&EPA
United States
Environmental Protection
Agency
Environmental Research
Laboratory
Duluth MN 55804
National Effluent Toxicity
Assessment Center
Research and Development
EPA/600/3-88/036 Feb. 1989
Methods for Aquatic
Toxicity Identification
Evaluations
Phase III Toxicity
Confirmation
Procedures
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EPA/600/3-88/036
February 1989
Methods for Aquatic
Toxicity Identification Evaluations
Phase III Toxicity Confirmation Procedures
Donald I. Mount
U.S. Environmental Protection Agency
Environmental Research Laboratory
Office of Research and Development
Duluth, Minnesota 55804
National Effluent Toxicity
Assessment Center
Technical Report 04-88
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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
This document, Phase III, describes procedures for confirming that suspected toxicants
are the true cause of toxicity. These procedures are applicable and necessary whether
or not Phases I and II have been used to identify the suspected toxicants.
There are two major reasons to require confirmation procedures. First, the effluent
manipulations used in Phases I and II will, for some effluents, create artifacts that may
lead to erroneous conclusions about the toxicants. In Phase III, manipulations of the
effluent are absent or minimal and artifacts are far less likely to occur. Sometimes,
toxicants will be suspected through other approaches which are not themselves
definitive. In either case, confirmation is necessary.
The second reason sterns from the definite probability that the substances causing
toxicity change from sample to sample, from season to season or some other
periodicity. Since toxicity is a generic measurement, just measuring toxicity will not
reveal such variability. Phase III procedures will reveal the presence of variable
causative agents. Obviously, this crucial information is essential so that remedial action
may be taken to remove toxicity.
Confirmation, whether using these procedures or others, should always be completed
because the risk is too great to avoid this step. Especially for discharges with little
control over the influent or for discharge operations that are very large or complex, the
probability that different constituents will cause toxicity over time is great.
Unlike Phases I and H, most of the approaches in Phase III are equally applicable to
chronic as well as acute toxicity. Effluent manipulation is minimal, and additives, for the
most part, are not used; therefore, chronic tests can be done.
A section is also included for guidance when the treatability approach (EPA, 1988A;
EPA, 1988B), rather than the toxicant identification approach, is taken. The treatability
approach requires confirmation as much as or more so than the toxicant identification
approach.
The reader should refer to Phase I for Quality Assurance/Quality Control (QA/QC),
Health and Safety, Facilities and Equipment, Dilution Water, Sampling and Testing.
in
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Abstract
Various procedures are described that provide evidence that the suspected toxicants in
effluents are the actual toxicants. These procedures include: correlation, symptoms,
relative sensitivity, spiking, mass balance! and miscellaneous procedures.
IV
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Contents
Page
Foreword Ill
Abstract ....!!.............!..... iv
Contents v
Figures '.'.'.'.'.'.'.'. vi
Tab'es vii
Acknowledgments vjjj
1. Introduction 1.1
2. Correlation Approach 2-1
3. Symptom Approach 3_1
4. Species Sensitivity Approach 4_1
5. Spiking Approach 5^
6. Mass Balance Approach 5_1
7. Deletion Approach - 7_1
8. Miscellaneous Approaches 8-1
9. Conclusions g_1
10. When the Treatability Approach Has Been Used 10-1
11. References _
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Figures
Number
2-1.
2-2.
2-3.
Correlation of whole effluent toxicity and one suspect toxicant for a POTW effluent.
Toxicity contribution for one of two toxicants in a POTW effluent
Toxicity contribution from two toxicants for a POTW effluent
Page
2-2
2-3
2-4
VI
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Tables
Number Page
6-1. Comparison of Whole Effluent and Methanol Fraction Toxic Units 6-1
VII
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Acknowledgments
I want to thank the staff of NETAC for their contributions to the preparation of this
document. Endless hours of technical discussion, generation of data, suggestion for
improvement, manuscript review and final editing and typing were all necessary and
useful inputs to produce this document. As for Phase I and Phase II, the administrative
and financial support of Nelson Thomas apd Rick Brandes were crucial.
