United States        Office of Water       EPA 823-P-99-001
          Environmental Protection    4304          December 1999
s»EPA   Response
          to Comments
          1998 Update of Ambient
          Water Quality Criteria for


Response to Comments
1998 Update of Ambient Water Quality Criteria for Ammonia
September 1999
U.S. Environmental Protection Agency
Office of Water
Office of Science and Technology
Washington, DC 20460
Office of Research and Development
Mid-Continent Ecology Division
Duluth, MN 55804

This document contains responses to public comments that EPA solicited through 63 FR
44256 (August 18, 1998), which announced the publication of the 1998 Update of Ambient Water
Quality Criteria for Ammonia. Because that criterion was published as guidance and not
regulation, neither the solicitation of public comment nor the response to public comment are
required by law or regulation. Rather EPA is publishing these responses to improve
understanding of the technical issues involved in deriving a criterion for ammonia. For this
reason, this document includes technical comments only. Although EPA considered or acted
upon the few policy-related comments submitted, discussion of this material is not included here.
These technical responses are published in conjunction with a revision of that ammonia
criterion, undertaken in response to public comment, and contained in the 1999 Update of
Ambient Water Quality Criteria for Ammonia, which supersedes the 1998 document. It is
important to note that except where indicated otherwise, all comment citations to page-, figure-,
and table-numbers refer to those of the 1998 Update. Page-, figure-, and table-numbers changed
in the 1999 Update.
This Response to Comments document and the two above mentioned criteria Update
documents are not regulations, and cannot substitute for the Clean Water Act or EPA regulations.
Thus, they cannot impose legally binding requirements on EPA, States, Tribes, or the regulated
The original comments are available in docket number W-98-20 at the Water Docket,
Environmental Protection Agency, 401 M Street SW, Washington, DC 20460. The technical
comments addressed in this document (in order of appearance) were submitted by (A) Jim
Schmidt, Wisconsin Department of Natural Resources; (B) Tom Sinnott, New York State
Department of Environmental Conservation; (C) Hall and Associates, Washington, DC, through
several municipalities, wastewater authorities, or associations of such entities; (D) John
Zambrano, New York State Department of Environmental Conservation; (E) John Sullivan,
American Water Works Association; (F) David Fowler, Milwaukee Metropolitan Sewerage
District; (G) Alan Anthony and Alex Barron, Virginia Department of Environmental Quality, and
(H) John Hall, of Hall and Associates.
These responses were written by Charles Delos and Russ Erickson. Comments or questions on
this material may be submitted to: Charles Delos, U.S. EPA, Mail Code 4304, Washington, DC
20460 (e-mail: delos.charles®epamail.epa.gov).

Comments and Responses
Comment A-O I
My only concern is with the use of EC2O
estimates for defining chronic values. The
intent of this value is clear, namely to provide a
better definition of the safe value used to define
chronic toxicity and particularly to generate a
value that can be used consistently between
species. However, my question is whether or
not the general EPA guidance (such as the
GLWQI) needs to be revised to allow this
estimate, since there is no mention of the use of
EC2O in any of the Federal guidance from
which Wisconsin’s standards are derived.
The concern, therefore, is not with consistency
within the ammonia database, but rather with
any implications the use of EC2O might have
for the chronic toxicity database and criteria of
other substances. We in Wisconsin would both
suspect and expect that EC2O values should be
able to be generated from the existing database
on any compound, since it is just a matter of
defining a dose-response relationship for each
toxicity test result. However, it is not clear why
this approach was not incorporated on more of
an across-the-board basis, such as in the
GL WQI. Our concern is more with consistency
between substances than just for ammonia
Another reason for Wisconsin’s concerns are
more internal, as Wisconsin and other Great
Lakes states are using 1C25 to define chronic
toxicity in whole effluent toxicity tests. This
matter may not be totally relevant when it
comes to ammonia, especially since there are
still on-going negotiations between the Great
Lakes states and EPA over the whole effluent
toxicity standards. However, the fact that
states are using a 25th percentile to define
chronic toxicity in the whole effluent toxicity
test regime as opposed to the 20th percentile
for ammonia is a consistency concern as well.
Wisconsin is not really in a position at this time
to suggest that the EPA chronic criteria for
ammonia be based on an alternative level such
as EC25 to be consistent with 1C25, but our
concern is mainly over any implication that this
approach be used for all substances (besides
ammonia) without going through a more
general review and approval scenario such as
GLWQI. This is because we see no reason why
the EC2O approach cannot be usedfor other
substances, and we just want to make sure that
the EC2O approach gets its own review rather
than face any implication that this is general
guidance that is “hidden” within the ammonia
criteria document.
The regression approach for obtaining an ECx
(effect concentration for “x” percent of
individuals) from concentration-response data
from chronic tests is consistent with the 1985
National Guidelines and the GLI Guidelines.
Neither guidance specifies the particular ECx or
the particular statistical technique. For the 1998
and 1999 Ammonia Update, EPA used the
EC2O, and a maximum likelihood nonlinear
regression approach, using a weighting scheme
to emphasize the data closer to the EC2O.
However, the document notes that there is an
element of risk management (choice of level of
protection) in such use. Consequently, states
and tribes are free to use other response levels
in setting criteria. States and tribes are also free
to use other regression techniques, or to
continue obtaining chronic values via the
traditional hypothesis testing approach.
A table of regression parameter values for all
toxicity tests used in the calculation of the CCC
has been added to the 1999 Update. This
allows readers to calculate any ECx (e.g., the
EC25) for each of the tests and to derive the
corresponding CCC, also as discussed in
response to Comment B-02. EPA does not
believe that there are substantial differences

between levels of protection provided by the
EC2O, the EC25, and traditional hypothesis
Comment B-O I
In the development of the proposed chronic
criterion for ammonia, the 1998 Update
departs from the 1985 Guidelines by selecting
the EC 20 as the appropriate threshold for
chronic toxic effects instead of using the
geometric mean of the lowest observed adverse
effects concentration and the highest no
observed adverse effects concentration. It is
unclear whether or not this change was made
for solely statistical purposes or f it is the
intention of the EPA to establish a precedent
that a 20% impact to survival, growth, or
reproduction is acceptable. No defense,
just!/Ication, or explanation for the selection of
the EC2O as the appropriate chronic effects
threshold, beyond its statistical utility, was
provided. The New York State Division of Fish,
Wildlife and Marine Resources (DFWMR)
believes that the EC2O is an inappropriately
high threshold that will not be protective of the
aquatic community, particularly during periods
of low flow when organisms are faced with
multiple stressors. If regression analysis is
used for determining chronic values, the
appropriate threshold is the Ed, or X-
intercept. The safety of aquatic communities
should not be compromised because of the high
variability of toxicity tests, particularly since
contaminant concentrations at or near CCC
levels are likely to be only one of a multitude of
stressors that aquatic organisms are faced with
during low flow conditions.
Before addressing the comment, EPA notes that
in oral conversation with EPA staff, the
commenter withdrew this comment. However,
EPA believes the issue is of general interest and
merits discussion.
As may be observed from concentration-
response graphs in Appendix 6 of the document,
the EC 1 would occur in the flat portion of the
concentration-response curve and therefore
would involve great statistical uncertainty. That
is, given the inherent random variability in
chronic toxicity results, there could be a wide
range of concentrations that might in fact cause
a one percent effect. The percentage of control
organisms affected is in fact often rather
variable and thus uncertain.
Perhaps more important, however, is that EC2O
is generally reasonably close to the traditionally
used chronic value, the geometric mean of the
NOEL (which statistically is not significantly
different from zero effect) and the LOEL
(which statistically is significantly different from
zero effect). EPA believes that experience has
shown that its other criteria, falling between the
NOEL and LOEL, are fully protective. Thus,
EPA believes that criteria derived from EC2O
would likewise be fully protective. EPA did not
intend any significant change in the level of
protection inherent in the SMCVs or GMCVs.
EPA only intended to provide a better technique
for obtaining chronic values: one that better
considered all the data within a study.
In conclusion, EPA does not believe that an
effect level as low as one percent among
sensitive species is generally necessary for the
protection of aquatic life uses. This is particu-
larly true when applied to chronic tests of early
life stages that naturally undergo a high rate of
mortality, dependent on population density.
Comment B-02
The 1998 Update document does state on page
71 that the selection of the EC 20 as a chronic
threshold is a risk management decision. The
document does not, however, provide the
necessary information for a risk manager to
select a more appropriate effects
concentration. The data points for each study
used to calculate the regression equation, or
the final regression equations for each study
were not provided It would have been very
simple for the document to include a table that
listed the data points for each study normalized

to ammonia nitrogen at pH 8 and adjusted for
temperature. It would have been simpler still
to include i/ic regression equation for each
study next to the corresponding graph in
Appendix 6 Then risk managers would have
the option of recalculating the ammonia
criterion for a different effects concentration.
EPA agrees. This information has been added
to in the 1999 Update. The reader may now use
the values of the regression parameters to
calculate ECs for other percentage responses. It
should be noted, however, that the weighting of
data in the regression technique was done with
the intent of estimating the EC2O.
Consequently, estimates for ECs comparatively
distant from the EC2O (e.g., EC5 or EC5O)
might not be the very best the test data are
capable of supporting.
Comment B-03
The text of the 1998 Update document does not
support the Office of Science and Technology
Policy Recommendations that a cold-season
ammonia criterion could be established that is
as much as three-fold higher than the criterion
applicable to the remainder oft/ic oft/ic year.
Ostensibly, the same reasoning could be
applied to any contaminant; that is, (1 early life
stages are the most sensitive, then a less
stringent standard can be applied during the
periods of time when early l(fe stages are not
present. Furthermore, a state can propose a
less stringent criterion any time for any
substance, providing that it is scient(flcally
just flabIe. The data in the 1998 Update
document explicitly do not provide adequate
scient j/Ic j usufication for an arbitrarily-based
three-fold increase in the chronic ammonia
criterion during cold weather. The
recommendation should either be withdrawn,
or the 1998 Update document should be
revised to provide explicit guidance as to how a
cold-weather ammonia standard should be
appropriately derived.
EPA does not agree that the information
presented in the 1998 Update does not support
the cold-season relaxation of the chronic
criterion where fish early life stages are absent.
Nevertheless, EPA agrees that the discussion
could be improved, and has substantially
expanded the discussion and increased the level
of analytical rigor for the 1999 Update.
Data indicate that survival ofjuvenile and adult
fish is a less sensitive endpoint than growth and
survival of early life-stage fish. In addition, the
data indicate that the sensitivity of invertebrates
decreases with decreasing temperature.
Consequently, it is appropriate for the chronic
criterion to increase when fish early life stages
are absent and water temperatures are low.
The new discussion is contained in the 1999
Update sections on temperature dependency and
seasonality of endpoints.
Comment B-04
The cold-season CCC assumed that the
toxicity of total ammonia to fish is
independent of temperature for each
endpoint. The chronic survival tests conducted
over a range of temperatures for juvenile
fathead minnows show that they are more
sensitive at colder temperatures (that is, 9.6 mg
NIL at 6 °C versus 19.3 mg NIL at 25 °C and
15.9 mg NIL at 30 °C).
EPA does not agree. Fathead minnow, with an
EC2O of 9.6 mg N/L at 6 .°C, is easily protected
by the 1998 CCC with the 3X winter provision,
and the ELS-absent 1999 CCC. Although EPA
would be reluctant, based on the non-significant
fathead minnow trend, to conclude that juveniles
and adults of all fish species become more
sensitive at low temperature, the seasonal
adjustments would still be justified, even if all
fish species exhibited the fathead minnow
temperature trend. For 1999, EPA took the
fathead minnow juvenile and adult GMCV to be
9.3 mg NIL. This is so far above the

temperature-adjusted Hyalella GMCV that the
fathead minnow GMCV could be cut in half
without affecting the CCC calculation.
Comment B-05
If the lower limit of each range were taken as a
point value, then three times the lower GMCV
limit of 3 mg NIL would not exceed the 9 mg
NIL lower limit of the LC 20 range. However, f
the upper limit of each range was taken as a
point value, than three times the upper GMCV
limit of 8 mg N/L (24 mg NIL) would exceed the
15 mg NIL upper limit of the LC2O range by a
considerable degree. If the geometric mean of
the ranges are used, three times the GM of the
GMCV range (4.9 mg NIL) exceeds the GM of
the LC 20 range (11.6 mg NIL). The very
highest a cold-water ammonia criterion should
be set at is only 2.4 times the year-round
chronic criterion.
If the summer CCC had been set at 3 mg NIL
rather than 1.27 mg NIL, then there might be
good reason to limit the winter adjustment to
2.4X rather than 3X. However, application of
the 2.4X ratio to the actual 1998 CCC (1.27 mg
NIL) would not seem appropriate because the
two most sensitive genera are not fish, but
invertebrates. For invertebrates, the reason the
winter criterion can be higher is that the data for
invertebrates show them to be less sensitive at
low temperature. Consideration of the available
fish LC2Os do not indicate that a 3X adjustment
would yield a toxicity problem.
Nevertheless, for 1999 EPA has redone the
entire seasonal assessment to provide a genus-
by-genus evaluation of chronic effect levels
under winter conditions.
Comment B-06
Regarding invertebrates, two most sensitive
species in the ammonia chronic database are
Hyalella azteca, and Musculium transversum.
All of the chronic tests were conducted at
temperatures ranging between 23.5 - 25°C. No
data are presented to suggest that the cold-
water chronic toxicity would be any different
than the warm-water toxicity. On page 76, the
document defends the concept that survival is
less sensitive under cold weather conditions by
using acute toxicity data. They cite one study
that found the 96 hour acute LC5O for
Musculium transversum was 1.9 times higher
at 15°C than at 21 °C, and 2.7 times higher at
5°C than at 21°C. The same study found that
for an amphipod, Crangonyx pseudo gracilis,
the 96 hour LC5Os were 6-fold higher at 12-
13°C and 8-fold higher at 4°C than at 25°C.
If the assumption is made that chronic toxicity
is similarly reduced by colder temperatures,
then raising the CCC can bejust /ied.
However, acute toxicity is not the same as
chronic toxicity. Ammonia is a metabolic
waste product. No organism can live on its
own waste, waste products must be excreted.
Long term, continuous exposure to ammonia
could have physiological impacts through
different pathways than those that cause short
term, acute toxicity. Even jf the toxicity
pathways are the same, the document
acknowledges on page 76 that: “The effect of
temperature on the rate of biochemical
processes might, however, affect the result of
acute tests more than the results of chronic
tests.” One could hypothesize a corollary to
the finding of the amphipod toxicity data
described above, that it would take the same
concentration of ammonia 6-8 times longer to
result in an LC5O at 5°C than at 25°C. Six to
eight times 4 days (96 hours) is only 24- 32
days, sign /Icant!y less than a typical winter.
EPA agrees that decreasing temperature would
probably reduce chronic toxicity less than acute
toxicity. Thus, in the 1999 Update the
relationship between chronic toxicity and
temperature has been taken to be less steep than
the relationship between acute toxicity and
temperature. EPA believes that the 1999
Update, in contrast to the 1998 Update, has
evaluated the available data as rigorously as is
feasible, and has accounted for the effect of

temperature on kinetics. EPA does not believe,
however, that it can endorse the comment’s
speculation that the same concentration that
causes toxicity in four days at 25 °C would
cause toxicity in 24-32 days at 5 °C.
Comment B-07
The proposed chronic criterion for ammonia is
under protective because of the use of the “less
than” values for Hyalella azteca and
Muscilium transversum in the chronic
database. Because these species are the two
lowest species in the chronic database, they
have considerable influence over the slope of
the regression that determines the FCV. One
way in which the uncertainty of the SMCVfor
Muscilium iransversum could have been
accommodated would have been to include the
Zischke & Arthur (1987) field data, which
showed a CVof 1. Inclusion of this data would
result in a GMCVoJ 1.90 instead of 2.62, and
the resulting FCVwould in turn be <1.15
instead of <1.27. While this approach is not
mathematically or statistically precise, it does
reduce the uncertainty of the Muscilium
transversum GMCV. Some type of uncertainty
analysis should also be applied to Flyalella
The GMCV for Hyalella is a less-than value
because the lowest treatment concentration
yielded more than 20 percent effect. This
species is being retested. If the results are
substantially different than those presented in
the document, then EPA will recalculate the
The GMCV for fingernail clam is a less-than
value because the relevant tests were of juvenile
long term survival. It was not an early life stage
test capable of reflecting survival, growth, and
reproductive endpoints. No test protocol of that
type exists for fingernail clam. EPA believes
that including the fingernail clam juvenile long-
term survival test in the data set improves the
reliability of the criterion and is to be preferred
over simply rejecting such tests as unsuitable for
deriving chronic criteria.
Lowering the fingernail clam GMCV by
including field results does not substantially
change the CCC. EPA has calculated the
GMCVs from lab data because they are
generally more precise than field data. EPA has
no basis for using the field data for fingernail
clam and not using the field data for other
species. In contrast to fingernail clam, the field
data for fathead minnow indicated higher effect
concentrations than the lab data.
EPA is also reluctant to modify its handling of
fingernail clam data on the basis of factors
related to the Hyalella test. Such attempts to
balance unrelated potential errors tend to appear
subjective and are difficult to explain.
Comment B-08
The document erroneously states that the
fingernail-clam chronic value is already based
on long-term survival ofjuveni(es so it is a
relevant end-point for cold-weather conditions.
The chronic value was based on two 42 day
studies during warm water conditions, and one
“similar” study. Forty-two days cannot be
construed to be “long-term survival “,
considering that the typical lifespan for the
species is 12-18 months. There is no basis for
assuming those tests in any way reflect survival
during cold weather conditions. Pennak (1989)
reports that when temperatures drop below
10°C, freshwater mussels like Musculium
transversum burrow into the substrate with
only thefr siphons extended. Even though the
animals are generally dormant, their siphons
do open infrequently, so they would be exposed
to ambient concentrations of ammonia during
the winter.
See response to Comment B-07 for discussion
of the use of the fingernail clam tests. Also note
that since the chronic criterion is applied as a
30-day average, 42-day average concentrations
would necessarily have to be somewhat less

than the 30-day average in real-world time
EPA does not agree with the comment’s
implication that the Update document assumed
that the animals were dormant in winter. In
fact, EPA assumed the that they were not
dormant; EPA intended to provide them with
the same level of protection in winter as in
summer, while recognizing the observed effect
of temperature on sensitivity.
Comment B-09
Similarly, according to Pennak (1989),
Hyalella azteca is a cold stenotherm. Although
amphipods are typically shallow water
organisms, some species can be found quite
deep in the water and most are well-adapted to
cold water conditions. There is no reason to
believe that they are entirely dormant during
cold weather. They can be present in very high
abundances, and f active in during winter
months, they would be exposed to potentially
toxic concentratins of ammonia.
As with Comment B-08, EPA does not agree
that the temperature adjustment assumes that
the organisms are “entirely dormant” at cold
temperature, any more than the pH adjustment
assumes that the animals are dormant at low
pH. The temperature relationship was derived
from the available toxicity data. Considerations
of what the organisms might be doing in winter
versus summer did not affect the Hyalella
GMCV, in part because the available toxicity
data do not allow distinguishing between
survival and reproduction effect concentrations.
Comment C-01
The Update contains no overall evaluation of
the level ofconservatism.. . .Such an analysis
would have clearly demonstrated that the
suggested criteria are much more restrictive
than the laboratory data underlying the criteria
In fact, the 1998 Update did provide such an
evaluation, at least with respect to the criterion
concentration values, in the form of Figures 9-
12 (or Figures 11-14 in the 1999 Update).
These figures compare all the acceptable acute
and chronic toxicity test data to the criteria
concentrations, and therefore would embody all
aspects of the criteria process that affect the
concentration calculations. There are issues,
such as averaging periods, which are not
embodied in this piot, which will be treated
separately later, but these figures do
demonstrate that the procedures used to derive
the criteria did not result in criteria
concentrations “much more restrictive than the
laboratory data underlying the criteria
Consider the 1998 Update’s Figure 10 regarding
acute toxicity. The FAV is supposed to
correspond to the fifth percentile most sensitive
taxa, so only a small percentage of the test
results should lie below the FAV. In the pH
7.5-8.5 range, several percent of the LC5Os, for
a variety of organisms, lie below the FAV. The
criterion is two-fold lower than the FAV to
provide a level of protection better than 50%
mortality, but several LC5Os are even below this
concentration. Thus, to characterize the
criterion as “much more restrictive” than the
laboratory data is unjustified.
At more extreme pH (<7.5 and >8.5), only a
few LC5Os are near or below the FAV and all
are well above the criterion concentration. This
might be a reason for the contention that the
criterion is too restrictive, and is certainly a
basis for criticism of the pH relationship later in
the Comments. However, this behavior is
exactly what is expected because there are few
data in these pH ranges. The FAV is intended
to be at the fifth percentile, so no LC5Os should
lie below it in these pH ranges, in which there
are not very many tests. The fact that there are
some LC5Os at or very near the FAV in these
pH ranges actually indicates the FAV is
appropriate. To demonstrate this point, a more

