be found. Attempts can be made
to neutralize or remove other toxicants before
performing this test, but few if any methods are
available in which there is certainty that additional
constituents would not be removed. If, after such
removal, this test shows ammonia to be the toxicant,
then ammonia is a principal part of the toxicity.
3.4 Zeolite Test
Principles/General Discussion:
Zeolites are naturally occurring or synthetically
created crystalline, hydrated alkali-aluminum sili-
cates. They have a general formula of Mn
0*Al2Oa.ySi02.zH2O; M = group IA or IIA element, h
= +2 for group IA, +1 for group IIA, y>2, and z =
the number of water molecules contained in the
interconnected voids or channels within the zeolite
(Windholz, et al., 1983). When zeolite is placed in
aqueous solutions, the positively charged group IA or
IIA elements (M" +) of the zeolite are mobile and can
undergo exchange with other cations in the water. As
such, zeolites have frequently been employed as ion
exchange resins for removal of the ammonium ion
NH4+ from aqueous solutions. Because of their
ability to exchange other cations such as heavy
metals, and their use as molecular sieves, filter
adsorbents and catalysts, zeolites have not been
suggested for use in Phase I. Zeolites can be
effective in Phase II, however, if Phase I results
implicate ammonia as the causative agent and
establish that other groups of toxicants (such as
organics and metals) are playing no role in effluent
toxicity.
3.5 Procedure
In this section of Phase II, toxicity tests and ammonia
measurements are made on whole effluent and
effluent having passed through a zeolite column.
Removal of toxicity by zeolite will add to the evidence
implicating ammonia as the toxicant.
To prepare the zeolite column, 30 g of zeolite is
added to 60 mL of high-purity water. Zeolite
particles should be of approximately the same size
(32 to 95 mm) to insure efficient ion exchange while
preventing channeling (e.g., effluent not contacting
the zeolite during flow through the column) or
excessive resistance to flow. Removal of extremely
large or small particles can be accomplished by
screening the zeolite with sieves or mesh screens.
The zeolite slurry is poured into the column and three
bed volumes of dilution water are passed through the
column. The last 10 mL of dilution water is collected
for use as a zeolite blank and should not be toxic.
Next, 200 mL of 100% effluent is passed through the
column at a rate of 2 mL/min. After the dilution water
remaining in the column has been flushed out by
effluent, post-column effluent is collected and
measured for ammonia and toxicity. Temperature and
pH should be recorded in order to provide the means
to calculate both total and un-ionized ammonia in
the sample.
Another aliquot of the effluent sample (not having
passed through the zeolite column) is used for
ammonia analysis and a toxicity test. These data will
be compared with the same data for the post-zeolite
column effluent to determine if the post-column
reduction in effluent toxicity is consistent with
ammonia removal through the column.
3.6 Interpretation of Results
The control for test organism survival, dilution water
quality, and other test conditions, will be provided
through the preparation of a single exposure toxicity
test on dilution water. Dilution water having passed
through the column will act as a blank for toxic
artifacts leached from the zeolite. Increased toxicity in
the post-zeolite effluent, relative to the whole
effluent, indicates the presence of toxic artifacts.
Since many cations will be exchanged, adding solids,
such as the YCT food (yeast-Cerophyl-trout food)
fed to Ceriodaphnia, is a wise precaution. Additional
measures to clean-up the zeolite (such as Soxhlet
extraction) or alternate uncontaminated sources may
need to be investigated. Column packing, effluent pH,
ammonia levels, and flow rate through the column
can all affect the efficiency of the cation exchange
process. Lowering effluent pH prior to zeolite
chromatography and/or lowering flow rate through the
column may also result in greater removal of
ammonia. Occluded gas between zeolite particles
may also impair the column's capacity to remove
ammonia. If this appears to be a problem, the zeolite
solution should be degassed using a vacuum prior to
pouring the column.
Zeolite can be regenerated, but fresh zeolite should
be used to pack new columns. If the equitoxic
ammonia test and zeolite test are consistent with
ammonia toxicity, confirmation should be started as
described in Phase III.
3-4
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Section 4
Cationic Metals
4.1 General Overview
The reduction in effluent toxicity afforded by addition
of EDTA indicates that certain cationic metals may be
present in the effluent at lethal levels. The suspect
causative agent(s) is chosen based on correlation of
effluent toxicity and metal concentrations, reference
metal toxicity data, and changes in toxicity observed
during manipulation of water quality characteristics.