VIII
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Section 1
Introduction
The final confirmation phase consists of a group of
steps intended to confirm the suspected cause of
toxicity. It will usually follow work completed in Phase
I and II (Mount and Anderson-Carnahan, 1988A and
1988B; hereafter referred to as Phase I and Phase II),
but the boundary may be indistinct. Phase III
procedures should also follow after the toxicants have
been identified by other means. Rarely does one step
or test conclusively prove the cause of toxicity.
Rather, all practical approaches are used to provide
the "weight of the evidence" that the cause of toxicity
has been identified. Described below are various
approaches that are often useful in providing that
"weight of evidence." These consist of correlation,
observation of symptoms, relative sensitivity, spiking,
mass balance estimates and miscellaneous
adjustments of water quality.
The final confirmation is needed not only to provide
data to prove that the suspected toxicants are the
cause of toxicity in a series of samples, but perhaps
more importantly, to assure that the cause of toxicity
is consistent from sample to sample over time. When
remedial action involves treatment changes, one must
be certain that toxicity from specific toxicants is
consistently present and that the suspected toxicants
account for all the toxicity. Treatment changes will not
necessarily result in removal of everything to an
acceptable concentration. If toxicity is caused by a
variety of toxicants being added at varying intervals,
the remedial actions that are practical may differ from
those needed when toxicity is caused by the addition
of the same constituents consistently.
There is a strong tendency to shorten or eliminate
this final confirmation because by the time it has been
reached, an enormous amount of testing may have
been done, the investigators are convinced of the
cause of toxicity and the final confirmation seems
redundant. To skimp at this stage is to invite disaster!
One cannot expect to fractionate and otherwise
change a complex mixture (that most effluents are)
and not produce artifacts or come to some false
conclusions about the toxicants.
Not all approaches will be applicable to every effluent,
and certainly, in time, additional ones will be
developed. They need not be done in a particular
order, but some, especially the correlation, require
substantial calendar time to complete and should be
initiated at the beginning stages. Judgment must be
made as to how many Phase III approaches should
be used and how many samples for each should be
completed. How completely this phase is done will
determine the authenticity of the outcome. Certainly
the confidence needed is dependent, at least in part,
on the significance of the decision that will be based
on the results. For example, if a suspected toxicant
can be removed by pretreatment or by a process
substitution, a higher degree of uncertainty may be
acceptable than if an expensive treatment plant is to
be built. Such considerations are subjective and
cannot be reduced to a single recommended number
of samples.
In Phases I and II, the permissibility of "rounding
corners" on methods and protocols to reduce cost
and allow more testing was discussed. This is all
reversed in this phase, because here the definitive
data that constitute the basis for decisions are
generated. In Phase III, the best test procedures
should be followed paying careful attention to test
conditions, replicates, quality of test animals,
representativeness of the effluent samples tested and
strict QA/QC analytical procedures including blanks
and recovery. Analytical measurements must be
clearly definitive for the identity of the toxicant as well
as for the concentration measurement. Sometimes,
small differences in toxicity must be detected and if
so, concentration intervals should be smaller to better
detect small differences. All of the data from Phases I
and II are preliminary relative to Phase III. However,
since the phases merge from one to another, stricter
QA/QC should begin in Phases I and II as soon as the
toxicants are suspected, so that the data can be used
in Phase III.
The following approaches have been useful in
previous toxicity identification evaluations (TIEs) in
our laboratory. They need not be done in any
particular sequence, and the list for possible
approaches will get larger as experience is gained.
Some techniques used in Phase III require keen
observations and extensive or broad knowledge of
both chemistry and toxicology. Above all, an ability to
1 - 1
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synthesize bits of evidence in a logical sequence is
essential. Our work shows that the more experienced
the investigator, the higher the success rate. This
work cannot be done successfully in a
compartmentalized fashion where workers do not
interact daily and certainly cannot be successful
without the personal involvement of a well-trained
staff.