detailed analysis, provided in Appendix I of this
response document, shows what scatter pattern
is expected if the p1-I relationship used here is
absolutely correct. This analysis shows a
pattern very similar to that in Figure 10.
For chronic toxicity, the 1998 Update’s Figure
12 provides a similar demonstration that the
criterion is not “much more restrictive than the
laboratory data”. There are several chronic
values for a variety of species near or below the
criterion line. Given the limited amount of data
available, the location of the criterion relative to
the data is reasonable and argues against the
alleged severe compounding of conservative
assumptions, at least with respect to the
procedures used to derive the criterion
concentrations. The fact that the data do not lie
near the criterion concentration at high and low
pH is again expected due to the limited amount
of data and the relative sensitivity of the species
As mentioned above, these figures do not
encompass all factors which might relate to how
conservative the criteria are, but do refute the
contention that the “criteria are much more
restrictive than the laboratory data underlying
the criteria calculation”, at least with respect to
the criterion concentrations. Conservatism due
to other issues such as averaging periods is
addressed later in response to specific
Comment C-02
The Update document also fails to correct the
long-standing misapplication of chronic
criteria under seven-day once in ten year
flows “.
EPA has performed rigorous analysis of first-
order serially correlated, log normally
distributed time series of concentrations, with
the intent of determining what percentage of
grab samples or 24-hour composite samples
would need to attain the CCC in order for the
30-day average not to exceed the CCC more
than once in three years.
EPA has previously noted, for example in
material supporting the Great Lakes Initiative,
that it believes that the once-in-three-year goal is
consistently sufficient to insure protection, but
not always necessary. Although the scope of
the ammonia project provided for a careful
assessment of the appropriate averaging period,
it did not provide for a re-examination of the
once-in-three-year goal. However, if it were
assumed that the once-in-three-year goal was
appropriate, the purpose of the time series
analysis was to determine the grab or composite
sample exceedance frequency (or the
percentage of time exceeding) that would
correspond to the 30-day average, once-in-
three-year goal. EPA recognizes the
incongruity of performing a sophisticated
analysis on a rudimentary endpoint, but believes
the results are still of interest, and relevant to
addressing the comment.
The time-series analysis indicated that to attain
the 30-day once-in-three-year goal, the
percentage of grab or 24-hour composite
samples that need to be below the CCC depends
on (a) the degree of serial correlation, and (b)
the amount of variability in the time series.
Serial correlation is measured by the correlation
coefficient between the logs of daily mean
concentrations on adjacent days. High serial
correlation means that the variations in
concentration occur smoothly. Low serial
correlation means that the concentrations
variations are abrupt and choppy. Low serial
correlation makes it more difficult to put
together a sufficient number of daily
exceedances to cause the 30-day averaging
period to exceed the criterion.
Variability is measured by the standard
deviation of the logs of concentrations. Because
the analysis is simply relating the frequency of
either grab or daily exceedances to the
frequency of 30-day exceedances (not to the

mean of the time series), it might not be
expected that the standard deviation would be
an important parameter. However, it should be
noted that the time series is taken to be log
normal, but the 30-day average is an arithmetic
mean, not a logarithmic (or geometric) mean.
The use of arithmetic means on log normally
distributed values yields a dependency on the
log variance. On a log scale the highs and lows
are equidistant from median in such a
distribution, but on an arithmetic scale the highs
are relatively further from the median than are
the lows. Consequently, as the log standard
deviation increases, it takes fewer peaks,
because of their magnitude relative to the
troughs on an arithmetic scale, to exceed the 30-
day arithmetic mean.
The procedure for counting 30-day average
exceedances is that used in the 1986 Technical
Guidance Manual for Performing Waste Load
Allocations, Book VI Chapter 1, Stream Design
Flow...”. There are many different ways of
counting multiple successive exceedances. The
results are affected by the counting procedure
used. EPA believes that this particular counting
procedure is not unreasonable, even though it
has no better relationship to the original
selection of the three-year recurrence goal than
other equally valid counting procedures.
The results of the analysis indicated that for log
normally distributed concentrations with log
serial correlation coefficient between 24-hour
composites of 0.86-0.94 or lower, and log
standard deviation of 0.5-0.8 or lower,
attainment of the CCC in 95% of grab samples
or 24-hour composites can be expected to allow
attainment of the 30-day once-in-three-year
goal. Available data suggest that the above
degree of serial correlation and variability are
reasonable for surface waters. What this means
is that maintaining concentrations below the
CCC 95% of the time will yield attainment of
the 30-day once-in-three-year goal.
Comment C-03
The Update document, using acute data,
concludes that fish (unlike invertebrates) are
equally sensitive to total ammonia at low and
high temperatures and therefore, constant total
ammonia criteria are necessary. Although
invertebrates could tolerate much higher
ammonia levels at lower temperatures, no
temperature adjustment was made in
formulating the acute or chronic criteria
because invertebrates were determined to be
“insensitive” based on acute data. However,
unlike the acute database, the two most
chronically sensitive species were
invertebrates, not fishes. Because the chronic
criteria were based directly on invertebrate
sensitivity to ammonia at high temperatures
(25°), the Update should have allowed much
less restrictive criteria at lower temperatures.
This is a clear error in the chronic criteria
derivation and has a major impact on the
appropriate chronic criteria at various
temperatures. The chronic criteria should be
temperature dependent.
EPA agrees that the 1998 Update was
problematic in not having a temperature
dependency. The analysis was revised for 1999,
such that the CCC increases as temperature
causes the sensitive invertebrate GMCVs to
increase, irrespective of whether the fish ELS
are present or absent.
Comment C-04
EPA improperly asserts that, depending upon
fish spawning, either a 1.27 mg/i or 3.81 mg/i
(total ammonia as N) chronic criterion is
necessary throughout the winter months. This
recommendation is not supported by any
information on the sensitive species that drove
the document (e.g., Hyalella and Muscillum -
fingernail clam). No data presented in the
Update indicate that chronic toxicity criteria
need to be more restrictive than 9.0 mg/i (as N)
in the winter when sensitive organisms do not
spawn and sign j/Icant growth is not occurring.
All available winter data on the four most

sensitive organisms (including bluegill)
indicate that a value of 9.0 mg/I indexed to pH
of 8 should be acceptable in the winter
See response to C-03. EPA does not agree that
the CCC could be 9 mg NIL. This would
substantially exceed the likely effect
concentrations for Hyalella. Refer also to
responses to C-34 through C-42.
Comment C-05
In generating the pH relationship, the Update
stated that it was “speculative to assign
different relationships for different taxa” and
there, EPA “used [ the] average generic shape
for the pH dependence” (Update @ 24 - 25).
Contrary to these statements, the chronic pH
relationship was ‘flattened out” (i.e., made
more restrictive than the acute pH
relationship) based on very limited data. This
generated more stringent chronic ammonia
criteria for pH values ranging from 7.0 - 7.7
even though a review of the very limited
chronic data does not indicate that more
restrictive criteria are necessary. Using test
results below pH 7.0 (a relatively rare stream
condition) to skew the criteria lower in the 7.0 -
7.7 range (a typical environmental condition)
is inappropriate. This lowered the criteria by
30%@pH 7.5 and 85%@pH 7.0.
EPA does not agree. First, although there are
limited chronic pH data, statistical tests on the
available data demonstrated significant
differences between chronic and acute
relationships, which is also very evident from
visual inspection of the data (see Appendix II of
this response document). Most importantly, the
statistical comparisons included acute and
chronic tests from the same study , for which the
ratios between acute and chronic effect
concentrations differed by several-fold across
the pH range. Such large differences should not
be ignored. Second, there is no inconsistency
here between the data standards applied to
generating chronic relationships and those used
to elect a generic acute pH relationship. The
Update recognized that acute pH relationships
differ among taxa, and even considered the
potential significance of the pH relationship for
Hyalella on acute criteria at low pH and ion
concentration. What “speculative” referred to
was not whether there was a basis for
concluding that certain taxa had different
relationships, but rather which specific
relationships, other than the pooled generic one,
should be assigned to various taxa, especially
those that were not tested for pH effects.
Relationships between pH and toxicity were not
available for the three most sensitive genera,
and the fourth most sensitive genus
(Oncorhynchus) has a relationship very close to
the generic one. Establishing different pH
relationships for different taxa was not justified
due to the lack of certain data and the minimal
effect such an effort would have on the criterion.
This issue is thus much different than that
regarding differences between acute and chronic
toxicity, to which the comment drew a parallel.
This issue of pH relationships is discussed in
more detail in the responses to Comment C-24
through -33.
Comment C-06
The pH relationship was developed based upon
fish, under the claim that they are the most
acutely sensitive species, but is then applied to
the invertebrate data which represent the most
chronically sensitive species. The invertebrate
data do not indicate any consistent pattern of
pH dependence (see, Hyalella acute data)
calling into question the pH dependence
assumption for chronic criteria.
The first sentence of the comment does not
correctly characterize the 1998 Update. First,
the pH relationships were not “developed based
upon fish”, but on a mix of fish and
invertebrates. Second, a “claim that they (fish)
are the most acutely sensitive species” had
nothing to do with the development or
application of the pH relationships.

With regard to the invertebrate pH dependence,
it is true that there is uncertainty, just as there is
uncertainty in any relationships for criteria,
including those proposed in the commenter’s
Appendix A. The Update attempted to
minimize uncertainty by using the average
trends in data most relevant to each question.
Nothing in this comment indicates that this was
not the case and most definitely does not make
the case for “conservative” assumptions. The
legitimacy and uncertainty of the pH
relationships is discussed further in later
sections of this response document.
Comment C-07
The chronic database was skewed with an
abundance of more sensitive species, causing
the calculation of a lower chronic criteria than
isjust /Ied by the Guidelines for Development
of National Water Quality Criteria for the
Protection ofAquatic Organisms and Their
Uses (USEPA 1985) (hereinafter “National
Guidelines ‘). The Update acknowledged that
the acute criteria database was more balanced
with a better representation of sensitive and
less sensitive species (Update @ 71). While
EPA recognized this fact, no action was taken
to properly balance the chronic criteria
calculation. This assumption lowered the
criteria by approximately 15 percent (15%).
The above chronic database issue can be viewed
from two standpoints: (a) relative to the
Guidelines minimum database, and (b) relative
to the acute database. The composition of the
Update’s chronic database is in accordance with
the National Guidelines, exhibiting the diversity
specified for the minimum database, with the
exception of one invertebrate, an insect. This
shortfall was handled by assuming that, if tested
in a full chronic test (in place of the available
subchronic test), such insect would have been
tolerant. This particular assumption cannot be
viewed as conservative. In addition, it may be
noted that the chronic database included two
genera from the tolerant family Daphnidae, and
thus could have legitimately had even one fewer
tolerant species.
On the other hand, when compared to the acute
database, the chronic database is dominated by
taxa with low or mid-range acute LC5O, and has
little representation from taxa having high
LC5Os. The Update document notes this fact in
the context of discussing a variety of factors or
uncertainties, some of which would raise the
criterion, and some of which would lower the
criterion. EPA does not feel that it has a basis
for saying that the taxonomic representation in
the acute database better corresponds to nature
than does the taxonomic representation in the
chronic database. Consequently, EPA does not
feel that it has a good basis for undertaking what
might well be criticized as a contrived
adjustment of N, the number of tested species.
In particular, EPA does not agree with the
commenter’s implication that such an
adjustment of N is called for by any provision in
the Guidelines, although such an adjustment is
not without precedent.
Because N was already adjusted upward by one
(for the tolerant insect), it is only three highly
tolerant species shy of matching the acute data
set. Increasing N by three would yield less than
a 10% increase in the CCC.
Comment C-08
Less restrictive acute and chronic criteria are
justjfled under cool weather conditions based
upon analysis of the studies that spec flcally
evaluated temperature dependence for the
more sensitive species. (See, Appendix A.) By
“lumping” all of the data together in Figure 4,
including those data and studies that were
insufficient to assess whether a total
ammonia/temperature dependence existed,
EPA caused the criteria to be more stringent by
afactor of 1.5 - 2.0 during the winter, even
where sensitive fish species may spawn.
EPA does not agree that the pooling method
used in the Update caused the criterion to be

more stringent at low temperature. This is
discussed more in later responses (C-34 through
C-42). Nevertheless, EPA agrees that the 1998
CCC was somewhat too low under certain
conditions, particularly when fish ELS were
present in cooler waters. This has been
addressed in the 1999 Update. It might be
noted, however, that under some other
conditions, 1999 CCC is more stringent than the
1998 CCC.
Comment C-09
The short term chronic criteria were arbitrarily
reduced from an acceptable seven-day average
value 2.5 times the thirty-day average to a
four-day average 2.0 times the thirty-day
average. There is no technical basis for this
reduction in variability factor and averaging
Some additional safety is provided by limiting
the 4-day average to a factor of 2.0 times, rather
than 2.5 times the 30-day average. However,
EPA agrees that a 4-day average 2.5 times the
30-day average is supported by the data. The
1999 Update has incorporated this change into
its recommendation. See also the response to
Comment C-54.
Comment C- 10
The chronic studies were generally long-term
studies (significantly greater than thirty days).
The criteria are typically applied to
environmental conditions that occur for
periods of seven to thirty days once every Jive
to ten years even though the document
indicates that a thirty-day exceedance once in
three years is acceptable. EPA has previously
acknowledged that applying criteria in this
manner is “very conservative.” Despite this
acknowledgment, the Update makes no attempt
to advise state authorities on appropriate ways
to convert the criteria to effluent limits without
imposing excess conservatism. (For example,
a 30/Q/3 flow should be used for permit
derivation with the chronic criteria. Use of this
flow basis ensures that even minor exceedance
of the criteria will not occur more frequently
than once in three years when permit
compliance is achieved.)
See responses to comment C-02 and C-59.
Comment C-Il
When treatment plant design conservatism is
considered (normally plants are designed to
operate at 50% - 30% of the permit limit and
typically better under low flow conditions), it is
apparent that the standards to permits process
will produce afurther safety factor of two to
EPA considers this to be outside the scope of
the criterion. EPA does not believe that it is
appropriate adjust the criterion to counteract
potential conservative biases of engineers
designing sewage treatment facilities. EPA
cannot assume that all permit limits result in the
addition of new or upgraded treatment
processes, in which a design engineer would
have an opportunity to exercise a conservative
Comment C-12
The Update asserts that acute criteria, based
upon no-mortality 96-hour tests, should be
applied as one-hour averages. There is no
basis whatsoever in the Update to support this
position, and this issue has major permitting
consequences. The restrictive acute averaging
period recommendation is cited by states to
reduce allowable mixing zones even though no
realistic acute threat is present. At most a 24-
hour averaging period should be applied to the
acute criteria with a caution that pulse or batch
discharges (a non-municipal discharge
scenario) need to be evaluated separately
considering the toxicological information in the
This issue is raised again in Comment C-60 and
will be discussed in detail there. However, it

should be noted here that the 1-hour averaging
period is specified in the National Guidelines,
which allows alternatives where appropriate.
As explained later, available data do not
demonstrate a basis for significant relief from
this default averaging period, and certainly do
not justify a 24-hour averaging period. This
comment also seems to imply that short
averaging periods create inappropriate
conservatism due to how they are implemented.
EPA recognizes the need for updating its
mixing zone guidance, which is contained in the
Technical Support Document for Water
Quality-based Toxics Control(1991).
The averaging period is an expression of how a
concentration time series should be restricted to
limit the possibility of transient exposures that
can cause greater effects than intended by the
criteria concentrations. Setting a longer
averaging period to compensate for potential
conservatism in how the criteria are applied is
not appropriate.
Comment C-I 3
The recommendation to apply the
EL S/invertebrate-based warm weather chronic
criteria during the winter months violates the
National Guidelines requirement that the
criteria must be well supported and not have a
sign jficant likelihood of over- or under-
protection. The Update acknowledges that
essentially none of the critical chronic criteria
development assumptions apply during the
winter months (e.g., the presence of sensitive
life stages of fish, the temperature adjustment
for invertebrates, etc.). The mixed database of
sensitive invertebrates and fishes clear4’
indicated that non-ELS fish l festages are much
less sensitive than ELS and that the
invertebrate temperature dependency
sign flcantly affects the chronic criteria
calculation. Therefore, EPA must state that the
ELS chronic criteria do not apply during low
temperature periods, and the Agency needs to
recalculate a winter criterion using valid and
applicable assumptions.
The National Guidelines do state general goals
of avoiding over- and under-protection, but also
give specific tests and procedures for setting
chronic criteria, including data needs for
adjusting criteria for various water quality
factors, which are followed by the Update. The
Guidelines do not recommend adjusting for
specific factors if there is not sufficient data to
do so.
EPA believes that the cold-season policy
provision in the 1998 Update addressed the
concern of the comment. Nevertheless, EPA
recognizes the problem that a lack of a
temperature-dependency caused, and has
provided for this in the 1999 Update. EPA
believes that the 1999 Update also fully
addresses the concern of the comment.
Comment C-I 4
The use of Hyalella data from Borgmann
(1994) (which were inserted by EPA into the
criteria calculations after the Peer Review) is
unauthorized because of high mortality
occurring in the controls. Only 66% of the
controls survived, and considerable variability
was exhibited in the control reproduction (30 to
65 young per flask) . The actual individual test
results only reported 60% survival in two of the
four tests! This indicates that (1) the stock was
diseased, (2) some factor other than ammonia
greatly influenced survival, or (3) there were
problems in running the test. EPA would never
accept such results for establishment of site-
spec flc criteria if submitted by a permittee. In
any event, EPA should not use this data to
establish the criteria, as it clearly fails
acceptability guidelines (no greater than 20%
mortality); thus, it should not be used to set a
national criteria.
EPA does not agree for the following reasons:
(a) Reproductive tests arc generally quite
variable, and variability per se is no measure of
acceptability. The relevant issue is whether,