Principles/General Discussion:
The structure of EDTA (ethylenediamine tetraacetic
acid) is shown below:
HOOC'
HOOC
COOH
COOH
This compound can be thought of as a Lewis base,
having the capability of donating electrons (through
the nitrogen and hydroxyl oxygen atoms) to Lewis
acids (such as metals) in order to satisfy the
coordinate requirements. EDTA is a hexadentate
ligand; there are six positions on the molecule
available to donate electrons. The metals that are
chelated by EDTA are a function of the number of
electrons needed by the cation to fill its outer orbital.
Generally, EDTA forms a 1:1 complex with cations of
+ 2 and + 3 valences.
Simply stated, one could predict the degree to which
certain metals are chelated by EDTA by comparing
the stability constants (f3 = [EDTA x metal]/[metal] x
[EDTA]) for the metal-EDTA complexes. Such a
comparison suggests which metal would win in a
competition for the electrons provided by the EDTA
ligand. One finds that EDTA strongly chelates Fe3 + ,
Cu2*, Ni2 + , Pb2 + , Cd2 + , C02 + , Zn2 + , AI3 + , and
Mn2 + , while Ca2 + , Mg2*, Sr2 + , Ba2 + , Tl+ and
Ag2+ are only weakly chelated by EDTA because of
their relatively low electrophilicity (Flaschka and
Barnard, 1967).
Unfortunately, the complexing effect of EDTA towards
metals in a complex aqueous solution cannot be
estimated solely on the basis of stability constants
(Stumm and Morgan, 1981). Complex solutions
contain other organic and inorganic ligands which
compete to different extents for the various available
cations. This becomes obvious when studying
apparently supersaturated aqueous solutions of
metals in which otherwise insoluble metals are
solubilized by their association with ligands.
The concentrations of other ligands, relative to that of
EDTA, as well as their specificity for particular metals
will greatly change the results from the above
prediction. For example, even when the stability
constant for EDTA and a metal is much greater than
the stability constant for the metal and a different
ligand, if the concentration of the competing ligand is
much greater than the EDTA concentration, no
substantial complexation of the metal by EDTA will
take place. The aquatic organism itself can be
thought of as a powerful and very specific organic
ligand, often acting as a sink for metals in aqueous
systems. While the oxygen donor atoms of EDTA
form general complexes, the ligands provided by
organisms are very selective as a result of the
geometric arrangements formed. This situation is
exemplified by the case of Hg2 + ; the EDTA Hg2 +
stability constant is quite large (21.8) compared to
that of other metals chelated by EDTA. Despite this,
addition of EDTA does not reduce mercury toxicity
because the aquatic organisms' ability to take up the
metal is greater.
Solution pH, temperature and ionic strength can also
affect the form in which a metal exists in an aqueous
solution. At low pHs, H+ competes with the metal for
EDTA binding sites; at high pHs, OH" competes with
EDTA and other ligands for a coordinative position on
the metal. Time may be an important factor in
determining what EDTA chelates and the extent to
which chelation takes place. Because equilibria may
be established relatively slowly, unstable complexes
may form and dissociate, changing the composition of
the solution over time. Addition of EDTA can result in
a redistribution of many or all of the metals 'in
solution. This can result when EDTA binds strongly
with a certain metal, in turn freeing up other metals in
4- 1
-------
the system. It should be pointed out that unlike metal
complexes with naturally occurring ligands (e.g.,
humic and fulvic acids), EDTA forms a very stable
complex. Finally, the degree of EDTA complexation is
also affected by dilution. Increasing dilution results in
fewer polynuclear complexes and a greater degree of
hydrolysis. This is most obvious with the less stable
EDTA complexes of unidentate metals such at Tl+.
Given the need for information on the solution's major
and minor ions, its oxidation/reduction state, the
acid/base and complexing components, and finally the
adsorbing surfaces, it becomes clear that even the
multiligand, multimetal models currently available will
not succeed in predicting the cations and cor-
responding quantities bound to the added EDTA.
Likewise, there exists no reliable method to detect or
isolate specific EDTA chelates, particularly when the
concentration of the metal of concern may be very
low. For this reason, utilization of general methods for
the detection of cations is desirable.
i.