1 -2
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Section 2
Correlation Approach
The purpose of this approach is to show whether
there is a consistent relationship between the
concentration of the suspected toxicant(s) and the
effluent toxicity. For the correlation approach to be
useful, toxicity of the effluent must be sufficiently
variable to provide an adequate range of LCSOs over
which to do the regression analysis. In our
experience, an effluent with insufficient variability has
not been encountered. The LC50 data versus toxicant
concentration: must be transformed to give a linear
relationship for regression analyses. More importantly,
if there is more than one toxicant and each has a
different toxicity, .the' concentration of each must be
adjusted for the different toxicity before they can be
summed. The easiest way to prepare such plots is to
convert all the data to toxic units (TUs). Effluent TUs
are obtained by dividing 100% by the LC50 in percent
of the effluent. The toxicant concentration is
converted to TUs by dividing the measured toxicant
concentration by the LC50 of the toxicant. If more
than one toxicant is present, the concentration of
each one is divided by the respective LC50 and the
TUs can then be summed (cf., discussion below on
additivity).
Figure 2-1 is an example of the regression from an
effluent from a publicly owned treatment work
(POTW) in which the suspected toxicant was
diazinon. The independent variable (x-axis) is the
TUs of diazinon and the dependent variable (y-axis)
is the TUs of effluent toxicity. The solid line is the
observed regression line obtained from the data
points, and the dashed line is the expected or
theoretical regression line. We know that if there is
1.0 TU of the toxicant in 100% effluent, then the
effluent should have 1.0 TU (LC50 = 100%). Likewise,
for 2.0 TU of toxicant the effluent TU should be 2.0,
et cetera. Thus, the expected line has a slope of one
and an intercept of zero. In Figure 2-1, the intercept
(0.19) is not significantly different from zero and the
slope is very close to 1 (1.05). The r2 value is 0.63
which, while not high, indicates that the majority of
the effluent toxicity is explained by the concentration
of the toxicant. In a small data set such as this one is,
one value that had about 5.0 effluent TUs lowers the
r2 value substantially.
Figure 2-2 is a similar plot for another POTW in
which diazinon was also the suspect toxicant. For
these data, the slope is 1.38, the intercept is 1.24 and
the r2 value is only 0.15, all indicating a very poor fit.
The r2 value being so low means a large amount of
scatter and, therefore, little can be inferred from the
slope and the intercept.
Based on this analysis, we returned to Phase I and II
procedures and discovered that two other
organophosphates, chlorfenvinphos (CVP) and
malathion, were present at measurable concen-
trations, and CVP was present at toxic concen-
trations. A new correlation was begun, measuring all
three chemicals. CVP and diazinon have nearly
identical LC50 values for the species used in this TIE.
Malathion is about one-fourth as toxic; therefore,
malathion did not contribute much toxicity because of
its low concentration and its being less toxic.
In addition to slope and intercept, some judgment of
the scatter about the regression line must be made.
This can be done statistically, but when the sample
size is large, the scatter can be very large and yet not
negate the relationship. A suggested approach to
avoid the effect of sample size on the significance of
scatter is to set a lower limit on r2. This value (often
expressed as percent) provides the measure of how
much of the observed effluent toxicity is correlated to
the measured toxicant. It is not dependent on
choosing the correct LC50 value of the toxicant. The
specific choice of the minimum value of r2 should be
made based upon the consequences of the decision.
One must recognize that experimental error makes an
r2 value greater than 0.8 or 0.85 difficult to obtain.
Therefore, where minimal chance of an incorrect
decision is needed, an r2 value of nearly 0.8 might be
used. Where an increased risk of an incorrect
decision (i.e., a lesser amount of the toxicity
accounted for) is acceptable, a lower value such as
0.6 might be used.
Since less than 1 TU cannot be measured in whole
effluent, such values are, of necessity, excluded from
the regression. However, when the TUs based on
chemical analyses are less than 1.0 TU and effluent
LC50 values are less than 1.0 TU, the data support
the validity of the regression. In this effluent there
was another toxicant present in a different fraction
that was not identified and was not always
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Ul
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I
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a
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6.00-,
5.00-
4.00-
3.00 -
2.00-
1.00-
0.00
Theoretical ~
Observed :
Legend: R2 = 0.63
Slope = 1.05
Y—Intercept = 0.19
I
I
0.00
1.00
2.00 3.00 4.00
Toxic Units of Suspect Toxicants
5.00
6.00
Figure 2-1. Correlation of whole effluent toxicity and one suspect toxicant for a POTW effluent.
measurable. This would explain part of the deviation
from the expected values.