with this variability, appropriate statistical tests
demonstrate a significant effect of the chemical
relative to the controls. This was clearly the
case here. This is certainly not an inconsistency
with the Guidelines.
(b) This comment does not give references or
specifics about its cited “acceptability
guideline”. In fact, no such guideline exists for
this test. There is a guideline of 20% mortality
for certain tests. This includes a 10 y test for
Hyalella, but the test is question here was for 10
weeks . If 20% mortality is acceptable in 10
days, is it unreasonable to have 13% more
mortality in another 60 days? These are
relatively short-lived organisms and 10 weeks is
a significant fraction of their life span. In
culture units that are reproducing and growing
vigorously, average adult mortality often
reaches and exceeds 20% over a ten week
period, and the tests in question included earlier
life stages which would typically have even
greater mortality.
(c) Several aspects of the tests argue against
undue influence of disease or other factors.
There was a clear, consistent, and steep dose-
response with ammonia concentration. The data
were combined from duplicate tests which were
consistent with each other. Another test starting
with adults (which did have only 20% control
mortality) showed 68% reduction in
reproduction over six weeks at the lowest
ammonia concentration tested. This reduction is
similar to the 78% reduction in reproduction
over ten weeks observed at the same
concentration in the tests starting with juvenile
organisms. (The juvenile test was used because
it (a) included lower concentrations which better
defined the effect concentration and (b) included
more life stages).
For the above reasons, using this study is not
only inconsistent with the National
Guidelines, but actually quite in accordance
with them and the use of the best available
Comment C- 15
It should be noted that the Peer Review process
concurred with EPA ‘s earlier recommendation
that [ the Borgmann (1994) Hyalella data]
should not be used because of the high control
mortality. It is inappropriate to use this data to
derive more restrictive criteria in light of this
prior position and the lack of subsequent Peer
Review of this data.
EPA does not agree. It is true that in the draft
Update that went to peer review, EPA did not
include the Hyalella data in the actual criteria
calculations. But this was not because of the
high control mortality. The primary reason was
that the EC2O was below the lowest treatment
concentration, which resulted in a large
uncertainty in the EC2O and violated the
prerequisitess adopted for EC2O estimation. A
secondary reason regarded uncertainty about
possible impacts of pH variation during the test
based on the pH ranges reported in the paper.
Furthermore, the draft Update specifically
stated that the calculated criterion should be
evaluated with respect to whether it afforded
protection to Hyalella based on this test.
Therefore, although the draft Update did not use
the EC2O of this test directly in the calculations,
it still gave credence to this test.
After the peer review, and in spite of one of the
peer reviewers concerns about the suitability of
the test, two things happened that changed the
EPA position. Concerns about the pH variation
were satisfied after receipt of more detailed data
from the paper’s author. This allowed the pH
associated with effects concentrations to be
adequately defined. More importantly, it was
realized that, even if the EC2O was too
uncertain to use in calculations, the lowest test
concentration represented a concentration which
almost certainly exceeded the EC2O. Using this
in the criteria calculations thus provided-a “anti-
conservative” (high) estimate for this organism
and for the criterion. Continuing to not use this
point would result in a criterion higher than
what valid information for a sensitive organism

indicated. To not use this information would be
inconsistent with the National Guidelines and
the use of the best available information.
EPA does not agree that such changes between
draft and final represent an improper response
to or handling of the peer review process. Peer
review is intended to elicit expert opinions
which will point out errors or raise issues not
fully addressed in the draft document and which
should be considered in further changes to the
document. It does not require that all peer
opinions or suggestions be followed - this is
often not appropriate or possible, in part
because there is often a difference of opinion
among the peers. In fact, there were a variety of
peer comments which, if followed, would have
made the criterion more restrictive, but which
were not adopted in the Update. Nevertheless,
although EPA is not bound to follow the course
of actions recommended by peer reviews, EPA
does consider it necessary to provide clear
justifications for not following peer
Peer review also does not preclude other
changes not raised in the peer process, even if
the peer group endorses an approach which is
then changed. Again, proper justification
should be given if the change appears to be
contrary to peer opinion. With respect to the
Hyalella test, EPA believes that it has clearly
documented sound reasons for the change in
Comment C-16
It should be noted that unpublished follow-up
studies on Hyalella, not included in the record,
indicated that increased ammonia levels were
acceptable (between 2.5 mg/I and 3.5 mg/i [ as
N] nominal exposure). However, the measured
ammonia levels were much less than nominal
exposures (35-60% less) calling into question
the validity of the test measurements and
procedures. (Note: nominal and measured
concentrations in Borgmann (1994) were
virtually identical over the ten week test
EPA believes that the new range-finding test
(from the Columbia Lab), not used in the
Update, shows Hyalella to be quite sensitive,
the effects concentrations being no more than a
factor of two higher than in the test used in the
Update, even based on nominals. If anything,
this argues against the contention that the earlier
Hyalella test was faulty in some way. Based on
measured concentrations, this new test shows
even greater sensitivity than the earlier test.
Comment C-i 7
The Update used organism reproduction as the
sensitive endpoint in the chronic analysis for
Hyalella. The Hyalella study authors
concluded that organism reproduction should
not be used for evaluation purposes because
of low sample size and high variability
between replicates. (Borgmann @ 332-333.)
Courts have repeated ruled that it is per se
arbitrary and capricious to use an expert’s test
results in a manner contrary to the conclusions
of the expert. A/may, Inc. v Cal 4fano. 569 F 2d
674 (D.C. Cir 1977). Thus, the use of the
Borgmann study (jf at all) should be limited to
chronic mortality endpoint analysis,
recognizing that growth is also nor adversely
affected at that level as stated by Borgmann.
This comment misrepresents the cited
statements by the author, which are as follows:
Reproduction was significantly reduced at
0.32 mM ammonia in the experiment with
young amphipods. This was first
observed in experiment 2 (the first 10
week experiment). Concentrations of
0.10 and 0.18 were, therefore, added in
the final 10 week experiment.
Reproduction was also lower at these
concentrations, but not significantly so
because of the low sample size and high
variability in reproduction between
replicates. Reproduction in the
experiment with adults was also reduced
at 0.32 and 0.56 mM, although only the

reduction at 0.56 mM ammonia was
statistically significant. The mode of
action of ammonia is, therefore, different
from that of metals of PCBs, which do
not cause significant reproductive
impairment at concentrations below those
causing chronic mortality (Borgmann et
al., 1989, 1990, 1993). Chronic
mortality is, therefore, not a reliable
indicator for use in estimating safe
concentrations of ammonia to Hyalella,
unlike the other toxicants studied so far in
our laboratory.
What the author actually states is that
reproductive effects at the lowest concentrations
tested were not significantly different from the
controls based on the statistical tests that he
employed, because of the low sample size and
high variability among replicates. This in no
way is saying that reproduction should not be
used for evaluation purposes and is certainly not
supporting the use of chronic mortality instead
of reproduction. In fact, the author clearly is
concluding that reproductive effects are present
at lower concentrations than mortality and
explicitly states that it is mortality that is not
reliable in estimating safe concentrations of
ammonia. One problem here is that the
statistical tests used by this author are
insensitive and cannot confirm that 50-70%
inhibition of reproduction is significant. This is
not uncommon in the toxicity literature and
more appropriate statistical evaluations show
these reproductive effects to be real, as
Borgmann clearly believes they are.
EPA also believes that it does not make sense
not to use a study which shows an organism to
respond to low concentrations because the
author did not statistically demonstrate that the
effects were present at even lower
concentrations. But even if this comment were
correct that reproductive effects should be
ignored, what would be the consequence to the
criterion? The EC2O for survival in these tests
is at the lowest test concentration, the same
concentration used in the Update. Thus, the
criterion would be exactly the same, except that
it would not be based on a “less than” value.
Comment C- 18
In addition to basic data acceptability issues
regarding the Hyalella test, there are a
number of confounding factors that preclude
use of this test. Both Borg,nann studies (1994
and 1996) confirmed that a host of water
quality factors, unrelated to ammonia,
influence the toxicity of ammonia to this
organism (potassium, hardness, bromide, and
sodium). The 1996 acute tests demonstrated
that water effects change organism sensitivity
by at least a factor often. In mid-west and
western streams where salt and hardness levels
are typically high, this organism would be
insensitive to ammonia at hardness ,greater
than 200 mg/l. Thus, whether or not this
organism is the “most sensitive tested” will
vary from site to site. Given this information,
the National Guidelines would require that the
criteria be a function of these various
parameters. The Update, however, concluded
that essentially no water efftcl ratio is relevant
to the criteria. Given the atypical response of
Hyalella, using this organism to drive the
national criteria calculation is inappropriate.
Minimally, the Update should recalculate the
criteria eliminating Hyalella for streams with
hardness greater than 200 mg/I.
This comment does not accurately reflect
available data and relationships. It is true that
certain ions do affect ammonia toxicity of
Hyalella. Ankley et al. (1995) reported that
acute ammonia toxicity to Hyalella decreased
with increasing hardness, and further noted that
this increase was greater at low pH. At pH 8.5,
the variation in LC5O was only 1.5-fold between
soft water (50 mg/L CaCO3) and hard water
(240 mg/L CaCO3), but appeared to be at least
10-fold different between soft and hard water at
pH 6.5. Borgmann (1994) also showed a large
difference in toxicity between waters with
hardness of 14 mgfL and 140 mg/L as CaCO3
(although this effect was also confounded by

pH, the effects of other ions were clear).
Borgmann and Borgmann (1997) showed that
these effects were due to sodium and potassium,
not hardness (and also not bromide as this
comment asserted). The effects of sodium and
potassium appear to be on the toxicity of
ammonium ion, not unionized ammonia, which
is why the effects are most pronounced at low
pH, where ammonium ion toxicity
predominates. This implies the following:
(a) EPA does not agree that the criterion should
be recalculated for hardness> 200 mgfL by
excluding the Hyalella data for this range. First
of all, hardness is not the factor of importance
here, so it would be inappropriate to adjust the
criterion in terms of hardness. Second, it would
be inappropriate to simply exclude one species,
because there is no reason to suspect that
Hyalella is “atypical” as the comment suggests.
These ion effects are consistent with
mechanisms for ammonia toxicity that are likely
true for other organisms as well, though not
necessarily to the same degree. In fact, other
authors have noted effects of such ions on
ammonia toxicity to fish (e.g., Soderberg and
Meade, 1992, J. Appl Aquaculture 4:83).
(b) If there is some adjustment to be made, it
should be based on sodium, and this adjustment
should also vary with pH. Borgmann and
Borgmann (1997). propose a model for this, this
model being a simple extension of the joint
toxicity model already used for the pH
relationship in the criteria, with the toxicity of
ammonium ion being sodium dependent rather
than constant. (Their model also includes a
potassium dependence, but the sodium
dependence will almost always predominate in
natural waters.)
(c) The data and model of Borgmann and
Borgmann indicate that increasing sodium
concentrations above that already present in the
Hyalella chronic reproductive test (0.6 mM, or
about 14 mgfL) would have relatively little
effect. Their data show, at pH 7.6, the LC5O at
10 mM sodium (230 mgfL) to be only 45%
greater than at 23 mgIL. Their model indicates
that if the chronic Hyalella test had been run at
tenfold higher sodium levels (140 mg/L), the
effect concentrations would only be about 30%
greater. So high a sodium concentration is very
unusual in receiving waters (the 14 mgfL in the
test itself is already at the median sodium
concentration for U.S. waters based on
NASQAN monitoring, and less than 10% of
waters have concentrations even over 100
mgfL). Very few waters would get relief, and
what relief should be given is limited and
(d) In contrast, applying this model to the
Hyalella chronic data would usually result in
greatly lower effects concentrations in waters
with low sodium concentration. Borgmann and
Borgmann (1997) showed 7-day total ammonia
LC5Os as low as 0.14 mg/L for low ion waters
(2 mg/L sodium) with pH of 7.4-7.8.
Furthermore, this model (and Ankley’s data)
suggest the pH relationship should be more
restrictive at low pH because these sodium
effects are more pronounced there.
(e) However, these effects are only well
established for Hyalella, and only for acute
toxicity. The limited data with fish are hard to
quantify. The key question would be what to
assume about the sodium dependence for other
species - the same as Hyalella, something
different, or none at all? There really is not
sufficient information to address that issue, but
whatever is assumed, the consequence might be
more restrictive criteria under many conditions,
with some modest relief when sodium
concentrations are high.
The comment is correct that this is an issue of
potential importance. It would be preferable to
have this factor accounted for in the criterion,
but the Update did not do so because sufficient
data to reliably quantify the effect of dissolved
ions on ammonia toxicity is lacking. It is
inappropriate to characterize the Update as
deviating from the Guidelines simply because it
does not account for all factors known to affect

toxicity. In fact, to adjust the criterion for this
factor the Guidelines would require more data
than is available in this case. The issue should
be what uncertainties are present because this
factor is not addressed and what uncertainties
would be introduced by modeling the factor
with inadequate information. Low dissolved
oxygen and low levels of chlorine are also
known to increase ammonia toxicity, which
would be relevant to P01W discharges, but
also were not accounted for because of
uncertainties regarding modeling them.
Comment C-I 9
Use of the 1981 Sparks and Sandusky
fingernail clam study is inappropriate. The
study acknowledged numerous flaws and
problems with the culturing of the organisms
and the conduct of the tests as follows: (1) the
authors had a history ofproblems with growth
and reproduction in the lab; (2) bacterial
slimes impacted the tests; and (3) growth of
harvested organisms only lasted two weeks!
With respect to the well water test used by the
Update, the study authors stated that the clams
in well water “didn ‘t grow at all” and were
“starving” due to inadequate food supply.
(Sparks and Sandusky ® 32-36.) Given the
authors clear statements that the tests were not
run properly (one must feed organisms in a
chronic test), it is apparent why these test
results were sign (/Icanlly less than similar tests
run at the same laboratory. This test result
should be stricken from the criteria calculation.
While this comment is correct about some
problems faced in the series of experiments by
these authors, it misrepresents much about this
study. The most significant errors involve the
quote and reference to page 32-36 in the report
and the implication that organisms were not fed.
First of all, the organisms were fed. Second, the
growth problems in this section referred to an
experiment early in the study in which feeding
was more limited than later in the study (when
the ammonia tests were run). Third, for the
experiments after which the feeding was
modified (including the ammonia experiment)
the organisms did grow in the control and this
growth continued throughout the tests (it did not
last only “two weeks”). Fourth, there was a
dose dependent inhibition of growth correlated
with ammonia exposure, including in the first
two weeks, further substantiating that the
control organisms were healthy enough to grow
in the absence of ammonia stress. Admittedly,
the control growth was still low compared to
tests in river water and can probably be
attributed to less food, but it is inappropriate to
characterize this as “starving” and it is sheer
speculation to suggest that the response to
ammonia is due to anything other than the
ammonia. Even if suboptimal nutrition did
increase susceptibility, this does not invalidate
the test - organisms in nature do not always
exist under optimal conditions or show optimal
growth either.
EPA also does not agree that problems in the
Sparks and Sandusky study were the reason
“test results were significantly less than similar
tests run at the same laboratory” (in the study of
Anderson et al.). There is no particular reason
to consider the early tests to be more valid.
They were conducted by the same organization
using very similar methods. If anything, the
later study improved feeding methods and
showed better growth in the control organisms
than the earlier study. Clearly, the nutritional
status is not a likely reason for the differences
between the two studies. Organism sensitivity
can vary for a variety of reasons, and the fact
that the earlier study had a higher effect
concentration should not be treated as evidence
that it is more valid.
There is no convincing reason to conclude that
the effects concentration from the Sparks and
Sandusky study is inappropriately low and
should not be used. While the control growth
was low, there was growth, there was good
control survival, and there was a consistent and
large dose-response relationship with ammonia
concentration. Such results should not be
ignored, and neither should the results from the

earlier study be ignored. The Update averaged
results from both studies, so the value used in
the criteria calculation represents a compromise,
moderate sensitivity.
The Update also considered other information to
evaluate whether the sensitivity indicated in the
Sparks and Sandusky study should be suspect
and whether the value used for criteria
calculations should be considered inappropriate.
It was found that other available information
suggests that this organism should be sensitive.
The mesocosm study at Monticello discussed in
the Update showed substantial effects on
fingernail clams at concentrations near or even
below that of the Sparks and Sandusky study,
and in this study the control treatment showed
high reproduction rates, indicating that the
organisms were thriving. The Update also
considered that these studies in the laboratory
did not include reproduction and early life
stages, which have been shown to be more
sensitive than juveniles to ammonia in studies
with other clams, so if anything the effect
concentrations would be expected to be too
high. Another factor considered was that the
study of Anderson et al. showed that ammonia
quickly decreases ciliaiy motion in clam gills at
low concentrations - such an endpoint is not one
that can be used directly for criteria, but
provides information on ammonia effects that
increases the credibility of observed effects on
growth and mortality.
Finally, another recent study substantiates low
effects concen trations for a similar clam species.
Hickey and Martin (1998, Arch. Environ.
Contam. Toxicol.) reported on 60 day tests of
Sphaerium novaezelandiae, a New Zealand
species closely related to the one used in the
Update, and a genus also found in the United
States. They reported total ammonia EC5Os for
survival of 3.8 mg NIL, for morbidity of 2.7
mgfL, and for reproduction of 0.8 mgIL. The
pH in these tests varied between ammonia
exposure levels and with time, so there is some
uncertainty as to what pH these effects
concentrations correspond to, but it is certainly
less than 8.0 and perhaps as low as 7.5. In any
event, these results indicate sensitivity as greIt
or greater than the Sparks and Sandusky study
and the Monticello study and the authors stated
that “the use of the U.S. EPA criteria would
provide minimal protection for S.
novaezelandiae for chronic ammonia exposure”.
Comment C-20
The National Guidelines @ 43 require that the
criteria accurately reflect facto rs that influence
organism sensitivity. EPA stated that
invertebrates exhibit a temperature dependence
with total ammonia. Therefore, the Hyalella
andfingernail clam data (the two most
chronically sensitive organisms) should have
been adjustedfor temperature, yet no
adjustment was made in developing the chronic
criteria. As discussed in more detail in the
temperature section below, failure to adjust the
chronic criteria to reflect changes in organism
sensitivity had a sign jflcant impact on the
chronic criteria calculation. Much less
restrictive chronic criteria would have resulted
for temperatures ranging 20-0°C, regardless
of whether or not early l fe stages offish were
present. Therefore, the Agency needs to
recalculate appropriate chronic criteria for
various temperatures (25 - 0°C).
For 1999 the CCC has been modified to account
for the expected temperature dependency of the
sensitive invertebrates, while still protecting
ELS of fish when present, orjuvenile and adult
fish when fish ELS are not present. EPA
believes that the National Guidelines provide for
the criteria derivation procedures used for
temperature and seasonally varying 1999 CCC,
but in no way require them.
Comment C-2 1
The chronic data set fails to meet the minimum
data guideline of eight species including
salmonids and an insect, in addition, the
exclusion of the extensive l /’e cycle test trout
database developed by Thurston (which would
have resulted in the calculation of higher

criteria) resulted in the calculation of unduly
restrictive chronic criteria. Failure to use the
lçfe cycle tests simply because earlier, less
extensive tests produced lower results is not
valid. Such an approach would only allow new
data to produce more restrictive criteria. If the
minimum requirements are not met, EPA needs
to use a d f [ erent calculation methodology.
Minimally, the chronic criteria should be
recalculated including the salmonid data and
adding several insect test results.
Regarding the salmonid data, it is true that this
data was not used in the calculations, for
reasons explained in the Update. The rainbow
trout tests varied substantially among four
different studies. The National Guidelines
would normally give primacy to the life cycle
(LC) test, but this primacy rule is based on the
presumption that life cycle tests are more likely
to include all sensitive stages and be a more
accurate reflection of risk. Because two of the
early life stage (ELS) tests were substantially
more sensitive than the life cycle test,
automatically giving primacy to the life cycle
test becomes questionable. This is not
equivalent, as the comment asserts, to only
allowing “new data to produce more restrictive
criteria”, but rather recognizing that LC tests by
their very nature usually are more sensitive and
that when ELS tests are actually more sensitive
it is legitimate to consider why. It should be
noted here that the National Guidelines stress
the importance of not simply adhering to the
specific recommendations, but doing what is felt
most scientifically sound given available data.
There are various reasons that those ELS tests
would show more sensitivity than the LC test,
and some of these reasons would mean that the
ELS tests should be treated on a par with the
LC test, or even given primacy. Depending on
how these data are handled, this species could
range from the most sensitive to moderately
tolerant. The situation was further complicated
by some tests providing only upper or lower
limits of effects concentrations. Nevertheless,
by comparing the CCC to the all the available
salmonid data, viewed as a whole, a decision
can be made as to whether the criterion should
be considered adequately protective or not for
salmonid waters, or whether the criteria should
be modified for salmonids upon separate
consideration of the rainbow trout tests.
EPA does not agree that its handling of the
salmonid data produced “unduly restrictive”
criteria. In fact, it had very little effect on the
criterion. If the rainbow trout data (either the
LC test result alone or the average of the values
from all rainbow trout tests) were used in the
calculations, the dataset would be one genus
larger and this would increase the calculated
FCV from 1.27 to 1.31 at pH=8, an increase of
only 3%.
Regarding the insect data, an acceptable chronic
test was not available, so it is true that the
minimum data set was not fully met.
Nevertheless, based on the other insect data
available, the Update assumed that, if an insect
were tested, it would likely be more tolerant
than the four most sensitive genera tested. The
criterion was calculated based on these four
genera, but increasing the total number of
genera by one to account for the presumed
tolerant insect.
This is justified as follows. First, the
assumption that a insect would be tolerant is
reasonable, given their high acute tolerance and
the fact that a subchronic test on an insect
suggested it was fairly tolerant. There is no
need to assume a specific tolerance, only that
the insect is more tolerant than the fourth most
sensitive genus. It should be noted that this
assumption is not “conservative”, which the
commenter was earlier concerned with, but the
opposite of conservative. Second, calculation of
the chronic criterion directly from chronic data
is the prefened method in the Guidelines,
although it is generally not done because of the
lack of chronic data. Calculation by acute-
chronic ratios is subject to more uncertainty,
especially in this case where acute-chronic
ratios are not available for some important taxa.