For the most part, there are two methods available for
analysis of cationic elements and inorganic
compounds. In AA analysis a light beam at the
wavelength of the metal of interest is passed through
an atomized sample. The light energy absorbed by
the atomized sample provides the measure of the
concentration of the metal in the sample. A flame
(direct aspiration procedure) or graphite furnace
(furnace procedure) is used to atomize the sample. In
inductively-coupled plasma atomic emission
spectroscopy (ICP), nebulized sample is directed into
a plasma torch. The resulting atomic line emission
spectra (characteristic of the metals in the sample)
are produced via radio fre-quency inductively
coupled plasma. The advantage to this latter
technique is that it can be used to simultaneously
determine multielements at trace levels in aqueous
solutions, but sensitivity and interferences are
sometimes problems. AA analyses, on the other
hand, require that the analyst choose, in advance,
those elements to be measured. The furnace AA
technique does offer the advantage of producing
detection levels which are typically an order of
magnitude lower than the levels resulting for ICP. The
need for the lower levels of detection provided by
furnace AA will be a function of the toxicity and the
concentrations of metals present in the sample.
In either case, a measure of the dissolved metals in
the sample (i.e., those constituents which pass
through a 0.45 iim membrane filter) is recom-
mended. The "dissolved" metals are in no way
synonymous with the "biologically available metals."
Beyond the use of the aquatic organism, however,
there is no technique to determine the "biologically
available" fraction of the total metal. Further, only
rudimentary techniques are available to specifically
identify the individual species of a metal (e.g., free
charged metal ions [Mn + ], inorganic ion pairs or
complexes such as aquoions, [M(H2O)n + m], hy-
droxoions [M(OH)pn-P + ], oxoions [MOnn' 2q + ],
organic complexes and chelates [M x EDTA], metal
species bound to high molecular weight organic
material [M x lipid] or metal species in the form of
highly dispersed colloids or sorbed on colloids [M x
clay]). Stumm and Morgan (1981) have listed some
general methods for assisting in identification of
individual species.
Given the concentration of metals present in toxic
effluent samples, a number of techniques, described
below, are available to implicate the toxicant(s).
4.2 Procedure
In order to compare the results of the metal analyses
and toxicity test, sample preservation with HNOa is
not recommended. Instead, immediate analysis is
recommended. An effluent LC50 should be
determined for the test species using whole effluent.
The dilution water used should be reconstituted water
of the same hardness as that of the effluent. A 250
ml_ aliquot of the sample is filtered through a 0.45 pm
filter. The filter should be prepared by rinsing with 200
mL of high purity water. An appropriate quantity of the
last portion of the high purity water passing through
the filter should be collected as an analytical blank for
metal contamination from the filter. A single exposure
toxicity test on 10 mL of the dilution water having
passed through the filter should also be performed.
The filtered sample should be tested for toxicity as
described above to measure the effect of filtration on
sample toxicity. The filtrate should be analyzed for
metals. If toxicity is reduced or removed upon
filtration (and effluent toxicity has not previously been
affected by C^Q SPE or filtration through a 1.0 pm
glass fiber filter) it will be necessary to measure
metals retained by the 0.45 urn filter. Methods for
determination of suspended metals are described in
Section 200 or Method 200.7 (EPA, 1983). If the AA
analysis is used for dissolved or suspended metals,
iron, copper, nickel, lead, cadmium, chromium,
cobalt, zinc, aluminum, and manganese should be
included in the analyses.
Initial determination of suspect metals based on a
comparison of metal analyses and toxicity test results
should be made. This can be followed for the
dissolved and/or suspended metals, as necessary,
based on toxicity test results. Because metals can be
readily purchased, tests on pure metals should be
done rather than to use published values. Side-by-
side tests with reference metal and effluent avoid
subtle error and the improved data are well worth the
effort. Literature summaries are also available (EPA,
1980; 1985B; 1985D; 1985E; 1985F; 1986; 1987A;
and 1987B). In addition to matching the hardness and
pH of the dilution water to the effluent sample by
addition of the appropriate ratios of MgCC-3 and
4-2
-------
CaCOs, the wastewater total suspended solids (TSS)
and TOC can also be approximated in the water used
to test the reference metal. TOC and SS from the
YCT food can be added such that the TSS level of
the dilution water equals that of the effluent sample.
The TOC of the dilution water can be further
supplemented through the addition of humic acid.
Some indication of the binding of metals to organics
in the effluent may be arrived at through hexane
extraction of an aliquot of the sample (Stary, 1964).
Theoretically, metals bound to organics soluble in
hexane should leave the effluent. The hexane can be
evaporated and the residue reconstituted and tested
for metals. Additionally, the reduction in metals can
be estimated by repeating the metal analysis on the
extracted effluent. The toxicity attributed to metals
associated with organics may be estimated by
performing a toxicity test on the solvent extracted
effluent. Traces of hexane must be removed by
aeration from the extracted effluent by aeration prior
to toxicity testing. The effects of aeration on sample
toxicity must also be considered in this analysis. In
any case, metals strongly suspected of causing or
contributing to sample toxicity should be tested in
pure solution as described above with the TIE test
organism species.