Depending on the specific type of variability,
correlation may be more definitive when two or morp
toxicants are present. For example, suppose three
toxicants are involved, as in Figure 2-3. If each
toxicant has the same LC50 and each is strictly
additive with the ratio of their concentrations
remaining the same, the slope will be the expected
but the intercept will be positive if all are not
identified. If the relative amounts (ratios) of each
toxicant vary from sample to sample, the slope and
intercept will be different from the expected if only
one toxicant is identified.
If the toxicity of one of the toxicants is substantially
different, and if the ratios of the three toxicants vary
from sample to sample, then the slope, intercept, and
r2 value will all be different from expected if all are not
identified. Much can be learned from studying the
interrelationship of slope, intercept, and the r2 value.
For example, a high r2 value and an intercept near
zero with a slope larger than 1 can be caused by
using an LC50 for the toxicant that is too large.
If the toxicant concentrations vary over a larger range,
a significant correlation will be easier to demonstrate
than for a narrower range. Great care must be taken
to understand the interactions of the toxicants, i.e.,
whether they are additive, and if so, whether they are
additive at different ratios of one to another. If they
are not additive, then the exact ratio found in each
sample must be tested in order to know the expected
toxicity.
Experience has shown that there is a strong tendency
to unconsciously assume that toxicity is always
caused by the same constituents. If this assumption
creeps into the data interpretation but is false, some
very erroneous conclusions may be reached. That is
why other steps (given below) in the confirmation
must accompany the correlation because they help to
reveal any changes in the toxicant(s).
'2-2
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10.00 -i
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CD
_
O
.C
6.00 -
2 4.00 -
o
•§
2.00-
0.00"
Theoretical
Observed
Legend:
R2 = 0.15
Slope = 1.38
Y—Intercept = 1.24
0.00
2.00
4.00
6.00
i i i i i i i | i i i i i i i i i |
8.00 10.00
Toxic Units of Suspect Toxicants
Figure 2-2. Toxicity contribution for one of two toxicants in a POTW effluent.
A difficult problem to be solved is to determine the
LC50 of the suspected toxicants in the effluent.
Rarely can one remove the toxicants from the effluent
and be sure that nothing else has changed or been
removed. Therefore, one cannot directly determine
the LC50 of the toxicant in the effluent.
Characteristics such as pH and hardness are readily
recognized as affecting ammonia and metal toxicity,
respectively. With non-polar organics, suspended
solids and total organic carbon may affect toxicity
even more. For these reasons, measuring an LC50 in
a dilution water may give quite different results than
would be obtained if the test were to be conducted on
an effluent.
When the LCSOs of effluent samples vary widely, a
particularly troublesome complication occurs
regarding the effect of these common effluent
characteristics on toxicity that takes place. If the
LC50 is high (e.g., 80% effluent) then the test
concentrations which determine the LC50 (e.g., 75%
and 100% effluent) will exhibit characteristics similar
to 100% effluent. But if the LC50 is 5%, then these
characteristics will more closely resemble those of
the dilution water.
Changes in pH may also cause problems, particularly
since most TIE tests will be done under static rather
than in flow-through conditions. Effluent pH,
especially of POTWs, usually will rise 1.0-1.5 pH
units (e.g., from 7.2-8.4) when the effluent stands in
test vessels exposed to air. This pH change would
approximately triple the sample toxicity caused by
ammonia. We have a large data base for ammonia
and we know just what characteristics to be
concerned about; for many other compounds we do
not have such a large data base. Therefore, great
care must be exercised to avoid misjudging the true
toxicity of suspected toxicants in the effluent.