To abandon direct calculation because the
database does not include one element which
probably is tolerant and will not be used directly
in the calculations is contrary to trying to use
procedures which will produce criteria most
reflective of the available data. Finally, EPA
can see no valid technical reason why the
chronic criteria would need to be recalculated
including “several insect test results”.
Comment C-22
The Update correctly notes that the chronic
criteria database is skewed by the inclusion of
a preponderance of sensitive species.
Therefore, a more reasonable estimate of the
chronic criteria needs to be developed by
utilizing a higher N value that offsets the
skewed database. An N of 12 would appear
reasonable based upon the acute data sets.
(See, Exhibit 2 comparing the sensitivity of
organisms contained in the acute and chronic
databases.) This would produce a total
ammonia chronic criteria (25°C atpll 8) in the
range of 1.5 - 1.75 mg/i (as N) consistent with
the reliable data sets (i.e., excluding Hyalelia).
EPA does not believe that this comment
correctly reflects what the Update stated. It is
true that the Update did note that the acutely
tolerant species are under-represented in the
chronic database relative to the acute database.
But this was one of several comments
speculating on how different factors, changes,
or options might increase or decrease the
chronic criterion, and did not specify that the
chronic database was in fact inappropriately
skewed to sensitive species. The differences
between the acute and chronic database does
not necessitate recalculating with larger N.
There is no basis for deciding that the acute data
set provides the preferred balance among
species. Both data sets display a reasonable
amount of diversity once the tolerant insect
assumption is accounted for in the calculations.
It cannot be known which better represents the
range of sensitivity that would be found in an
assemblage of taxa in the field, and for that
reason, while the Update mentioned the
possibility of further adjusting N, did not carry
through with it. (See also C-07.)
Comment C-23
EPA stated that any chronic criteria should be
limited by the bluegill test result from Smith
(1984). As indicated by the Peer Review
comments (which EPA ignored), the sole
bluegill study by Smith should not be used to
arbiirarily decrease chronic criteria because it
is a single unver fied test, critical DO
information was not available to ensure test
reliability, significant pH variability occurred
over the duration of the test, and bluegill were
not confirmed to be a highly sensitive species
based upon EPA ‘sfield research. It is
irrational to spend hundreds of thousands of
dollars to investigate organism sensitivity,
including closely related species sensitivity,
and then to ignore all of the data by using one
organism to set a national criteria. As stated
in the Peer Review, absent confirmation that
bluegill sensitivity sign /Icantly different than
closely related species, the genus mean chronic
value should be used in the criteria derivation
as, on balance, the data indicate that this
species will be adequately protected
This comment seems somewhat moot, since
neither the 1998 nor 1999 criteria do this, but
the Update does note that this issue could arise
if modifications to the criterion would raise the
criterion above the chronic effect concentrations
for the bluegill. If this happened, reliance of the
criteria on just one test on bluegill, having a
result quite’ different from a related species
(green sunfish) having two chronic tests, would
be an important issue.
EPA does not agree with several of the
statements made in this comment:
(a) EPA does not believe that the actual pH in
this test (as opposed to the reported pH) was
excessively variable. The investigators
evaluated the mean and variability of pHs by

first converting pHs to hydrogen ion
concentrations, determining means and standard
deviations, and then converting back. Doing
such calculations on a hydrogen ion basis is
more appropriate for certain calculations, and
during the era this test was done it was thought
to be desirable for this type of data, although it
is usually no longer considered so. But to
provide a meaningful measure of the variability
of pH is not just a matter of converting the
hydrogen ion standard deviation back to the pH
scale, which is what the authors attempted to do
here. Thus, the mean pHs in this report are
valid, but the standard deviations are not. The
original data is not available, but it is now
thought that the likely error in converting these
values back to the hydrogen ion scale inflated
the likely true low variability in the pH
(probably around 0.2 units). These tests were
run in exposure systems and water used for
many similar tests, which almost always had
low variability in the pH.
(b) While the D.O. data are not available,
various tests run with this or similar systems in
the laboratory maintain D.O. levels well above
values of concern. Give little likelihood that
D.O. was unacceptably low, the absence of such
data is a not a good reason for discounting this
(c) The comparison by the Comments of these
results to those for bluegill in the Monticello
channel are not valid because the Monticello
experiment only tested survival arid growth of
juvenile bluegills. The Monticello results
showed a 40% reduction in growth ofjuveniles
at the highest exposure concentration (ca. 7 mg
NIL). No mortality was observed due to
ammonia, which is consistent with laboratory
tests which indicate juvenile bluegill mortality
requires higher concentrations. (However, it
should be noted that with such substantial
sublethal effects, mortality in the Monticello
streams would likely have occurred at
concentrations not much higher.) The chronic
ELS test includes a more sensitive lifestage, and
even that lifestage would be expected to show
only small effects at the second highest (2 mg
NIL) exposure level in the channels.
Consequently, the Monticello experiment does
not refute this experiment. The fact the bluegill
juveniles are no more sensitive than many other
fish species does not necessarily imply bluegill
ELS can be no more sensitive than many other
(d) Lastly, the invocation of the peer review is
also of doubtful relevance here. Only one of
five reviewers objected to using the test, and the
only stated reason was that it was an unrepeated
test that was more sensitive than other members
in that genus. This comment is therefore
misleading in its second sentence, which implies
that the peer review comments raised all the
listed concerns (including the D.O, pH, and
field study issues discussed above) and that this
was more than the opinion of one reviewer.
The issue that does remain is whether one test is
a sufficient basis for setting a criterion with the
importance of ammonia, and with a data set
such as is available for ammonia, where most
species had multiple chronic tests available for
defining their SMCVs. The use of an
unrepeated test is in fact consistent with the
Guidelines, as are the use of unrepeated tests for
the four most sensitive genera, which can also
have a significant effect on the criterion.
Nevertheless, verification of tests that are
critical to the criterion is a legitimate issue that
should be considered if reliance on the single
bluegill test became an issue.
At lower temperatures the temperature-
dependent 1999 CCC (with ELS present) does
take on values above the bluegill ELS SMCV.
Because bluegill early life stages prefer rather
warm water (e.g., the ASTM chronic testing
protocol calls for 28°C water), and because
bluegill spawning generally occurs during only
the warmest weeks or months of the year, the
risks to bluegill are probably subdued, although
not necessarily negligible if the Smith et al.
results were to be accurate.

Comment C-24
The Update @ 24 acknowledges that the
relationship appears most applicable to fishes
while other organisms (i.e., invertebrates) do
not appear to respond in the same manner.
Consequently, the Update acknowledged
uncertainty with the pH relationship and
eschewed an empirical approach to the data
that “uses the average generic shape for the
pH dependence.”
In fact, no such statement or acknowledgment is
made by the Update. The Update 24 notes
variability among the taxa, especially at low pH,
but does not make any distinction between fish
and invertebrates. In fact, there is variation
within both fish and invertebrates, and both
groups contain species with pH dependence
similar to, greater than, and less than the generic
acute pH relationship. For chronic toxicity, the
pH relationship is based on a fish and an
invertebrate, both of which show a dependence
similar to each other and to the chronic pH
relationship used in the Update (see Appendix II
of this response).
Comment C-25
Although the Update (@24) stated that
individual test results should not be used to
mod jfy the pH relationship, the relationship
was mod /Ied for chronic toxicity to be more
restrictive in the range ofpH typically relevant
to municipal facilities discharging to low flow
streams (pH 7.0- 7.7).
EPA believes that this comment has
misinterpreted the Update. What the Update
actually stated was that it would be speculative
to assign different relationships to different taxa.
Perhaps this was not explained as well as it
could have been, but the issue it intended to
address was whether different pH relationships
could or should be assigned to each species or
genus or larger taxonomic group, or whether a
single average relationship should be used. This
involved not just deciding what the pH
relationship was for the species included in
Figure 6 (1998) or Figure 8 (1999), but for
other species as well. Three of the four most
acutely sensitive genera did not have pH data
and it was not evident what individual
relationships in this figure would be most
appropriate to them, other than an average one.
The fourth genus already had a relationship very
similar to the average relationship for all
species. An average pH relationship therefore
seemed most appropriate and relevant to the
actual criteria calculations. Assigning different
pH relationships for each taxa was
“speculative” and served no useful purpose.
However, this does not mean that these
relationships could not be developed. Clearly,
channel catfish exhibited a relationship different
from most other fish, and a separate model
could be developed for this species. But it
would have no impact on the acute criterion
because this species is not among the more
sensitive species at any pH.
Comment C-26
[ The d j’ference between the acute and chronic
pH relationships] was based primarily upon a
single, unconfirmed test result using
smallmouth bass.
In fact, the shape of the chronic pH relationship
reflected both the smailmouth bass and daphnia
studies, weighted roughly equally. Appendix II
of this response provides graphs showing the
similarity between the two studies. It is
furthermore inappropriate to characterize this
relationship as depending on a single test,
because each study involved multiple tests, both
acute and chronic, that showed consistent
Comment C-27
No analysis was presented to assess (1) how
well the acute toxicity/pH relationship fit the
available chronic data, (2) the needfor the
reduction in light of the relative sensitivity of
the organism, or (3) whether invertebrate data

confirmed the need for a more restrictive
Regarding item (1), the Update on page 26
reported that the regression analysis of the
chronic data showed its relationship to deviate
significantly from the acute relationship. A
graphical presentation of the differences is
provided in Appendix II of this response, as
well as some additional information on the
statistical results.
Regarding item (2), which EPA interprets to
refer to the fact that the taxa used for the
chronic pH relationship were not among the
four most sensitive genera. The argument being
presented apparently is that, if the more
restrictive flattening at low pH is based on
relationships established for tolerant organisms,
this restriction is not needed for criteria based
on more sensitive organisms. While it would be
preferable to have pH relationships for the most
sensitive organisms, in the absence of such
information “the need for the reduction” rests
simply on what assumption is most appropriate
regarding the sensitive taxa: (a) the relationship
established for chronic toxicity in other
organisms, even if they are more tolerant, or (b)
the pH relationship that is based on even more
tolerant, acute endDoints . The available chronic
relationships provide the most relevant
information, and accord to the National
Guidelines regarding what data such
relationships should be based on. It should also
be noted that there are other options, possibly
including basing the chronic pH relationship for
Hyale Ha on the acute pH relationship for
Hyalella. But this acute data also shows that the
slope of the pH relationship should be more flat
(than the average acute pH relationship) to
extrapolate to low pH from pH 8, where the
chronic Hyalella test was conducted. Thus,
even based on the acute pH data, it can also be
argued that there is a “need for the reduction”.
Regarding item (3), the chronic pH relationship
data included invertebrate data, which followed
nearly the same trend as the fish chronic data.
Again, Appendix II has figures demonstrating
Comment C-28
The chronic data presented in Figure 12
(Update @ 70) do not support a sigm/Icant
decrease in the slope of the pH relationship
below pH = 7.7. EPA expended considerable
effort to demonstrate that the overall pH
relationship for acute toxicity was a good fit for
the data trends (Update Figure 7 27).
Because of the conflicting information on the
causes of ammonia toxicity (total versus un-
ionized ammonia), the approach employed was
“somewhat empirical” and designed to fit the
available data. (Update @ 7, 21 - 29.) The
decrease in slope used in the chronic pH
relationship was based on toxicity tests for only
two organisms (Update Figure 8 28) and no
comparison was made to demonstrate that the
acute relationship was a “badjit” of the
chronic data. The data do however indicate
that chronic toxicity may ‘flatten out” (e.g.,
become less restrictive) above pH = 8.5. EPA
should revise the criteria to ‘fit the data”
consistent with the methodology claimed to be
The Guidelines specifically note that if the
acute-chronic ratio varies with the water quality
characteristic, the acute relationship should not
be used. This is the situation here, as discussed
further in Appendix II.
The Guidelines also speci ’ that a chronic
relationship should be developed from chronic
data, if sufficient data exist for at least one
species. In fact, there were two data sets here
that showed similar pH relationships for chronic
toxicity data that differed significantly from the
acute pH relationship based on regression
analysis. Again, these differences are presented
in more depth in Appendix II of this response.
The smallmouth bass data set is particularly
compelling because it includes parallel acute
and chronic data sets which show acute-chronic

ratios to increase consistently and significantly
with decreasing pH. This is completely contrary
to any assertion that these relationships should
be the same. This does not preclude the
possibility that, once tested, the chronic pH
relationships for the sensitive organisms will be
different, but in the absence of such information,
the best assumption is that the relationship for
chronic toxicity should have a less steep slope
for extrapolating to lower pH.
EPA does not agree that Figure 12 (1998) or
Figure 14 (1999) supports a steeper slope at
low pH and a shallower slope at high pH. This
issue is further discussed in Appendix I of this
response. Briefly, if data arc plotted against a
relationship such as in Figure 12 and most of
the data are near the middle of the pH range, by
simple probability the data in the middle are
more likely to include values iii the tails of the
distribution and thus bulge further down. In
contrast, the small number of tests at low and
high pH would be less likely to produce results
in the tails and the data would cluster closer to
the overall mean among taxa. Focusing on the
lower boundary of the data in each pH interval
creates the appearance that slopes are greater at
low pH and smaller at high p1-i than they
actually are. This same appearance is evident in
the acute data in Figure 10 (1998), but as the
simulation in Appendix I demonstrates, this
appearance is exactly what would be expected if
the pH relationship were absolutely true for all
the data. For the chronic data in Figure 12
(1998), it is clear that the data at low pH and
high pH are for more tolerant organisms. Thus,
EPA believes that the appearance of greater
slopes at low pH and smaller slopes at high pH
are based on confounding comparisons between
sensitive and tolerant organisms.
The key issue here is what pH relationship the
sensitive taxa should be assumed to follow. The
Comments would seem to suggest that even
though two chronic data sets are available that
show similar relationships to each other and
show clear differences from acute data, they are
not to be believed and that the acute
relationship, because of its larger data set,
should be followed, even though it is clearly a
worse fit to the available chronic data sets. EPA
believes that it is better to use the available
chronic relationships. Furthermore, if acute
data were used to set the pH dependence for
chronic toxicity, there is an acute dataset for the
most chronically-sensitive organism, Hyalella.
As these comments themselves note elsewhere,
the pH dependence for Hyalella is less than the
average for all species, which means that even if
acute pH data were used, the criteria should still
be more restrictive at low pH. To suggest that
the average acute pH relationship is a better
choice to apply to the sensitive chronic data is
not supported by the available chronic data or
the available acute data for Hyalella. This does
not mean that, once tested, the chronic pH
relationships of the sensitive species might not
be found to be different than assumed, or even
like the acute pH relationship, but lacking this
information, EPA believes it is making the best
use of the available data.
Comment C-29
The organisms that drove the chronic criteria
(invertebrates) appear to be less affected by
changes in pH than do fishes (Ankley 1995).
Based upon Figure 6 in the Update (@25), the
pH relationship will sigm/Icantly under-predict
the acceptable total ammonia for these
organisms. There is no basis to conclude that
using the acute toxicity/pH relationship will
under-predict toxicity to sensitive invertebrates.
This comment is a bit confusing, primarily
because the example it brings up actually argues
against rather than f using the acute pH
relationship. EPA interprets the invertebrates
referred to in the first sentence to in include
Hyalella, the species studied by Ankley et al.,
but also fingernail clam, the other invertebrate
that drove the criterion. It is correct that
Hyalella toxicity is less affected by pH than
most fishes and than the average pH
relationship. But this is not necessarily true for
other invertebrates. Chironomus also is less
affected by p1-I than the average, but

Lumbriculus is more affected by pH. Daphnia
and Macrobrachium seem to follow the average
relationship very closely, with a little more
flattening at low pH. What might be true for
fingernail clams is completely unknown. But at
least in the case of the most sensitive organism -
Hyalella - the acute data do indicate a flatter pH
relationship and it will be assumed that this
organism serves as the example here.
To state flatly, as this comment does, that the
pH relationship under- or over-predicts toxicity
is not entirely appropriate: either can occur
depending how the relationship is used. (That
is, if the slope is flatter than it should be, using it
to go from low pH to high pH overpredicts
toxicity, but using it to go from high pH to low
pH underpredicts toxicity).
Nevertheless, since the chronic criterion is
heaviLy influenced by tests run near pH 8, EPA
understands the commenter’s preference for
using a steeper acute slope rather than the
shallower chronic slope to extrapolate this data
to lower pH, a pH region of interest to many
municipal dischargers. But the above comment
cites the even shallower slope Hyalella data in
Figure 6 of the 1998 Update (or Figure 8 in
1999) as supporting such an action. In fact, the
opposite is true. As this comment itself notes,
the acute Hyalella data on Figure 6 show less
dependence on pH (lower slopes) than does the
average acute pH relationship. Therefore, using
the average acute pH slope to extrapolate to
lower pH will under-predict toxicity (over-
predict toxic concentrations) rather than over-
predict toxicity (under-predict toxic
concentrations) as the Comment suggests. This
is shown in Figure 6. The solid lines denote the
model fit to just the Hyalella data, while the
dotted lines denote the average acute pH
relationship. The dotted lines arc near the
observed data trends near pH 8, but are above
the data at pH 6.5-7.5. This means that acute
toxicity at low pH for Hyalella is under-
predicted by the average acute pH relationship -
toxic concentrations are over-predicted, not
under-predicted as this comment claims. This is
also evident in comparing the acute Hyalella
data to the acute criterion equations - these data
lie progressively closer to the criterion at lower
pl-I (i.e., Hyalella is more tolerant than other
organisms at higher pH, but becomes more
sensitive relative to other organisms at lower
The fact that the acute pH relationship under-
predicts relative toxicity for this organism was
pointed out in the Update and it was noted that
at low pH this organism is among the most
sensitive acutely, at least at low sodium
concentrations. Consequently, the facts
presented in this comment actually argue
against using the average acute pH relationship
for describing the chronic toxicity of Hyalella.
Comment C-30
Winter, not summer, is the most important
period to ensure that the criteria are properly
applied The Update acknowledged that low
temperature chronic ammonia requirements
should be based upon survival endpoints where
ELS considerations are not present. There is
no technical basis presented to believe that the
acute toxicity/pH dependency should d ffer
from the chronic pH dependency when both are
based on this same effect (i.e., mortality).
Because survival is the endpoint of concern in
cold-season situations where early life stages
are absent, the question here is whether (a) the
expected chronic pH relationship for protecting
survival under winter conditions should be the
same as (b) the measured chronic pH
relationship (survival, growth, and reproduction
endpoints), or (c) the measured acute pH
relationship (survival/mortality endpoints only),
both from warm temperature tests.
Early life stage (ELS) mortality was the main
response in the smalimouth bass chronic data
set. Even based solely on ELS mortality, the
chronic pH relationship is similar to that used in
the Update and is substantially different from
the acute pH relationship in the same study.