The correlation between the toxicity of a sample and
its concentration(s) of metal(s) over time (Phase III)
can also be used to narrow the list of suspect
causative toxicants prior to confirmation. The effects
of water quality characteristics on metal toxicity must
be included over the sampling period. One solution to
this problem is to collect several samples over a short
time span (e.g., six grab samples in 24 hours). The
correlation coefficient for sample metal concentration
(or summed toxic units of metals) versus sample
toxicity is calculated for each sampling event (i.e., 24
hour period). The population of correlation coefficients
for such multiple sampling events may give results
less affected by hardness, suspended solids and
TOC. The assumption in taking this approach is that
water quality characteristics affecting metal toxicity
will vary less during short time periods. Obviously,
metal concentration must vary enough to provide a
sufficient range for correlation. When one reaches
this stage, Phase III work is being started and Phase
III methods should be consulted. Symptoms, species
sensitivity, spiking, water quality adjustments and
correlation are all applicable Phase III approaches to
confirm the cause of toxicity.
Where there is also non-metal toxicity, this toxicity
identification and then its confirmation must be
integrated with the metal work data.
4-3
-------
-------
Section 5
References
Alford-Stevens, A.L., J.W. Eichelberger, and W.L.
Budde. 1985. Method 680. Determination of
Pesticides and PCBs in Water and Soil/Sediment
by Gas Chromatography/Mass Spectrometry.
Physical and Chemical Methods Branch, U.S.
Environmental Protection Agency, Cincinnati, OH
45268.
EPA. 1980. Ambient Water Quality Criteria for Silver.
EPA-440/5-80-071. Environmental Protection
Agency, Environmental Research Laboratory-
Duluth, Duluth, MN.
EPA. 1983. Methods for Chemical Analysis of Water
and Wastes. EPA-600/4-79-020. Environmental
Monitoring and Support Laboratory, Cincinnati, OH.
EPA. 1985A. Ambient Water Quality Criteria for
Ammonia. EPA-440/5-85-001. Environmental
Protection Agency, Environmental Research
Laboratory-Duluth, Duluth, MN.
EPA. 1985B. Ambient Water Quality Criteria for
Cadmium. EPA-440/5-84-032. Environmental
Protection Agency, Environmental Research
Laboratory-Duluth, Duluth, MN.
EPA. 1985C. Ambient Water Quality Criteria for
Chlorine., EPA-440/5-84-030. Environmental
Protection Agency, Environmental Research
Laboratory-Duluth, Duluth, MN.
EPA. 1985D. Ambient' Water Quality Criteria for
Chromium. EPA-440/5-84-029. Environmental
Protection Agency, Environmental Research
Laboratory-Duluth, Duluth, MN.
EPA. 1985E. Ambient Water Quality Criteria for
Copper. EPA-440/5-84-031. Environmental
Protection Agency, Environmental Research
Laboratory-Duluth, Duluth, MN.
EPA. 1985F. Ambient Water Quality Criteria for Lead.
EPA-440/5-84-027. Environmental Protection
Agency, Environmental Research Laboratory-
Duluth, Duluth, MN.
EPA. 1986. Ambient Water Quality Criteria for Nickel.
EPA-440/5-86-004. Environmental Protection
Agency, Environmental Research Laboratory-
Duluth, Duluth, MN.
EPA. 1987A. Ambient Water Quality Criteria for Zinc.
EPA-440/5-87-003. Environmental Protection
Agency, Environmental Research Laboratory-
Duluth, Duluth, MN.
EPA. Draft 1987B. Ambient Water Quality Criteria for
Aluminum. U.S. Environmental Protection Agency,
Environmental Research Laboratory, Duluth, MN.
Flaschka, H.A. and A.J. Barnard, Jr. (Eds.). 1967.
Chelates in Analytical Chemistry. Marcel Dekker,
Inc., New York, NY. 418 p.
Mount, D.I. 1988. Methods for Aquatic Toxicity
Identification Evaluations: Phase III Toxicity
Confirmation Procedures. EPA/600/3-88/036.
Mount, D.I. and L. Anderson-Carnahan. 1988.
Methods for Aquatic Toxicity Identification
Evaluations: Phase I Toxicity Characterization
Procedures. EPA/600/3-88/034.
Peltier, W. and C.I. Weber (Eds.). 1985. Methods for
Measuring the Acute Toxicity of Effluents to
Freshwater and Marine Organisms. Third Edition.
U.S. Environmental Protection Agency. Cincinnati,
OH. EPA-600/4-85-013.
5- 1
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