Total organic carbon (TOC) and suspended solids
(SS) may have great effects on organic chemical
toxicity. Effluents are likely to be very complex and
variable and the role of TOC and SS may be quite
different than in surface water. For example, SS may
be composed largely of bacterial cells in one effluent
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6.00-,
5.00-
- 4.00 -
o>
UJ
£
o
.c
"o
a
o
'x
3.00-
2.00-
1.00-
Theoretical — — —' —
Observed
Legend: R2 = 0.73
Slope = 0.82
Y—Intercept = 0.46
0.00 f i i i i i i i i i | i i i i i i i i i | i l l l II I l l | l ll l ll II l | l l M M l M I I II i I I I I I I
0.00 1.00 2.00 i 3.00 4.00 5.00 6.00
Toxic Units of Suspect Toxicants
Figure 2-3. Toxicity contribution from two toxicants for a POTW effluent.
sample and largely of clay from storm runoff in
another. The rate of equilibrium for adsorption of
toxicants to solids as well as the equilibrium itself may
be different because of the different organic content
Whether sorption takes place only on the surface or
internally in the solids as well, may affect toxicity;.
Sometimes TOG may be largely dissolved and at
other times it may occur in the form of fat droplets, oil
films, or as a coating on SS. Any of these forms may
have a different effect on the toxicity of an organic
chemical.
These factors impact heavily on toxicity mea-
surements. We cannot just measure SS, TOG, or
dissolved organic carbon (DOC) and correct for them
as we might do for hardness or pH. These require a
different approach. When there are two toxicants, one
affected by pH and hardness and the other affected
by SS and TOG, the complexity may seem over-
whelming. ;
An approach we have not yet tried, but which seems
promising, is to collect sets of samples, each
collected during a short period (e.g., 24 hours) and do
a separate correlation for each set. The basis of this
suggestion is that effluent characteristics that affect
toxicity would likely be more similar during short time
periods than during long time periods. Our experience
suggests that POTW effluents vary sufficiently in
toxicity over short time periods to provide a useful
range. The down side of this approach is that the
analytical and toxicological error of measurement may
make obtaining a high r2 value very difficult on the
small number of samples. The statistics would have
to be carefully developed to use this approach.
Simply testing the individual slopes and intercepts
separately, probably would not work.
Correlation should be accompanied by other Phase III
approaches, described below, because sometimes
additional toxicants will occur (especially in POTW
effluents). When this occurs and one recognizes that
it has occurred, these samples can be omitted from
the correlation or their toxicity can be appropriately
corrected before use in the correlation.
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Section 3
Symptom Approach
Different chemicals may produce similar or very
different symptoms. Probably no symptom of
intoxication is unique to only one chemical. Therefore,
while similar symptoms observed between two
samples means the toxicants could be the same,
different symptoms means the toxicants are definitely
different in the two samples. By observing the
symptoms displayed by the test organisms in the
effluent and comparing them to the symptoms
displayed by test organisms exposed to the
suspected toxicants, failure to display the same
symptoms means the suspected toxicants are
probably not the true ones.
Written descriptions of symptoms are usually not
helpful. Behavior is difficult to put into words so that a
clear image of behavior is obtained. Compendiums of
symptoms are not available for aquatic organisms.
The best approach is to expose organisms to the
known (or suspected) toxicant(s) and observe how
the organisms react. By the time of the final
confirmation, toxicity tests with the suspected
toxicants will have been conducted using pure
compounds. If so, symptoms should already have
been observed. A prudent investigator will note the
symptoms seen during earlier testing because they
can be very helpful in later work as well.
The intensity of exposure can change the symptoms.
One must compare symptoms at concentrations that
require about the same period of onset. This is most
easily done by using exposure concentrations that are
some multiple of the LC50. In this way both the
unknown (effluent) and the known toxicants (pure
compound) can be set at the same toxicity level. This
does not mean that observations of the organisms
should be delayed until the normal length of the test
has elapsed. With some toxicants, the test organisms
will show distinctive symptoms soon after the
exposure begins, whereas later, symptoms are often
more generalized and less helpful. For other
toxicants, a sequence of different symptom types are
displayed by the test organism over the exposure
period and the sequence may be more definitive for a
given chemical than the individual symptoms. In few
cases will the symptoms be unique enough to
specifically identify the toxicant, but symptoms
different from those caused by the pure suspected
toxicant are convincing evidence that the suspected
toxicant is not the true one.