Consequently, although the comment does not
fully articulate it, the real question is whether (a)
the juvenile and adult chronic mortality is closer
to (b) ELS chronic mortality, or to (c) juvenile
and adult acute mortality, with respect to the pH
Survival versus time curves for ammonia
generally show a very rapid acute response
which quickly tails off, followed by a gradual
prolonged mortality. This pattern suggests two
different modes of mortality - one acute and the
other chronic. Chronic mortality can involve
somewhat different, more systemic, disruptions
than acute mortality, which might well be more
closely related to growth and other sublethal
effects. Consequently, EPA believes that in the
absence of the needed studies ofjuvenile or
adults to ascertain the pH relationship for their
chronic survival, it is not unreasonable to link
the their pH relationship to that for ELS chronic
survival and growth. Nevertheless, EPA
acknowledges the uncertainty, which can only
be reduced through additional testing. See also
the response to Comment D-05.
With the formulation of the 1999 CCC,
however, the issue is much less important,
perhaps even moot. Where fish ELS are absent,
the 1999 CCC is controlled by the Hyalella
GMCV and its predicted temperature
relationship. The juvenile and adult fish
GMCVs are too high to directly affect the CCC.
Consequently, it might be of greater interest to
conduct testing to determine whether
temperature affects the invertebrate pH
relationship in any way.
Comment C-3 1
The chronic toxicity versus pH data for
Ceriodaphnia and smalimouth bass presented
in the Update @ 28 more closely fit the acute
relationship between pH 7.0 - 8.5, indicating
that mod /I cation of the toxicity/pH relationship
was unjust fled Analysis of the pooled chronic
data indicated that the acute pH relationship
generally fits the chronic data as well as the
suggested more restrictive chronic relationship
(see, Exhibit 3). EPA needs to conduct
additional tests before proposing to establish a
more restrictive chronic criteria approach.
First, EPA does not agree that the commenter’s
Exhibit 3 presents an “analysis”. This exhibit is
a graph comparing the chronic data to the acute
pH relationship. It provides no visual or
mathematical comparison of the two possible
relationships: certainly nothing that
demonstrates “that the acute pH relationship
generally fits the chronic data as well as the
suggested more restrictive chronic relationship”.
Second, it is clear from this graph that the acute
relationship greatly under-predicts toxicity at pH
7 and below. Even at pH>7, the acute
relationship underpredicts toxicity for
smallmouth bass by almost two-fold at pH 7.25,
and comes this close only by overpredicting
toxicity at pH 7.8 and above. For one of the C.
dubia sets, similar errors occur.
Third, it is true that if the analysis is confined to
pH>7, the acute relationship fits the data better
than it does for the whole pH range. However,
it still provides a worse fit than the chronic pH
relationship in this range.
Fourth, the criteria cannot restrict itself to
pH>7. Lower pHs are common enough that
they need to be accounted for in the relationship.
Fifth, the data at pH<7 clearly demonstrate a
flatter relationship than the acute relationship.
This behavior not only reflects on what is
happening at pH<7, but also what the
relationship should be at higher pH, at least in
the pH 7.0-7.5 range. In other words, the clear
flattening at pH<7 indicates that some flattening
should be present at pH>7. A good relationship
tries to smoothly and appropriately account for
all of the data. Appendix II of this response
presents additional graphs and information that
show the improved fit using the chronic pH

Comment C-32
The Update discussion on the pH relationship
states thai “it would be speculative to assign
d jferent slopes for d fferenr taxa” and that
data from relatively insensitive organisms
should not impact the criteria derivation.
(Update @ 24, 26.) The smalimouth bass are
not among the most acutely or chronically
sensitive organisms. In numerous places
throughout the Update, EPA stated that criteria
adjustments should not be made based upon
test results from less sensitive species.
Therefore, imposition of the chronic toxicity/pH
relationship based solely on smalimouth bass
was not technically just fIed and adds levels of
conservatism to an already conservative
approach without demonstrated need.
This comment misinterprets statements in the
Update. The statement regarding assigning
different slopes to different taxa being
“speculative” was discussed in response to
Comment C-05. What was “speculative” was
assigning anything other the average acute pH
slope to describe acute toxicity for species for
which pH relationships were not established,
which was true for the most acute sensitive
genera. What was done with the chronic pH
relationship is compatible with EPA’s
statement. It would be “speculative” to assign
anything other than the average chronic
relationship to the sensitive chronic species. It
would be particularly “speculative” to assign the
acute relationship to these species, given that the
available data give clear indications against this.
The assertion that EPA stated that “data from
relatively insensitive ox ganisms should not
impact the criterion derivations” is a
misinterpretation. This statement referred to
species that were observed to deviate from the
average acute pH relationship at low pH, and
simply noted that accounting for such deviations
was not important because those particular
species were tolerant and therefore not included
in the four most sensitive genera used for the
final criterion calculation. However, this
statement was not trying to imply that data from
tolerant species should not impact the criteria
deviation in any way. In fact, the data from
those species were used in the pH analysis to
derive the average acute pH relationship.
Again, this is consistent with what was done for
the chronic pH relationship - the available data
was used to derive an average relationship
based on all organisms regardless of their
tolerances, and the average relationship is
assumed to apply to the more sensitive genera in
the absenceof contrary information.
EPA also does not believe that “in numerous
places throughout the Update, EPA stated that
criteria adjustments should not be made based
upon test results from less sensitive species.”
The Comments have not cited particular
statements in the Update relevant to such data
usage. Finally, the comment is not correct
attributing the chronic pH relationship solely to
smallmouth bass.
Comment C-33
In summary, the proposed more stringent
chronic total ammonia criteria between pH 7.0
to 7.7 is not just /ied based on the available
data and will lead to unnecessary nitr!/Icarion
requirements, particularly during the winter
months when pH is naturally decreased. This
is a critical pH range for municipal facilities,
and a typical pH range encountered in surface
waters throughout the country. The pH
relationship should not be skewed to fit pH
outside this range to the detriment of
dischargers to waterbodies within the range.
EPA should not claim that the pH dependent
criteria have been well established based upon
the acute criteria and then make further more
restrictive adjustments based on limited and
conflicting chronic data. A (1) single chronic
study from a (2) relatively insensitive fish
(smailmouth bass) at (3) atypical pH
conditions that (4) did not drive the acute or
chronic criteria calculation should not be used
to modify the pH relationship that was
developed from using over a dozen

independently conducted tests. Moreover, the
critical time period for application of the
chronic criteria is in the winter, and there is
no scientjfic basis to conclude that organism
response to acute and chronic mortality
endpoints exhibit a different pH profile
during this period.
As indicated in the Update document, an
empirical approach should be used to establish
the pH dependency applied to the criteria given
(1) the complex interactions being assessed,
(2) the inconsistency of organism responses
among the most sensitive organisms, and
(3) the lack of sufficient data. In light of the
inadequate and conflicting chronic database,
the acute pH relationship should be appliedfor
pH between 7.0 and 7.7 and more closely
reflect the acceptable chronic exposures from
Figure 12 in the Update. (See, Exhibit 4,
Figure 12 with mod /Ied pH slope.) The pH
relationship above pH 8.0 should be flattened
out consistent with the available data presented
in the Update.
This comment summarizes points made the
previous comments. To summarize the
responses to those comments, EPA believes
there is a good basis for using the chronic data
indicating that chronic pH relationships differ
significantly from acute pH relationships, even
though the acute pH relationship is based on
more data. Again, Appendix II provides some
additional analysis regarding this, but these
differences were discussed in the Update. It is
conceivable that more chronic testing will
produce different relationships, just as more
acute testing would produce taxa-specific
assessments for the sensitive organisms. Any
relationship will have some uncertainties, and
the best that can be done is to base relationships
on the most relevant data, as the Update did.
See also the response to Comment D-05.
Comment C-34
The discussion on temperature dependence
focuses on the more recent research and
concludes that “the temperature dependence is
incompletely resolved and more research is
needed, especially regarding chronic toxicity.”
Update @11. This conclusion sho uld have led
the Update to acknowledge that the same
concerns raised in the 1984 ammonia criteria
have never been resolved. The Update should
acknowledge that no data confirm that existing
EPA-approved un-ionized ammonia
approaches are, in fact, under-protective.
The quoted statement is accurate. This
statement and others in the Update, do in fact
acknowledge that concerns raised in the 1984
ammonia criteria are not yet fully resolved. But
it is unclear what significance the comment
places on this. Unresolved issues do not mean
that no action should be taken, nor that the
Update should not have been done to improve
data treatment where possible.
At this point EPA is not committing to either
agree or disagree that there are no data
indicating that some previously approved un-
ionized ammonia criteria could be under-
protective of aquatic life uses. As guidance, it is
beyond the scope of any criteria document,
including the Update, to proscribe the range of
acceptable regulatory criteria in different states.
See also the response to Comment D-06.
Comment C-35
The temperature dependent discussion focuses
on the 1987 DeGraeve study and concludes
that un-ionized ammonia sensitivity increases
with decreasing temperature. While these
statements are basically accurate, the
conclusion that both acute and chronic
toxicity expressed as total ammonia is fixed
regardless of temperature is clearly not
accurate based on the DeGraeve acute data.
Total ammonia LC 50 was demonstrated to
increase ssgn flcantly from 30°C to 0°C for
both channel catfish and fathead minnow.
(See, Update Figure 3.) Moreover, the
discussion on Arthur (1987) is misleading.
That author basically concluded that he could

not verify EPA ‘S assumed temperature
relationship (See, Appendix A which includes
an analysis of the Arthur study which
demonstrates there is a total ammonia toxicity-
temperature dependence.)
Regarding the DeGraeve study, EPA does not
agree that the “total ammonia LC5O was
demonstrated to increase significantly from
30°C to 0°C for both channel catfish and
fathead minnow”. Although the LC5O
increased somewhat with decreasing
temperature, standard linear regression
techniques conducted on the DeGraeve fathead
data showed that the slopes could not be judged
to be significantly different from zero at the 5%
confidence limit, given the trends and
uncertainty in the data. Any dataset, even if the
real slope is zero, will generally have some
trend up or down. For the channel catfish data,
there was a trend significant at the 5% level, but
this was the only dataset of many which showed
a significant trend, including others using this
species. When separately examining many
datasets, it is not unexpected that random
variations will show “statistically significant”
effects even if there was no real trend. In any
event, trends should be based on all available
data, not just selected data sets. This does not
mean that there are no real trends with
temperature, at least for some species, but
rather that the data as a whole do not support
the use of a particular trend for fish.
Regarding the Arthur study, the Update
presented a factual summary of what the tests
for each species showed, including recognition
that only three of five fish followed the
temperature relationship presented in the 1984
document and that the insect data showed a
different temperature relationship. If any of this
presentation was misleading, some specifics
should have been given. Arthur et al. did not
state any overall conclusion about EPA’s
1984/1985 temperature relationship.
Comment C-36
The purpose of the acute toxicity/temperature
dependence regression was to evaluate
temperature impacts on toxicity. Therefore,
the data relevant to this evaluation required
species testing over a sufficient temperature
range to test the hypothesis. Other
confounding factors (e.g, pH changes) needed
to be minimized during the tests (i.e., only
change one dependent variable). The acute
criteria pooled data analysis (Update Figure 4)
inappropriately included many studies that
failed to conduct tests over a sufficient range of
temperature to ensure that test variability was
not a confounding factor. Therefore, ab initio,
the analysis framework selected in the Update
would be unable to determine a definitive trend.
Numerous studies were conducted over less
than a 10°C d(fference, and many exhibited
excessive variability at identical temperatures.
(See, Thurston and Russo [ 1983] where
acceptable total ammonia levels variedfrom 15
to 50 mg/i at 12°C.) At least a 20°C range in
the testing with a number of intermediate
exposures should have been required, and tests
exhibiting excessive variability should have
been screened out of the temperature effects
analysis as likely indicative of other factors
influencing test results.
This comment, and the following two, address
the analysis in the Update of the temperature
dependence of acute ammonia toxicity to fish.
This comment also presents a conception of
what is desirable in data sets for temperature
effects. This includes a wide temperature span,
minimization of confounding variables, and low
variability of results at any particular
temperature. Qualitatively, these are all worthy
attributes, and whether certain datasets with
limited temperature range, high variability,
and/or few data inappropriately influenced the
results is a legitimate concern. However,
addressing this question requires specific
consideration of what the effects of various
types of datasets probably are. The questions
that should be asked is (a) whether a data set,

even if it has a limited range or size, or has
some variability, will still make a net positive
contribution to the overall analysis, and (b)
whether that contribution is appropriately
weighted relative to other data sets. EPA does
not agree with the comment about what data
sets are appropriate for inclusion in the overall
Appendix ill of this response presents analyses
which show that the pooled analysis used in the
Update is an appropriate framework for
integrating diverse sets, that the type of data sets
used in the Update provide positive, appropriate
contributions to the analysis and should not be
excluded, and that the Comments are wrong
that restricting analysis to a few sets with a
broader range, and ignoring other data, gives
better estimates.
In this comment, it is asserted that a data set
should be included in the analysis only if it has a
range of at least 20 C. No justification is given
for this specific range and no specific rationale
is given for why certain data sets used in the
Update should not be used. It is suggested that
tests should cover “a sufficient range of
temperature to ensure that test variability was
not a confounding factor.”
EPA, however, believes that test variability per
se is not a confounding factor. If the test
variability is due to factors that are correlated
with temperature, then these factors are
confounding (unless temperature were viewed a
surrogate for them). This is the reason that pH
correlations with temperature were addressed in
the Update. Test variability will reduce the
power of the test to detect effects, but this is
entirely appropriate and in keeping with the
nature of the data, and does not preclude data
sets with a limited temperature range, such as
that of Thurston and Russo, from contributing
useful information. The analysis in Appendix
III of this response shows that such sets make
an appropriate contribution to the analysis.
Comment C-37
Use of regression analyses and then
extrapolating beyond the domain of the
experimental data (by pooling results) is not
statistically appropriate when only aftw
closely spaced data points exist in the
individual studies. The regression lines
through the individual data sets are highly
speculative given the limited data and limited
temperature ranges. Pooling the data does not
increase the certainly that no correlation with
temperature has been demonstrated.
This comment questions the practice of using
data sets with few data points and a limited
range as part of the temperature analysis. EPA
agrees that the individual regression lines
through such sets are uncertain, but disagrees
that including such data sets in a pooled analysis
is inappropriate. The pooling that EPA did here
is no different than the pooling the commenter
did to get an average slope in his Appendix A.
The fact that some of the data sets cover only a
small part of the range being analyzed does not
make their use inappropriate. Whatever its
range, each data set provides an estimate for the
slope, which will be weighted in accordance
with its uncertainty, so that small data sets with
limited ranges will not contribute much to the
overall estimate for the slope. But they still
make a positive net contribution to information
and do on average improve the overall estimate
of the average slope, and the comment is wrong
to assert that pooling of such sets does not on
average improve overall uncertainty. The
analysis in Appendix III provides a
demonstration that such sets should be included.
Comment C-38
Several tests used in EPA ‘spooled analysis
contained an excessive number of replicates
that gave undue weight to the single test result
(e.g., Thurston Fathead Minnow . 1983). Other
data such as Cary (1976) were so variable at
identical temperatures as to render analysis of
temperature effects impossible. With respect to
the studies that were spec flcally designed to

test for a temperature relationship (West and
DeGrave), it is apparent that the allowable
level of total ammonia does increase with
decreasing temperature. Appendix A presents
an analysis of the available data that could
reasonably be used to evaluate temperalure/
toxicity trends. This analysis supports the
Inclusion of a temperature adjustment to the
acute (and therefore chronic) criteria. Thus,
the conclusion that total ammonia toxicity
should remain constant regardless of
temperature appears misplaced. At least a
50% increase appears Just (/Iedfrom the more
extensive studies for temperatures ranging 25 -
This comment, which asserts that some data
sets with many replicates are unduly weighted
in the analysis, provides an opportunity to
explain how data influence the pooled
regression analysis, and why EPA believes that
such influence is appropriate.
It is true that data sets with more data will be
weighted more in the analysis, but unless there
is some independent objective criterion to
weight sets differently, this is proper because
more data provide more information on the
parameters being estimated. However, in the
pooled analysis the actual influence each data
set has on the slope is not just a matter of
number of data, but their range and variability.
The Thurston fathead minnow study is one of
the data sets that the comments earlier noted has
a limited range and high variability. As such,
the uncertainty in its slope is increased relative
to data sets with greater range and/or less
scatter, and thereby its weight is less th n the
number of data might indicate. As Appendix III
demonstrates, the type of analysis and data sets
used in the Update do combine into
appropriately weighted estimates of the slope.
The factors of concern to the commenter, range
and variability, do in fact affect the influence a
study has on the pooled slope.
Thus, the Cary data set, which the comment
terms “impossible” to analyze because of its
variability, can be analyzed by the same
techniques used in the comnienter’s Appendix
A, and it validly assists in estimating the slope.
The variability of the data might make that its
slope more uncertain than those other data sets,
but it still can make a legitimate contribution to
the overall average slope, if weighted
appropriately to its uncertainty. Besides, the
variability of this set is not particularly great
compared to general variability found in many
toxicological evaluations, including the data sets
of West et al. used in the commenter’s
Appendix A. Excluding one low point, the
range of the data is only a factor of two once the
data are adjusted for pH. Again, Appendix III
includes examples that show that the overall
certainty of the slope estimate will be improved
by inclusion of such data sets.
This comment finally asserts that the data sets of
West et al. (actually finally published as Arthur
et al.) and of DeGraeve et al. constitute
acceptable sets and refers to analysis in
Appendix A which proposes temperature
relationships based on this data (although oddly
the fathead data from DeGraeve Ct al. is not
used in this analysis). This Appendix is
critiqued below. Briefly, this analysis does not
provide appropriate data selection or treatment,
and does not demonstrate a temperature
relationship with nonzero slope is justified for
acute toxicity to fish. Additionally, the assertion
that an acute adjustment automatically
establishes the same chronic adjustment is
unfounded. If the commenter asserts that the
data of DeGraeve et al. establishes a significant
acute relationship that warrants relief at low
temperature, then to be consistent the
commenter should also accept that the chronic
data of DeGraeve Ct al. indicate a need for a
more restrictive relationship for chronic toxicity.
Comment C-39
Fathead minnow (ident fled as a sensitive
species) repeatedly has been confirmed in

acute tests to tolerate higher total ammonia
levels at lower temperatures. (See, Thurston,
DeGraeve, and West) (DeGraeve ‘s chronic
study offathead minnow did not counter this
position as two critical test results (15 and
20°C) did not produce an EC2O endpoint.) The
available minnow data would support afactor
of 1.5 to 2.0 increase in the total ammonia-
based criteria as temperatures decrease to
EPA does not agree. The comment cites
Thurston’s work as demonstrating effects of
temperature, yet when pH effects are accounted
for (as the commenter earlier noted how
important confounding effects might be), the
slope of the data is virtually zero. This
comment is also inconsistent with earlier
assertions that this study did not have a
significant enough temperature range.
DeGraeve’s data shows a slope for fathead
minnows, but it is at most a factor of 1.25 over a
25°C range (not 1.5 to 2.0), and statistical tests
do not show that this is significant. West’s data
(published as Arthur et al.) is caveated by its
own authors regarding a variety of confounding
factors that might be responsible for the effects.
The claim that the temperature effect is a factor
of 1.5 to 2.0 rests almost solely on a single test
in Arthur et al. at extremely low temperature
which even the commenter acknowledges (in
Appendix A) might inappropriately skew the
EPA does not agree with the assertion that the
chronic data of DeGraeve does not counter the
commenter’s position. The fact that the tests at
15 and 20°C did not produce results does not
prevent at comparison of the high (25 and
30°C) and low (6 and 10°C) temperature
results. If the comment is correct that fathead
minnows are more tolerant at low temperatures,
this effect should be apparent in this data,
regardless of the 15 and 20°C tests. In fact, the
apparent effect is in the opposite direction, and
EPA does not agree that an objective analysis
can ignore this data.
In fact, the temperature trend lines for both
acute and chronic toxicity are not statistically
significant and the Update treated both as no
effect. Interestingly, the acute-chronic ratios do
show more of an effect of temperature than
either the acute or chronic data alone, reflecting
the opposite trends of the acute and chronic
Comment C-40
EPA ‘s conclusion that temperature effects need
to be “sufficiently large” to be included in the
criteria derivation is not an appropriate basis
for decision-making. The 1984 ammonia
criteria included a temperature effect even
though it was relatively small (about afactor of
1.5). This amount of criteria adjustment is also
the same range of effect encountered in
dissolved versus total recoverable metals
criteria. This level of increase is very
sign /lcant to municipal dischargers and could
easily mean the d /Jerence between the need to
construct nitr /Ication facilities and not having
to do so (e.g, permit limit of 10 mg/I versus 15
mg/i in the winter). Because conservative
assumptions have a multiplicative effect in the
calculation ofpermit limits, it is essential that
all relevant adjustments be included regardless
of how small they seem individually
EPA did not conclude that temperature effects
need to be “sufficiently large” to be included in
the criteria derivation. The criteria for inclusion
was whether a statistically significant effect
could be demonstrated. At one point, EPA used
the phrase “not particularly large” to simply
describe in general terms the temperature
dependence of some data sets, but this clearly
did not relate to any decision criteria. The
Update also noted the size of possible
temperature effects when uncertainties
represented by the adopted relationship were
discussed, but again this was not the basis for
any decisions. It should finally be noted that the
temperature dependence for total ammonia in
the 1984 criteria was at most a factor of 1.2 (not

any decisions. It should finally be noted that the
temperature dependence for total ammonia in
the 1984 criteria was at most a factor of 1.2 (not
1.5) over the temperature range from zero to the
TCAP, and usually less.
Nevertheless, in response to comments EPA has
re-evaluated the invertebrate temperature
dependence, and has for 1999 produced a
temperature dependent chronic criterion.
Comment C-4 1
The conclusion that the criteria should not be
temperature dependent is based upon studies
of fishes. The Update acknowledges that
invertebrates do no: follow this pattern and
become less sensitive as temperature decreases
(i.e., afixed un-ionized ammonia level appears
appropriate for invertebrates). The manifest
problem with the Update is that two
invertebrates not fishes, controlled the chronic
criteria derivation. Thus, using the acute fish
data to claim that the invertebrate-driven
chronic criteria could not be made less
stringent as temperature decreases is plainly
erroneous. The fish and invertebrate data sets
should have included the relevant adjustments
in calculating the criteria.
EPA agrees that this comment raises legitimate
concerns about temperature dependence. The
available acute data for invertebrates do indicate
decreased sensitivity at low temperature and the
1998 Update recognized this. The 1998 Update
did not “claim that the invertebrate-driven
chronic criteria could not be made less stringent
as temperature decreases”, but rather clearly
indicated the need to make modifications at
lower temperatures and provided a policy
statement encouraging this.
Nevertheless, for the 1999 revision of the
Update, EPA recognized the need for a more
thorough analysis of the available data, and has
in fact generated a temperature-dependent CCC,
based in part on the invertebrate temperature
Comment C-42
Appendix A [ of the commenter ‘s submission]
contains a reevaluation of the relevant chronic
criteria test results for the four most sensitive
species, utilizing the available data on
temperature impacts for the most sensitive
organisms. That analysis confirms that the
recommended criteria should be adjusted for
temperature between 25 - 15°C as the
sensitivity of invertebrates to ammonia changes
sign flcantly over this range. For example,
total ammonia sensitivity is expected to change
by afactor of 3.3 over this range for the “most
sensitive” amphipod (Hyalella) (assuming us
used in the criteria derivation). At 15°C,
Hyalella is not even among the four most
sensitive species.
Based upon a revised analysis that properly
applies the temperature effect to the relevant
species and uses a pH dependency based on the
acute relationship, the total ammonia (as N)
chronic criteria (N = 12, ELS present) is best
described as follows:
8. Osu
7. Ssu
3.1 mg/I
7.4 mg/i
3.5 mg/i
8.5 mg/i
This analysis presumes that the questionable
Hyalella data remain in the database. Thus,
the above recalculation of the criteria should
be considered conservative.
In summary, the acute and chronic criteria,
expressed as total ammonia, should increase as
temperature decreases. The acute criteria
should be adjusted upward at low temperatures
by at least a factor of 2.0 (between 25 and
5°C), consistent with the 1984 criteria analysis
and the most recent studies. The chronic
criteria should be increased even more
(without considering the early i fe stage issue)
because invertebrates are much less sensitive
as temperature decreases (factor of 7 between
25-0°C). Making all of these adjustments that
are supported by the data would substantially

increase the base criteria (25°C, pH 8.0, ELS
present) under cold weather conditions even
ELS are present.
Conceptually, this analysis has merit. It
incorporates some of the considerations that the
1998 Update suggested for low temperature,
but includes other considerations as well that
make it applicable to all temperatures.
However, this analysis also has many
shortcomings and questionable features, which
will be discussed in detail subsequently.
Consequently, EPA does not agree that the
values presented in the comment are appropriate
for its national criteria.
Comment C-43
[ The commenter ‘5] normalized data plots
include a trend line in Cartesian coordinates,
determined by least squares regression
The commenter’s regression model presented
on his page A-2 is appropriate and is the same
used by the EPA in the Update. The use of
least-squares linear regression with this model
(after log transforming it) and dividing by the
estimated LC5O at 20°C to normalize the data
after the initial individual regressions are also
appropriate. Nevertheless, EPA notes the
following concerns:
(a) No mention is made of the statistical
significance of these regressions. Datasets with
so few data will often have a substantial mean
trend just from random variation. Appropriate
statistical tests are needed tà determine if the
mean trend is not attributable to random chance.
(b) After conducting individual linear
regressions of logLC5O versus temperature and
normalizing the data based on these regressions,
the commenter plotted data on linear axes and
the regression of the normalized data is
conducted of LC5O versus temperature, not
logLC5O versus temperature. This is yields
certain problems. First, it violates the
assumption of homogeneity of variances
required in least squares regression. In the
absence of information to the contrary, the
dependent variable should be logLC5O.
Second, the regression to determine a common
slope for this data should be run on
unnormalized data using standard pooled
regression technique. Normalizing data based
on initial regressions and then running another
regression which builds on the earlier
regressions can increase error. Third, a linear
relationship of LC5O versus temperature would
not seem to be a reasonable a priori assumption.
Temperature relationships in biology are more
commonly power functions such as used in the
individual regressions. Furthermore, a linear
relationship of LC5O versus temperature here
results in very low LC5Os just a couple of
degrees above the 25°C, going to negative
LC5Os at 29°C and above. This raises
questions as to the appropriateness of the use of
the arithmetic scale for LC5Os. The arithmetic
scale causes a steep two-fold change in LC5O
between 20 and 25°C. Nevertheless, despite
EPA’s preference for the log scale for the
LC5Os, EPA does recognize that within the
range of values of the observed data the linear
scale does provide a reasonably good fit for the
Comment C-44
[ The commenter ‘s] trend line for [ three]
invertebrate [ species] shows sensitivity
decreased by a factor of six from a temperature
of 25°C to approximately.5°C.
This and other data do in fact make a case for
acute sensitivity to invertebrates becoming
greater with increasing temperature, and was so
acknowledged in the 1998 Update. As noted in
the previous comment, however, EPA believes
a regression with log LC5O is better a better
approach than with LC5O (arithmetic scale.).
Also, the regression analysis to determine the
mean trend should more properly be conducted
on the un-normalized data. (Normalization

should be just a method for better displaying the
data.) The regression analysis should also take
into account known uncertainties in the LC5O
estimation if at all possible, not relying just on
residual errors, which can underestimate true
Beyond the regression technicalities, however,
the key issue here is the implicit assumption
used in the Comments that acute toxicity and
chronic toxicity temperature relationships are
the same. In fact, there are good reasons to
expect less decrease in chronic toxicity with
decreasing temperature.
First, in the DeGraeve fathead minnow data, the
slope on a total ammonia basis is -.0052 for
acute (a factor of 1.35 higher at 5°C than at
30°C) but +.0057 for chronic toxicity (a factor
of 1.39 lower at 5°C than at 30°C). The
difference between acute and chronic toxicity
therefore increases by almost a factor of two
over 25°C. Importantly, even though separately
the trends in the acute and chronic data cannot
be said to be different from zero with 95%
confidence, the acute and chronic slopes can be
said to differ from each other with 95%
Second, that acute toxicity is more protected by
low temperatures than chronic toxicity is
expected based on the physiology of toxicity.
Part (not all) of the effect of temperature will
simply be to slow various processes down,
delaying (beyond the end of the test) rather than
eliminating toxicity. Such an effect will be
greater for short duration tests,and thus most
evident in acute LC5Os. For these reasons,
simply applying acute temperature relationships
to chronic toxicity is questionable. Therefore,
although EPA believes that invertebrates will-be
more tolerant on a chronic basis at lower
temperatures, it does not believe that the trend is
quite a strong as indicated in the commenter’s
Figure 9.
For the 1999 Update EPA used log LC versus
temperature plots, and projected the chronic
invertebrate temperature slope to be the
invertebrate acute slope minus the fathead
minnow ACR temperature slope. (In general,
for any data set the acute slope minus the ACR
slope will equal the chronic slope.) In doing
that, the key assumption EPA is making is that
the ACR slopes for fish and invertebrates are
the same. The ACR is mathematically or
numerically related to the kinetic coefficient
describing the rate at which toxicity occurs.
Kinetic coefficients vary with temperature;
chemical texts often present a “rule of thumb”
for estimating how a 10°C temperature change
will affect rates of a variety of chemical
processes. Equating the fathead minnow and
invertebrate ACR versus temperature slopes is
thus akin to using the measured fathead minnow
ACR slope for defining the rule of thumb on
how temperature affects the relevant toxico-
kinetics for a variety of species.
Comment C-45
The [ commenter ‘ 5] trend line for vertebrates
exhibited reduced temperature sensitivity as
compared to invertebrate data, and the results
were variable for various test species. Of the
five test species, only Pimephales exhibited a
large desensitivity at low temperature. The
effect is relatively small when Pimephales is
The comments do not report on the statistical
significance of the slope in Figure 10.
Additionally, this slope is affected by regressing
LC5O rather than log LC5O against temperature,
because points are not necessarily weighted
appropriately. In any event, of these five fish
species, four show essentially a flat relationship
and one shows a relationship that depends
almost entirely on one point at low temperature.
Without that point, the pooled relationship is flat
and with that point the relationship is still not
statistically significant, with just the one large
residual at low temperature suggesting the
possibility of a temperature effect.

Comment C-46
DeGraeve et at. (1987) studies on channel
ca JIsh suggest that this species exhibits a two-
fold decrease in ammonia sensitivity as
temperature decreases from 30°C to 0°C.
EPA does not agree. First, there is no analysis
or presentation to demonstrate that there is a
significant effect of temperature in this data,
other to assert a factor of two. Second, the
comments do not specify the form and slope for
whatever equation they propose to apply to
channel catfish. Third, although the commenter
previously emphasized the data of West/Arthur
et al., the comments omit mention that
West/Arthur’s slope for channel catfish is
virtually zero in the figure appearing in the
comments. Fourth, the comment ignores the
fact that, for fathead minnow, the DeGraeve
study also had a slight negative slope with
temperature for acute toxicity, but a positive
slope for chronic toxicity. EPA does not see any
justification for applying the acute channel
catfish temperature relationship to chronic
Comment C-47
[ In adjusting for the influence of temperature
on invertebrate sensitivity, the commenter
made no adjustment for life stage differences.]
EPA agrees that no adjustment for life stage or
endpoints is needed. Regarding the two most
sensitive invertebrates, the endpoints and
lifestages tested are arguably relevant to much
lower temperatures. For the fingernail clam, the
tests were of juvenile survival, which would be
of concern for lower temperature seasons. For
Hyalella, the most sensitive endpoint was
reproduction, but the effect concentration used
was also an EC2O for survival in ten week tests
starting with juveniles, and six week tests
starting with adults showed similar mortalities
to juveniles at higher concentrations. Thus the
effect concentration does relate to endpoints and
life stages relevant to lower temperatures. This
does not eliminate the need.to correct for any
effect of temperature on these chronic
endpoints, but it does establish a basis for
saying that a life stage correction is not
Comment C-48
The commenter has offered an analysis, which
EPA has summarized below, including a few
inferences where procedures were not clearly
[ (a) For all temperatures, invertebrate toxicity
was adjusted for temperature using the
relationship in Figure 9. Based on table
values, slope is apparently -0.34 mgJL/°Cfor
Hyalella, -0.39 mg/L/°Cfor Musculium, the
steepness increasing with increasing
[ (b) For temperatures for ELS present, fish
chronic toxicity (except for channel ca flsh)
was adjusted using the relationship in Figure
10 with the low temperature fathead minnow
point excluded Based on table values, slope is
apparently -0.080 mg/LI° C for Lepomis,
-0.0092 mg/L/° Cfor Pimephales, the steepness
increasing with increasing tolerance.]
1(c) Channel ca flsh chronic toxicity was
adjusted by an incompletely spec /Ied
temperature relationship. Based on table
values, relationship is of log LC5O vs
temperature with slope of -0.0101°C.]
1(d) When early fish life stages are absent, life
stage adjustments included setting the bluegill
chronic value was set to 15 mg NIL, not
adjusting the channel ca ’/Ish at all, and setting
the chronic value for other fish species to
three-fold their early life stage value. No
temperature relationship was apparently
applied in this temperature range other than
for channel catfish, but the multiplier was
apparently applied to whatever temperatures
the tests were at, not to some common
temperature, which makes these values have

inconsistent ratios relative to the temperature-
dependent values with ELS present.]
[ (e) The border between ELS present and
absent was set at 15° C.]
[ When fish early life stages are present, these
procedures resulted in a chronic criterion at
25°C similar to the Update value, but
increasing at 15°C to slightly more than twice
the Update value. This increase is almost
entirely due to the temperature relationship
assumed for invertebrates, but there is also a
slight effect due to the temperature dependence
assigned to fish.]
[ When fish early life stages are absent, these
procedures result in a chronic criterion at
15°C about three times the Update value and
at 0°C about six times the Update value. These
increases are due to both the assumed
temperature dependence for invertebrates and
the assumed I fe stage dependence for fish. At
15° C, these criteria match the three-fold
relaxation suggested in the Federal Register,
and are about twice as high at 0° C.]
The fundamental outline of the framework is
reasonable. Rules are adopted for the
temperature dependence of different endpoints
and for how relevant endpoints shift among
different temperature ranges. These rules are
used to calculate a set of chronic values at each
temperature, which in turn are used to calculate
the chronic criterion at that temperature. An
abrupt change occurs at the transition
temperature for ELS presence.
However, as already discussed, many of the
rules or relationships applied by the Comments
are of questionable validity. The data do not
support using any temperature adjustment for
chronic endpoints for fish. The temperature
relationship for chronic toxicity to invertebrates
is likely exaggerated due to the use of a
questionable regression model and analysis, and
not considering the applicability of acute
temperature relationships to chronic toxicity.
The estimated LC2O for chronic juvenile
bluegill survival is likely high. EPA believes
that with more reasonable relationships, the
increases in criteria at lower temperatures
would be less at most temperatures.
One particularly important issue is the transition
temperature between fish early life stages being
present and absent. The comments present no
justification in terms of fisheries biology for the
selection of 15°C, and EPA does not know of a
rationale for this type of selection. For this
reason the 1999 Update does not assume a fixed
temperature threshold for delineating the
presence or absence of fish ELS nationwide.
Even among warm water fisheries this
temperature may vary somewhat, depending on
Comment C-49
The National Guidelines require the
calculation of non-ELS criteria given that the
basic assumptions used to derive the criteria
are inapplicable during the winter. Hyalella
andfingernail clam data tested at 25°C, which
drove the chronic criteria calculation, are
clearly not applicable to low receiving water
EPA does not agree with this interpretation of
the National Guidelines. The National
Guidelines specif that chronic criteria should
be based on tests which include early life stages,
but do not indicate that such criteria should be
modified when early life stages are not present.
Nevertheless, EPA agrees that the ammonia
chronic criterion can and should take into
account this factor. The 1998 and 1999
Updates do provide for less restrictive criteria at
low temperatures.
Comment C-50
The applicability of mod fled criteria should
not require a determination that no fish spawn
in the winter First, fish did not drive the

chronic criteria calculations. Second, f non-
sensitive species spawn in significant numbers
in winter months, the available data confirm
that less restrictive criteria should apply.
EPA agrees that presence or absence of early
fish life stages should not be the only factor
affecting temperature-related adjustments. EPA
has made changes such that the 1999 Update’s
CCC provides for a temperature dependence
even when ELS are present.
Comment C-Si
States should not be prevented from adopting
reasonable winter criteria based upon different
interpretations of the data (which EPA readily
admitted was incomplete). The Update
indicated that up to a seven-fold increase in the
criteria was supported by the test results to
sensitive fish under low temperatures.
EPA’s criteria documents present EPA’s
recommended criterion. They do not set a
binding norm. They do not prohibit other
alternatives or define the acceptable range for
such alternatives. In fact, the information
provided in the 1998 and 1999 Updates was
intended to assist in efforts to develop and
evaluate alternatives.
With regard to the second sentence above, the
Update did not indicate that such an increase in
criteria was supported by the cited test results.
Although the 1999 Update does not estimate
any fish GMCV below 8.8 mg NIL when ELS
are absent, the temperature-adjusted Hyalella
GMCV remains well below this value.
Furthermore, the National Guidelines generally
set the criterion below the lowest GMCV in this
sized data set, as the commenter recognizes in
Comment C-48.
Comment C-52
EPA should not place the burden of resolving
criteria issues on states and municipal entities
when EPA itseiffailed to conduct sufficient
research to resolve the ammonia winter criteria
issue (as it committed to do 14 years ago). The
Agency ‘s implementation policy constitutes an
“unfunded mandate “for municipal entities to
conduct long overdue federal research. Such a
mandate must be reviewed pursuant to the
federal Unfunded Mandates legislation.
EPA ‘s attempt to provide relief under winter
conditions is, for all practical purposes, no
relief at alL To allow any adjustment, a
comprehensive biological study would be
required, which is beyond the means of many
small municipal entities. Rigorous analysis of
theoretical impacts should not be required
where it is apparent that (1) the data in the
criteria document fully support less restrictive
criteria, (2) the receiving water is generally
incapable of sustaining sensitive aquatic life
(e.g., seasonal ammonia discharge from a
small lagoon to an intermittent stream), or (3)
sensitive life stages will not exist.
Because the Unfunded Mandates Reform Act
(UMRA) applies only to rules and not to
guidance documents, the UMRA does not apply
The comment does raise legitimate issues about
how much biological information should be
needed under different circumstances. For the
1999 Update EPA has dropped the
recommendations about follow-up biological
surveys, although EPA continues to encourage
States and Tribes to use biological surveys for
water quality assessment, including that for
water receiving ammonia discharges.
Nevertheless, because the criterion has different
values when fish ELS are present and absent,
States and Tribes should reasonably be able to
define these periods in order to appropriately
use this provision. EPA believes that it is more
efficient to approach this on an eco-region-wide
basis rather than a site-specific basis.