A second caution regarding mixtures of several
toxicants. Mixtures of toxicants can produce
symptoms in test animals different from the
symptoms of the individual toxicants composing the
mixture. When more than one toxicant is involved,
one must not only include all the toxicants, but
include them in the same ratio as the whole effluent.
Often the toxicant of the mixture at the highest
concentration relative to its LC50 will cause most of
the symptoms. Just as for single toxicants, the
mixture concentration causing the same endpoint in a
similar exposure period should be compared. Spiking
effluent with the suspected toxicants and comparing
the spiked and unspiked sample, both near their
LC50 concentrations, is a good approach.
Symptoms may be quite different among different
species of organisms; therefore the use of two or
more species provides increased definitiveness in the
observations. For both species, one must compare
symptoms at concentrations that are equitoxic. The
greater the difference in sensitivity, the more
important this becomes. The concentration in mg/L is
unimportant; the important consideration is that
equitoxic concentrations are compared. Suppose, for
example, species A and B have LC50 values for a
suspected toxicant of 1 and 80 mg/L. Then
concentrations of 2 and 160 mg/L might be used to
compare symptoms of species A and B, respectively.
If the onset of symptoms is rapid, then perhaps 1.25
and 100 mg/L (1.25XLC50) should be tried. Since
symptoms vary with the exposure intensity, using
various multiples of the LC50 (i.e., 0.5, 1, 2) can add
additional confirmation, if the same set of symptoms
are seen in both series. If more than one toxicant is
involved, and the ratio of the two species' LC50
values for toxicant A is markedly different than for
toxicant B, C, D, ..., then the definitiveness of using
symptoms is even greater.
Time-to-mortality at equitoxic concentrations can
be used as a "symptom" type of test. Some
chemicals cause mortality quickly and some cause
mortality slowly. If for two effluent samples, one kills
quickly and the other kills very slowly, the toxicants
are probably not the same.
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Section 4
Species Sensitivity Approach
In addition to a comparison of symptoms displayed by
species, the LC50 values can be compared for the
effluent of concern and the suspected toxicants,
using species of different sensitivities. If the
suspected toxicants are the true ones, the LC50
values of effluent samples with different toxicity to
one species will have the same ratio as for a second
species of different sensitivity. And further, the ratio
for each species should be the same as for known
concentrations of the pure toxicant. The same ratio of
LC50 values for two species implies the same
toxicant in both samples of effluent. Obtaining the
same effluent toxicity ratio among various effluent
samples for each species as is obtained by exposure
to comparable concentrations of known toxicants,
implies that the suspected toxicants are the actual
ones present. However, if other effluent
characteristics affect toxicity and they vary, the ratios
could also be affected.
The common notion that goldfish are resistant and
trout are sensitive is very misleading and should be
discarded. Many species are more sensitive to certain
groups of toxicants than trout. Of course, there are
generalizations that can be made. For example,
sunfish (Centrarchids), frequently are much more
resistant to metals than goldfish, minnows, and
daphnids. Daphnids tend to be more resistant to
chlorinated hydrocarbon insecticides than fish and
more sensitive to organophosphate insecticides.
These differences must always be verified for the
suspected toxicants; generalities can only be used as
an initial guide to species selection. Differences of
10-100X are easy to find in some chemical groups
and difficult to find in others. If several toxicants are
involved, interpreting the results and designing the
ancillary experiments is more difficult. If successful,
the power of the result is much greater than for a
single toxicant. The difference in sensitivity between
Ceriodaphnia and fathead minnows has, on several
occasions, revealed either a change in the toxicants
present in a series of effluent samples, or the
presence of other toxicants in addition to the ones
suspected.