In the Federal Register Notice announcing its
1999 Update EPA is providing additional
implementation guidance on this issue. EPA is
not expecting States, Tribes, or municipalities to
perform intricate analyses of voluminous data.
EPA believes that much of the needed
information on spawning temperatures or
periods and subsequent development periods is
already known by state fisheries biologists.
EPA favors a straightforward approach using
available data and expert opinion.
It should be noted that the 1998 Update
recommended a 200 percent increase in the
chronic criterion concentration during cold-
season periods with fish ELS absent. In
contrast, the 1999 Update, because the
temperature dependency was built into its fish
ELS-present formulation, provides no more than
an additional 62 percent increase when fish ELS
are absent. At temperatures greater than 7°C
the percentage increase is less, and at
temperatures greater than 14.5°C, the criteria
concentrations for fish ELS present and absent
are identical.
Consequently, for properly implementing the
fish ELS provision, it is most important that
States and Tribes not misclassif ’ fisheries with
fall- or winter-spawning salmonids as warm-
water fisheries. Of intermediate importance is
the proper identification of temperatures or
dates for the onset of spawning for cool-water
spawning fish. Relatively least important are
the temperatures or dates for spawning of
warm-water spawning fish, because the
uncertainties here have relatively the least
potential for substantially affecting the pollution
control decision.
Comment C-53
In this draft Update, EPA confirms that thirty-
day averaging is acceptable so long as the
four-day average value is not more than two
times the thirty-day value. In reaching this
conclusion, EPA relied upon a ‘/Ield study”
conducted by the Agency’s Monticello
Laboratory (actually art ylcial streams) with
variable pH and temperature conditions, as
well as limited laboratory data
The Monticello data (now appearing in
Appendix 8 of the 1999 Update) were not used
to set the four-day averaging period or how
much higher the criterion could be for that
averaging period. The Monticello data were
used to determine at what concentrations,
relative to criterion concentrations, effects were
observed in these streams. As such, the
averaging periods used in the criterion were
used in summarizing the stream data to provide
an appropriate basis for comparison. The
Monticello data were cited as support that the 4-
day 2.0-2.5 x CCC provision was desirable, but
was not the basis for setting the values for this
provision. Rather, the basis for this provision is
discussed in the 1998 or 1999 Update section
titled “CCC Averaging Period” or “Chronic
Averaging Period.”
Comment C-54
Based upon this information, EPA indicated
that seven-day averages could be 2.5 times the
thirty-day no effect level without causing
demonstrable impacts but, for “consistency,”
EPA lowered both the averaging period (to
four days) and the acceptable variability (to
2.0). (Update @ 78- 79.)
See the response to Comment C-09.
The basic issue here is that averaging periods
cannot be equated with test periods, which
merits some further explanation to help
responses to this arid later comments.
Several general properties of laboratory toxicity
tests and results should be recognized when
considering how to apply them to field data and
how to set averaging periods. First, laboratory
tests are generally conducted under fairly stable
conditions with regard to toxicant concentration
and other environmental variables, at least
relative to typical field conditions. Second, tests

are also generally conducted for fixed, standard
test periods and the endpoint is expressed
relative to the end of that period. Third, many
studies have demonstrated that if concentrations
fluctuate significantly during the test period,
effects are generally greater than if
concentrations are held constant at the same
mean value as the fluctuating concentrations.
Fourth, many endpoints do not need the entire
test period to be affected, so that the test could
be much shorter and still elicit the endpoint and
result in the same effects concentration. Fifth,
when tests of different lengths are run, effects
concentration do not vary in inverse proportion
to the test length; for example, if a 96-hr LC5O
is 1 mg/L, for most toxicants the 24-hr LC5O
would not be four-times the 96-hr LC5O, but
generally much less, and in some cases no
different than the 96-hr LC5O.
The length of toxicity tests therefore should not
be equated to an appropriate averaging period
for concentrations derived from that test, simply
because an averaging period equal to the length
of the tests allows for concentration fluctuations
which can elicit greater effects than the
laboratory test, while still having the same
average concentration as the laboratory test.
For example, if a 96-hr LC5O is I mg/L,
concentrations averaged over this period could
fall below I mgfL, but contain periods with
much higher concentrations, which would elicit
greater effects than intended. The averaging
period should be set small enough so that
concentration fluctuations expected within the
averaging period are not great enough to cause
undesirable effects. However, averaging
periods should also not be made smaller than
necessary, because they will restrict the long-
term mean exposure, which should not be lower
than necessary.
Another aspect of averaging periods that must
be considered when comparing them to toxicity
test lengths is that toxicity tests are for isolated
exposures - with a beginning and an end -
whereas field exposures typically are not. An
averaging period does not circumscribe an
isolated exposure, but rather pertains to the
worst exposure in a longer time period, which
would generally be preceded and followed by
exposures below, but still near, the criterion
concentration. Thus, in the example above, if
the averaging period is made to be 24-hr rather
than 96-hr, this is not the same as an isolated
24-hr exposure, but of an exposure that
averages 1 mg/L over the worst 24-hr period
and probably is a substantial fraction of I mgfL
for a much longer period.
It should finally be noted that the derivation of
an alternative averaging period is acknowledged
by the Guidelines, although they do not set forth
specific procedures for deriving either the
default national value or alternative values for
the averaging period. In its 1998 and 1999
Updates EPA is recommending a chronic
averaging period 7.5 times the default
Guidelines value. For the usual four-day
averaging period they recommend a criteria
concentration 2 or 2.5 times greater (in 1998
and 1999 respectively) than the CCC obtained
through the Guidelines.
Comment C-55
Municipal faci lities are required to receive
thirty-day and seven-day average permit limits
unless it is impracticable to derive such limits
(see, 40 CFR § 122.45(d); accord, In Re: City
ofAmes, Iowa, NPDES Appeal No. 94-6, April
4, 1996). Thus, use ofa four-day average
approach is not consistent with applicable
NPDES rules.
EPA does not agree that the criteria averaging
period is to be delimited by the permit averaging
period. NPDES rules are not germane here.
The averaging period of criteria is and should be
set for biological reasons, and can be and have
been appropriately translated to other periods
under various flow situations. The Technical
Support Document for Water Quality-based
Toxics Control covers this subject area. In
addition, EPA can provide individual technical
assistance to states and tribes that have

questions about appropriate translation
Comment C-56
When long-term life cycle tests (sixty days or
greater) are used to calculate “no effect”
levels and then set as maximum thirty-day
exposures, a safety factor is built into the
criteria derivation process. (See, Update
discussion @ 75 that Diamond’s 21-day and
14-day test results under-predict chronic
impacts because longer exposures cause
greater impacts for equivalent concentrations.
This is basic dose/response toxicology.)
Borgmann ‘ 5 Hyalella chronic toxicity study
indicated that the four-week EC5O
concentration was approximately nine times
higher than the ten-week results. Adding a
further variability limitation without
quantifying the safety factor already
incorporated into the thirty-day average
chronic criteria is inappropriate.
While it is true that effect concentrations will
generally decrease with duration, EPA disagrees
with the comment in several ways.
(a) The comparison using Borgmann’s data is
inappropriate. The four-week LC5O is only
slightly higher than the ten-week LC5O (0.95
versus 0.77 mM). They are both much higher
than either the LC20 or the EC5O for
reproduction, but those differences are a matter
of endpoint, not duration of exposure.
Therefore the cited factor is not germane to the
issue at hand.
(b) Few of the tests in the chronic database in
the Update are of durations of 60 days or
longer, although the two most sensitive
organisms have tests with this duration.
(c) The ammonia chronic averaging factors do
not necessarily, or even likely, impose
significant safety factors. Reducing averaging
periods will reduce long-term allowed
concentrations, but this does not necessarily
provide a margin of safety with respect to the
actual desired level of effect . In fact, as
explained above, when concentrations fluctuate
substantially, the averaging period needs to be
smaller than the test period to prevent greater
effects than are represented by the concentration
derived from the laboratory tests. In this regard,
a 30-day period relative to a 60-day period is
not a large restriction. If concentrations do not
fluctuate enough to be of toxicological concern,
the issue is relatively moot, since 30-day
averages will be near the 60-day averages, so
again there is no significant safety factor. In
fact, if exposures are relatively constant beyond
60 days, effects could possibly be even greater
than intended because of the “basic
dose/response toxicology” noted in this
comment, and a 30-day averaging period
provides no margin of safety at all.
Comment C-57
EPA has repeatedly stated that field results
should not be used to generate national criteria
because of the uncertainty associated with the
actual exposures, variable pH, and other
critical water quality factors (e.g., dissolved
oxygen, ‘temperature, predation, disease, etc.)
that are impossible to accurately quant fr.
Relying on the Monticello laboratory result to
generate criteria is inconsistent with EPA ‘s
own conclusion that such data are inherently
EPA does not agree. In fact, the.Güidelines do
provide for consideration of field data, and such
information has been used to set criteria,
although the Update repeatedly states that
Monticello data were not directly used because
they were field data. Care must be taken in
applying such data for the reasons given in this
comment, but EPA has not made any absolute
policy or conclusion as asserted here. Such an
absolute policy would be irresponsible, since it
would preclude use of data which could be
relevant. Furthermore, as previously stated, the
Monticello data were not used to set the
averaging period. They were mentioned in the

section regarding the chronic averaging period,
but only as part of various data that might
reflect on the consequences of different
averaging periods. The averaging period and
factor were based on certain laboratory data.
Comment C-58
National criteria recommendations are only
supposed to be developed when sufficient
reliable information is available. The
Averaging Period section of the Update is
replete with statements that insufficient data
are available to provide a defensible short term
averaging period recommendation (“Rigorous
definition of this excursion restriction is not
possible with the limited data available.”
(Update @ 78)). This weakly supported
national criteria recommendation should
therefore be withdrawn.
EPA does not agree. The Update did recognize
where uncertainties exist and where conclusions
about averaging period varied among different
studies. This is only proper, because any
actions taken should be evaluated in terms of
possible uncertainties. However, just because
uncertainty is present does not mean that some
action is not justified. The quoted sentence
should be judged in terms of the context in
which it was made, which was after
summarizing the 7-day fathead minnow tests
which showed effect concentrations 2.5-fold
higher than the 30-day tests. The Update noted
that these tests do provide the best indications of
how much excursion above the 30-day test
results might be permissible. However, the
Update also noted that excursions of the
exposure concentration should be restricted to
preclude eliciting effects based on these 7-day
tests. The quoted sentence merely pointed out
that an exact, rigorous definition was not
possible, because data for variable exposures
within such 7-day tests are not available. But
this uncertainty is not so great as to preclude
y steps. It is certain, at least in the context of
these tests, that the restrictions should be more
stringent than using a 7-day averaging period
with a 2.5 fold concentration factor, even if how
much more so cannot be rigorously specified. It
is also reasonably certain that the 4-day average
with no factor specified by the Guidelines is
unduly restrictive. This leads to a final point
that should be made. If EPA considered the
uncertainty in this restriction too great, EPA
would not proceed with recommending the 30-
day average period. EPA would not
recommend the 30-day averaging period
without corollary considerations of a shorter
averaging period.
Comment C-59
EPA makes no statement regarding the
appropriate stream design flow to use f a
steady state approach is employed in permit
development. This is a major departure from
all prior criteria documents that included a
steady state flow recommendation. In light of
historical (mis)statements that the proper
design flow for applying chronic ammonia
criteria is a 7/QuO flow, this issue must be
addressed in the Update to avoid widespread
misapplication of the new criteria.
A thirty-day once in three year exceedance
frequency is equivalent to allowing insiream
concentrations to be above the criteria 2.8
percent of the time. EPA’s 1991 TSD
recognizes that the design flow must properly
reflect the allowable frequency and duration of
criteria excursions. (See, 1991 TSD 79.
“The design flows used in steady state
modeling should be reflective of the CCC and
CMC durations and frequencies. “) Based
upon derivation of “never-to-exceed” permit
limitations (i.e., compliance is assured on a 99
percentile basis), there is no need to include
further safety factors in selection of an
appropriate chronic criteria design flow
because compliance with the permit assures
that instream concentrations will never be
greater than the criteria, as long as stream
flows exceed the selected dilution flow. A
30/Q/3 flow may be used because, by definition,
a flow lower than this flow does not occur more
frequently than once in three years or 2.8

percent of the time. (See, Exhibit 5, EPA
opinion that for ammonia, at least a 30/QuO
flow should be used to apply the chronic
ammonia criteria.)
Given the known safety factors that must be
included in treatment plant design (e g., any
ammonia limit less than 10 mg/I essentially
requfres construction offull-scale nitr /Ication,
resulting in actual effluent quality of one-half
to one-tenth of the allowable effluent quality),
there is no reasonable basis to claim that a
7/QuO design flow should be selected for
chronic criteria application in steady state
model applications. It must be noted that plant
performance under drought conditions is
optimum (i.e., well below permitted levels) due
to stable and reduced treatment plant flows,
regardless of the duration of the low flow event.
Therefore, the Update should recognize that
proper application of the thirty-day chronic
criteria requires use of a 30/Q/3 flow, unless
spec j/Ic information on the discharge indicates
that a more frequent exceedance is likely to
occur considering all relevant factors (e.g.,
plant performance under drought flow
conditions, highly variable instream
conditions, or intermittent discharge concerns).
EPA agrees that it is appropriate clarify the
appropriate design flow, in order to reduce
confusion about the implications of the 30-day
averaging period. If a design flow approach is
used, then the either a 30Q5 or a seasonal flow
exceeded 95 percent of the time is appropriate.
EPA does not agree with the comment that a
30-day once-in-three-year exceedance goal
ordinarily means 30/(365 x 3) = 0.028 = 2.8%
excursion frequency. In time series having a
realistic degree of serial correlation, which
indicates the degree of smoothness in the day-
to-day changes in concentration, the allowance
excursion frequency is somewhat higher than
this. Analysis of long time series indicates for if
the correlation coefficient between the logs of
daily composite samples is not more than 0.86-
0.94, an observed range for samples from larger
rivers, and the log standard deviation of grab or
composite samples is not more than 0.5-0.8,
also an observed range for ambient samples,
then the 30-day once-in-three-year goal actually
allows 24-hour composite samples to exceed the
criterion approximately 5 percent of the time.
This assumes that the criteria exceedances are
counted in the manner used in the 1986
Technical Guidance Manual for Performing
Waste Load Allocations, Book VI Design
Conditions, Chapter 1 Stream Design Flow for
Steady State Modeling.
The 2.8% frequency presented in the comment
might be valid if concentrations changed as 30-
day wide step functions. However, in realistic
time series, many excursions of the criterion
concentration are not of sufficient duration and
magnitude to cause the 30-day average to
It is also worth noting that the protection of 95
percent of the species 95 percent of the time is a
commonly used goal in ecological risk
assessment and management, and is the
recommendation of the 1998 SETAC expert
workshop Reevaluation of the State of the
Science for Water Quality Criteria
While EPA recognizes that design flows are
commonly used in deriving effluent limits, it
should be noted that flow is only one of the
important parameters. For the 1999 ammonia
criterion, temperature and pH are also
important. If a design condition approach is
used, then appropriate seasonal values for these
parameters also need to be selected.
Time-variable modeling is a better way of
dealing multi-parameter variability and
correlation. Where it is important to find the
most cost-effective alternative for protecting the
aquatic life use, then it is appropriate to account
for seasonality and day-to-day fluctuations of
effluent quality, streamfiow, and pH, and the

seasonality of temperature and fish early life
stage presence/absence.
Comment C-60
EPA also continues to recommend that a
stringent one-hour averaging period is
necessary for application of acute criteria even
though the acute criteria derivation has no
relationship to a one-hour exposure period.
Acute criteria are based on 96 hour no
mortality test concentrations. No information
presented in the Update supports the needfor a
one hour application of a 96 hour “safe” level.
The stringent one-hour averaging period
recommendation continues to confuse state
authorities that develop NPDES permits (e.g.,
one California Regional Board has begun to
include “one-hour” and “instantaneous
maximum “permit limits to implement EPA ‘ S
acute criteria recommendations; Iowa, Kansas,
and other states have implemented arbitrarily
restrictive mixing zone policies even on small
streams (ten feet wide) that completely mix
rapidly; Minnesota regulations preclude
greater than 1:1 mixing, even on the
Mississippi River, to address acute toxicity
“threats”). All of these policies refer to the
‘fast acting toxicant” assumption that was the
basis for asserting a one-hour averaging
period was needed. Because there is no
technical basis for this recommendation, it
should be withdrawn and replaced with an
appropriate averaging period that is
reasonably related to the criteria derivation
and not unduly conservative (e.g., 24-hour
averaging period).
EPA notes that the comment did not cite any
information indicating that ammonia was not
fast-acting in acute exposures. EPA is receptive
to evaluating any such information.
The 1-hour averaging period was not addressed
in the Update because of resource limitations
and because the previous ammonia document
had already noted that ammonia is fast acting,
so that it was not an issue likely to resuJt in large
departure from the Guidelines values.
Furthermore, as stated for other issues above,
the criteria are meant to provide an expression
of what exposures are of biological concern.
Although EPA is aware that misapplications of
criteria provisions may occur, as part of this
project it is not prepared to address all such
implementation issues. If these exposures are
misapplied or misinterpreted, the remedy should
not be to provide a less toxicologically
appropriate expression of exposure, but rather
to improve implementation. Averaging periods
should not be considered as the sole determining
factor in mixing zones, but one of several
factors that determine mixing policy intended to
provide a desired level of protection. This is a
valid issue, but not one that criteria documents
by themselves can solve.
EPA, nevertheless, does not believe that the
ammonia acute averaging period should be as
long as 24-hours. As discussed above -
regarding the chronic averaging period,
averaging periods often need to be much shorter
than the laboratory test duration because (a) test
endpoints often do not need the entire test
period to occur and (b) variable exposures will
generally elicit greater effects than the relatively
constant exposures typical of Laboratory tests.
For acute ammonia toxicity to most fish, 24-
hour LC5Os typically are the same, or only
slightly above, 96-hour LC5Os. Thus, a 24-hour
period applied to the CMC would still be
vulnerable to greater-than-intended effects from
any fluctuations within that period. In the field,
such fluctuations can be more substantial than in
the laboratory due to heterogeneous exposures
and diel pH variations. That the averaging
period should be much shorter than 24-hours is
evident from several studies:
(1) Ball (1967, Water Research, 1:767-775)
This study showed that LC5Os for rainbow trout
at 24 hours and beyond were the same; i.e., the
threshold LC5O is closely approached within 24
hours. The 3-hr LC5O was only about 50%
higher than the threshold LC5O, the 4-hr LC5O

only 30% higher, the 6-hr LC5O only 20%
higher, and the 12-hr LC5O only 10% higher.
These data do not account for the fact that
mortality from short exposures can be
somewhat delayed beyond the exposure.
Because of this, it is likely that the LC5Os at the
short durations are even closer to the threshold
(2) McCormick et at. (1984, Environmental
Pollution, Series A, 36:147-163) These
workers reported that LC5Os for green sunfish
at 24 hours and beyond also were not
significantly different -- the 24-hr LC5O was 0%
to 15% higher than the 96-hr LC5O, depending
on pH. The 3-hr LC5O was only 6-20% higher
than the 24-hr value, the 6-hr LC5O only 5-15%,
and the 12-hr value only 3-8% higher. Again,
delayed mortality is not accounted for in these
(3) Lloyd (1961, Water and Waste Treatment
Journal, 8:278-279) Relationships reported in
this study indicated that the two-hour LC5O for
rainbow trout is only 50% greater than the
threshold LC5O. Again, delayed mortality is not
accounted for in this relationship, so that the
short-duration LC5Os are probably somewhat
inflated relative to the threshold LC5O.
(4) Thurston et al. (1981, Water Research,
15:991-917) These workers reported that 96-hr
LC5Os for rainbow trout for a pulsed exposure
which alternated 6-hours on/6-hours off were
only 40% higher based on peak concentrations
than for a continuous exposure. Therefore, with
such fluctuations, the average c oncentration
should be <70% of that for a continuous
exposure. The averaging period would have to
be no more than eight hours, and can only be
that long because concentrations within the
pulse are relatively constant.
(5) Bailey et at. (1985, Aquatic Toxicology and
Hazard Assessment, 8th Symposium, ASTM,
pp1 9 3-212) In this study, the threshold LC5O
for bluegill was reached by 24 hours. The 8-hr
LC5O was approximately 50% higher than the
threshold and the 1-, 2-, and 4-hr LC5Os were
approximately threefold higher. This species
therefore showed a slower response than the
aforementioned studies. However, despite this
slower response, pulsed exposures in this study
resulted in LC5Os approximately equal to the
threshold LC5O when based on a two-hour
averaging period and only about twice the
threshold LC5O when based on a one-hour
averaging period.
The continuous exposures among these studies
all suggest that the averaging period for
ammonia should not be longer than a few hours,
even without corrections for delayed mortality;
otherwise, variations within the averaging
period could conceivably be great enough so
that lethal conditions are reached. The Bailey et
at. pulsed exposures furthermore directly
indicate that the averaging period should be no
longer than two hours even for a fish that does
not react particularly fast.
An approach for better quantifying the
averaging period is to take the inverse of the
Mancini (1983, Water Research 10:1355-1362)
kinetic constant. Mancini did report a constant
for ammonia based on the study of Bailey et al.
discussed above. His estimated constant -
(0.1 6/hr) suggests an averaging period of 6
hours, again for an organism that is not
particularly fast-responding and for data which
does not account for all of the delays in
mortality. The 3-hour data from McCormick et
al. indicate that the constant is in the 0.4-1 .0/hr
range, suggesting a 1.0-2.5 hour averaging
period is needed. Again, if delayed mortality
was taken into account, this could be even
shorter. Not all fish respond this fast, but
enough do to indicate that the averaging period
should be comparatively short.
Comment D-0 I
In allowing a threefold increase in the
criterion when ELS are absent, EPA
qualitatively assesses that invertebrates will be
protected during cold water temperatures. The