Comparison of sensitivity among species has another
very important use. Some species may evidence
toxicity from an effluent constituent that the TIE test
species did not. If this happens, then the above
comparison will be confused, but at least there will be
a warning. To decipher this possibility, one will need
to revert back to Phase II and even to Phase I to
characterize the additional toxicant and identify it with
the new species. A second Phase III may be needed
for this toxicant and species. It is important not to
assume that the resident species have the same
sensitivity as the TIE test species. For freshwater
discharges to saltwater, this is critical, because
Phases I and II are most easily done on freshwater
organisms because the effluent is freshwater. But the
concern is for marine organisms and so their
protection cannot be assumed (cf., Section 8,
Phase I).
4- 1
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Section 5
Spiking Approach
In spiking experiments, the suspected toxicants are
increased in concentration in the effluent sample and
toxicity is measured to see whether toxicity is
increased in proportion to the increase in concen-
tration. While not conclusive, if increased toxicity is
proportional to an increase in concentration, consid-
erable confidence is gained about the true toxicants.
Two principles form the basis for this added
confidence. To get a proportional increase in toxicity
from the addition of the suspected toxicant when it is
in fact not the true toxicant, both the true and
suspected toxicants would have to have very similar
toxicity and would have to be strictly additive. The
probability of both of these coinciding is small.
Removing the suspected toxicants from the effluent
without removing other constituents or in some way
altering the effluent is usually not possible. The
inability to do this makes the task of establishing the
true toxicity of the suspected toxicants in the effluent
difficult. For many toxicants, effluent characteristics,
such as TOG, SS, or hardness, affect the toxicity of a
given concentration. Some characteristics, such as
hardness, can be duplicated in a dilution water, but
certainly not TOG or SS because there are many
types of TOG and SS, and these generic
measurements do not distinguish between them. For
example, effluent TOG will usually occur as both
dissolved and suspended. In POTW effluents, the
source of the TOG is likely to be largely from biologic
sources, both plant and animal. For POTWs, bacteria
are likely to be a large component. If there have been
recent storms, oil from runoff might be high.
Simulating TOG from such variable sources is next to
impossible. TOC is not solely the result of organic
chemicals. For suspended solids, shape, porosity,
surface-to-volume ratio, charge and organic
content, all, or any, will change sorption, and none of
these is measured by the standard methods for
measuring suspended solids.
In a simple system, such as reconstituted soft water,
one can expect for most chemicals that doubling the
concentration will double the toxicity, at least in the
LC50 range. If solubility is being approached or there
are effects from water characteristics such as SS,
then the toxicity may not double or conceivably could
more than double. For example, if a chemical of high
log P is largely sorbed on solids, doubling the total
concentration may more than double the toxicity
because the added chemical is in solution.
Equilibrium may not become estab-lished during the
test period and especially not before the test
organisms are added.
If several toxicants are involved, then their interaction
(additivity, independent action, synergism) must be
measured or otherwise included. Since ratios may be
as important as concentration, the best way to spike
when multiple toxicants are involved is to increase
each toxicant by the same multiple of the LC50, e.g.,
by doubling each. In this way the ratios of the
toxicities remain constant. When two or more
toxicants are not additive, getting predicted results is
very strong evidence. Interpreting spiking data may
require a very high level of competence in both
toxicology and chemistry; otherwise the data could be
very misleading. Using more than one species of
differing sensitivity is effective in adding confidence to
the results.
When matrix effects are complicated, other types of
spiking can be done to reduce the effects of the
effluent matrix characteristics. If a method exists for
removing the toxicants from the effluent, such as the
SPE procedures, the extracts or fractions can be
spiked in addition to spiking effluent, using the same
principles as described for effluents. The advantage
in this approach is that matrix characteristics such as
SS and TOC will be absent or much reduced and will
not affect spiking experiments as much. The
disadvantage is that proof that the extracts or
fractions contain the true effluent toxicants must be
generated. Some approaches for doing this are given
in the next section (Section 6). The use of this
approach is especially applicable to fractions from the
SPE or the HPLC fractions for non-polar organics. In
these, the constituents are separated from much of
the TOC, SS and hardness, so that spiked additions
are more likely to be strictly additive than is true in
the whole effluent. Suggestions and precautions
about ratios and all other previously discussed
concerns apply here as well. Spiking fractions,
however, does not provide the same confidence
about the cause of toxicity in the effluent as spiking
the effluent directly provides. The mass balance
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