invertebrate chronic values, however, could be
adj usted for temperature, and the final chronic
value could be recalculated using the
Guidelines. The result is a chronic value
higher than EPA ‘s threefold increase. We
recognize that there is uncertainly in applying
ihe available invertebrate temperature
relationships for chronic toxicity. Did EPA
reject the quantitative approach to the
invertebrate data because of the greater risk
when the criteria become even less stringent.
Or is there sufficient confidence in the
temperature relationship to further increase the
criteria? A discussion of the degree of risk of
using the temperature relationship for
adjusting chronic values would be helpful for
our decision making.
EPA acknowledges the shortcomings of the
qualitative approach of the 1998 Update. For
the 1999 revision, a quantitative approach has
been used. The key uncertainty is the potential
difference between acute and chronic
temperature relationships. Based on kinetic
considerations, which are reflected in the
observed difference between acute and chronic
temperature relationships for fathead minnow,
there is an expectation that temperature
reductions will have less influence on reducing
chronic toxicity than acute toxicity. This is
because it is expected that reducing temperature
causes some reduction in acute toxicity simply
by delaying the toxicity beyond the end of the
96-hour test duration, rather than causing
reduction in long term sensitivity. As discussed
in the 1999 Update, a limited amount of data on
temperature versus ACR can be brought to bear
on the question. See also the response to
Comment D-06.
Comment D-02
In New York State, ELS will be present in April
and perhaps late March when ambient water
temperatures are between 5 and 18 degrees.
Nitr /Ication, however, is difficult or expensive
to accomplish at that time. Accordingly, a
discussion of the invertebrate temperature
relationship should include its potential use
when ELS are present.
EPA agrees and has added temperature
considerations when ELS are present.
Comment D-03
We note that f-fall ‘s comments also present a
temperature relat ions hip for fish. This
relationship appears to have some merit, and
we concur with the comment that the analysis
of Figure 4 of the Update, where the data from
multiple species are combined, can give ala/se
:mpression of no temperature dependency.
EPA does not agree. See responses to
comments C-34 through C-52 (in particular C-
38 and -39) and the discussion of Appendix [ II.
Comment D-04
Perhaps temperature should be restored to the
basic criteria as was the case with the pre-
Update criteria. An EPA discussion and
response would be appreciated.
EPA agrees. The Update has been revised to
incorporate temperature relationships, both for
the case of ELS present and the case of ELS
Comment D-05
It was suggested by Hall and Associates that
the chronic pH relationship should have been
based on the acute model because the acute
model was derived with an extensive data base
and the chronic model was based on only two
species. It was shown by Hall in his comments
that this limited chronic data reasonably fits
the acute model, suggesting that the more
extensively supported acute model is a better
estimate of an applicable model for chronic
effects. An EPA discussion and response would
be appreciated.

EPA does not agree that it should have applied
the acute pH relationship to chronic toxicity,
given the chronic data available for smallmouth
bass and Ceriodaphnia. See the responses to
comments C-24 through C-33, and the
discussion of Appendix 1. Nevertheless,
considering the limited number of available
chronic data points, EPA recognizes chronic
slope would be sensitive to additional toxicity
testing, were it to be done. EPA also recognizes
that there may be some good ways of obtaining
a chronic p 1-I relationship that might use the
extensive acute data base, while still accounting
for the acute-chronic differences indicated by
the smallmouth bass and Ceriodaphnia data.
Finally, EPA recognizes that in the face of
uncertainty, selecting among alternative
approaches is a risk management decision, for
which States retain flexibility.
Comment D-06
Lastly, it appears that ammonia criteria will
continue to contain uncertainty as suggested in
the Update document. Accordingly, EPA
should allow flexibility for varying approaches,
particularly with respect to temperature, when
approving criteria at the state level.
EPA agrees. Nevertheless, EPA must be able
to defend its approval of State and Tribal
standards. Consequently, States and Tribes can
enhance the flexibility available to them by
carefully considering the available information,
and clearly articulatina the rationale for
alternative approaches.
Comment E-O1
The data presented for the consideration of the
CCC being 3-fold higher in the cold season is
based solely on the relationship between
pollutant concentrations and effects on aquatic
life and does not appear to take into account
the effects of human health. Treatment of
ambient waters for drinking water purposes
needs to be considered for effects on public
health and safety.
Nutrient loading, such as ammonia, can lead to
increased plant material in water bodies, which
will increase the Total Organic Carbon levels.
[ After disinfection in potable water supplies]
this will lead to higher levels of disinfection by-
The Ire atability of ambient source water varies
with temperature. The effectiveness of chlorine
as a disinfectant is reduced in colder
temperatures. Allowing levels of ammonia to
increase in the cold season... would create an
increased chlorine demand which would create
an increased disinfectant load which would
create and increase the disinfection-by-
products produced
A WWA feels that the Agency should consider
the potential to affect the quality of [ drinking
water] source waters, and more importantly
public health in the evaluation of cold-season
ammonia levels.
The comment is correct in noting that the
aquatic life criteria derivation-does not involve-
human health considerations.
EPA’s water quality standards program has
maintained separate lists of human health
criteria and aquatic life criteria. Human health
criteria are not derived with the intent of
protecting aquatic life. Aquatic life criteria are
not derived with the intent of protecting human
States must adopt criteria to protect beneficial
uses of their waters: protecting potable source
waters, protecting human consumers of fish,
and protecting the aquatic life itself. However,
the criteria for these distinct uses differ from
each other. Where concentrations of a pollutant
impair both drinking water uses and aquatic life
uses, separate criteria should be adopted to
protect these uses.

Not all waters are classified for all purposes. If
EPA were to roll drinking water considerations
into its aquatic life criteria, thereby creating an
multi-purpose criterion, then it would have
difficulty justifying the necessity for this
criterion in waters that are not classified for
drinking water use. Thus, EPA believes it
better to tailor the criteria to the use. The
concerns expressed in the comment should be
addressed through human health criteria for
protection of source waters.
Regarding one technical detail in the comment,
certain points should be made about the concern
about ammonia as a nutrient in aquatic systems.
First, it should be noted that algal growth in
nearly all fresh waters is more limited by
phosphorus than by nitrogen. Consequently,
increased nitrogen loads do not necessarily
stimulate additional plant growth, because the
nitrogen is already present in excess. Second,
ammonia criteria have no effect on nutrient
nitrogen loads or concentrations. The so-called
“ammonia removal” from wastewater does not
remove much nitrogen, it merely oxidizes
ammonia to nitrate, which is then discharged.
Comment F—O 1
The EPA conclusion that ‘the CCC does not
vary with the type offish present,” is not
adequately supported.
EPA did not intend to imply that site-specific
recalculation of criterion, based on species
present, and excluding species absent would not
change the criterion. Rather, what EPA
intended to convey was that for the coarse
fisheries distinction sometimes recognized by
national criteria, cold-water, salmonids present,
versus warm-water, salmonids absent, the data
did not support different values for the CCC, in
part because the two most sensitive tested
species were invertebrates, not fish, and in part
because the chronic sensitivity of salmonids did
not appear to differ substantially from some
warm water fish.
Comment F-02
The EPA assumption that the species which
lack chronic test data are those which are
relatively tolerant of ammonia in the acute
database is incorrect. In particular,
invertebrate data are lacking, yet some
invertebrates are among the organisms most
sensitive to ammonia.
EPA does not believe it made the above
assumption. EPA believes that the
appropriateness of the ammonia criterion (and
other aquatic life criteria) rests on the
assumption that the tested species are
representative of the large number of untested
species that would be found in the field: that is,
that the tested species are not biased toward
either greater or lesser sensitivity than the
multitude of untested species.
Comment F-03
It is inconsistent to conclude that ammonia
toxicity “does not appear empirically to vary
with temperature” (Notice, p. 44256) while
simultaneously recognizing that, at least for
some species, toxicity of ammonia appears to
decrease with decreasing temperature (Notice,
p.4 1257).
EPA agrees. This inconsistency has been
resolved by the 1999 Update, by quantifying
(rather than merely acknowledging, as in 1998)
the temperature dependency of ammonia
toxicity on invertebrates. The 1999 CCC values
are temperature dependent.
Comment G-O 1
The update identj/Ies several issues that could
be more fully addressed if additional data were
available. 1 urge EPA to conduct the needed
research to further clarjfy those issues where
the available data are meager, and where the
uncertainty associated with those data are
likely to have the greatest impact on the
criteria. The following list recommends the

research needed to more fully address the
issues I believe are most important to Virginia.
1. Long term survival tests with sensitive
juvenile and adult fishes and conducted at low
temperatures for long enough durations to
assess toxic effects under conditions relevant to
establishing a cold season criteria.
2. Additional studies to better define the
sensitivity of Hyalella, the bluegill and the
fingernail clam as these appear to be among
the most sensitive species.
3. Additional tests with sensitive species to
better define the effect ofpH on chronic
4. Additional tests to determine what affect
osmotic stress may have on chronic toxicity of
sensitive species.
5. Chronic tests with freshwater clams in the
family Unionidae to act as surro gates for
endangered species in the same family.
EPA agrees with that studies in the above areas
would be of interest. In addition, during
formulation of the 1999 CCC it has become
apparent that additional data on the invertebrate
temperature relationship would be of interest.
Comment H-O1
EPA does not possess technical studies
demonstrating that chronic effects of ammonia
occur at less than 9.0 mgIL for warm water
fisheries during non-spawning/low
temperature periods (e.g., temperature less
than 10°C).
EPA does not agree. For cold-weather survival
of the invertebrate Hyalella, EPA estimates a
pH=8 GMCV of 3.82 mg NIL at 10°C and of
4.63 mg N/L at <7°C. This is based on the
Borgmann (1994) test at 25°C, adjusted by the
1999 Update’s invertebrate chronic temperature
relationship. This temperature relationship was
established from the Arthur et al. (1987)
invertebrate acute temperature-dependence
data, modified by the acute-chronic ratio’s
(ACR) temperature dependence, which was
estimated from the DeGraeve et al. (1987) data
for fathead minnow ACR temperature behavior.
For survival of warm-water juvenile and adult
fish (ELS absent), EPA in the 1999 Update has
estimated pH =8 GMCVs in the range of 8.78
to 9.55 mg N/L for four genera, independent of
temperature, and therefore applicable to the
<10°C condition specified in the comment.
Lastly, it should be pointed out that the period
during which fish ELS are present is longer than
the spawning period, because it includes the
time needed for development of embryos and
larvae into juveniles. The term “spawning
period” should thus not be substituted for “fish
early life stage present period.”
Comment H-02
There are no data demonstrating that a 1.27
mg/L chronic criterion (at pH 8.0) is required
for either warm or cold water fishery
protection when [ fish] ELS are not present or
temperatures are less than 15° C.
EPA believes the statement is correct. See the
response to H-0 1. For the 1999 Update the
CCC was set at 85 percent of the Hyalella
GMCV, adjusted for temperature and pH. EPA
believes that this should provide protection to a
high percentage of taxa, tested and untested. In
contrast to the 1998 Update, the 1999 Update
CCC (pH=8) is substantially above 1.27 mg
N/L at all temperatures below 15°C, thereby
satisfying the concern of the comment.
Comment H-03
Invertebrate data support the use of a
temperature adjustment for chronic criteria for
temperatures less than 25°C, and it is
acceptable to base that adjustment on the

available acute temperature effects
EPA agrees with the first clause. A temperature
adjustment has been added to the 1999 chronic
criterion. The adjustment applies above 25°C
as well as below.
EPA based the chronic adjustment primarily on
the invertebrate acute temperature dependency
data (per the comment’s second clause), but
modified this to account for the expected
temperature dependency of the acute-chronic
ratio (as stated in response to H-01).
Comment H-04
Available data do not demonstrate that existing
warm water fishery un-ionized ammonia
standards used by many states ranging 0.04 -
0.07 mg/L, typically applied at 7Q10 or 30Q10
flows, are not protective where pH is above 7.0
All decisions about the protectiveness of
proposed or existing State or Tribal standards
are made on a case-by-case basis, considering
the applicable laws and regulations, and the
available technical information. This guidance
does not pass judgement on State or Tribal
standards. EPA thus declines here to relate test
results to the protectiveness of such standards.
lower pH increases the likelihood of observing
an un-ionized effect concentration below some
stated threshold.
At pH>7, none of the data presented in the 1999
Update’s Figures 8, 10, 12, or 14, or Table 5
corresponds to effects at un-ionized
concentrations <0.04 mgIL. Considering only
the data in Table 5 of the 1999 Update (or
Table 2 in 1998), two studies show effects
below 0.07 mg/L. These are Sparks and
Sandusky (1981) with fingernail clam, and
Smith et al. (1984) with bluegill.
Again, it must be emphasized that these facts do
not by themselves indicate whether particular
standards are or are not protective of aquatic life
Comment H-05
Available data on salmonid fisheries do not
indicate that cold weather, non-spawning
periods require total ammonia chronic criteria
less than 3-4 mg NIL (atpH 8.0).
At temperatures between 11 °C and 0°C, the
1999 Update CCC (pH=8), with fish ELS
absent, falls in the range 3.05 to 3.95 mg NIL.
Consequently, if the term “fish ELS absent
period” is substituted for “non-spawning
periods” in the above comment statement (as
discussed in H-0l), then EPA agrees.
Nevertheless, it is within the scope of this
document to address a related, purely technical
question of whether there are any studies
showing chronic effects below the particular
concentrations specified in the comment.
Although certain studies, Rice and Bailey
(1980) with pink salmon and Broderius et al.
(1985) with smalimouth bass, have shown
chronic effects below 0.04 mg/L un-ionized
ammonia, both of these tests were at pH<7, and
are thus not applicable to the pH>7 condition
stipulated in the comment. When expressed as
un-ionized ammonia, effect concentrations
decrease with decreasing pH. Thus, testing at
The distinction between spawning period and
fish ELS absent period is greatest in fall-
spawning salmonid fisheries, where the
salmonid ELS are present all winter.
Comment H-06
Available data for salmonidfisheries do not
indicate that the 0.02 mg/I un-ionized ammonia
chronic criterion used by several states is

See H-04 for discussion about why EPA
declines to respond here to questions about the
protectiveness of State or Tribal standards.
Nevertheless, parallel with H-04, EPA can
address a related, purely technical question of
whether any chronic effects on salmonids have
been observed below 0.02 mgfL un-ionized
ammonia. Rice and Bailey (1980), testing at
4°C and pH 6.4, found effects on pink salmon
at un-ionized ammonia concentrations less than
0.02 mg/L. See H-04 for discussion of the
significance of this low test pH.
Parallel with H-04, this fact does not by itself
indicate whether particular standards are or are
not protective of coidwater fisheries uses.
Appendix I - Evaluating Adherence of pH Relationship to Data at Low and High pH
One assertion in the comments was that the pH relationship was overly conservative at low and
high pH. Part of the support for this assertion were observations regarding deviations of the data from
the pH relationships in Figures 10 and 12 of the 1998 Update (Figures 12 and 14 in 1999). These
observations included noting that the criterion fell
further below available data at the extreme pHs than
in the pH 7.5 to 8.5 range, that the data appeared to
be less curved than the pH relationship used, and that
the slope of the data at low pH was greater and at 1000
high pH was less than the pH relationship. This *
appendix demonstrates that such observations do not
raise legitimate doubts about the pH relationship - in
fact, they are what is expected if the pH relationship
is perfectly valid.
The figure at the side is a simulated dataset in
which the pH relationship used in the Update is
assumed to be true. The dotted and solid lines are
the FAV and CMC, respectively, from.Figure 10.
The abscissas of the data points are the pHs of the
acute data presented in Figure 10 of the Update, so
that the pH distribution of the data is the same as
presented in the Update. The ordinates of the data
points were randomly drawn from a log-normal
distribution with a log-mean 0.3 greater than the
FAV and a log standard deviation of 0.25, which
results in a scatter in the pH 7.5-8.0 range similar to
that seen in Figure 10 of the 1998 Update (Figure 12
6 7 6 9
. :i.
p 14

in 1999). (That is, plotted LC5O = l0”(log(2 CMC) + NRnd), where NRnd is normally distributed
with mean 0.3 and standard deviation 0.25, and where CMC is from Equation 12 of the 1998 Update
or Equation 13 of the 1999 Update.)
In this simulation, it is given that the curved pH relationship is valid. Yet, although the plotted
“data” have been calculated from pH relationship (with added random variability), the appearance of
the data has features that some comments argued to refute the pH relationship. It appears that the data
can be described by a less curved line. Many of the data are below the FAV in the pH 7.5-8.5 range,
but data are well above the FAV at the more extreme pH.
This type of behavior is expected and is a matter of simple probabilities. The criterion should
be farther below available data at the extreme pHs simply because there are fewer data there. For a pH
range with hundreds of LC5Os, many data points would be expected to be below the fifth percentile,
but with few LC5Os, none would be expected to be below it.
The simulated data set here is similar in appearance to the actual data in Figure 10 in the 1998
Update (Figure 12 in 1999). The exact appearance of the simulation data set varies depending on the
values of the random numbers generated. (That is, different seeds for the random number generator
yield somewhat different simulation plots.) Nevertheless, the basic characteristics of their appearance
are the same, with the low values from the center of the pH distribution falling closest to the criteria
line. This indicates that the 1998 Update’s Figure 10 data plot is consistent with rather than at odds
with the acute pH relationship.
Appendix II- Differences Between Acute and Chronic pH Relationships
Some comments were critical that the Update uses a different pH relationship for chronic
toxicity than for acute toxicity. Concerns include the fact that the chronic pH relationship is based on
just two datasets, in contrast to 15 data sets for the acute pH relationship. In various places, the
comments assert that such limited data provide an inadequate basis for adopting a different pH
relationship for chronic toxicity than already established for acute toxicity. The comments also make -
various claims about the acute pH relationship being appropriate for chronic applications. The
comments ignore that the Update did establish statistically significant differences between acute and
chronic toxicity pH relationships, even with the limited data. Various specific comments are addressed
in the main body of this response. This appendix presents more details on the differences between
acute and chronic toxicity than was provided in the Update in order to better explain the basis for using
different relationships.
Smalimouth Bass
One of the two studies used for the basis of the chronic pH relationship was that of Broderius
et al. (1985), who conducted both early life stage chronic tests and acute tests on smalimouth bass at
four different pHs. The acute data from this study are presented in 1998 Update Figure 6 and the
chronic data in 1998 Update Figure 8 (or Figures 8 and 10 in 1999). The Update reported that the
parameter estimates from the regression analysis of chronic data differed significantly from the

parameter values determined from
the regression analysis of the acute
data, but provided no detailed
comparisons of the acute and chronic
data. Some further comparisons will
be given here. In the figure at side,
both the acute and chronic data from
this study are plotted side by side.
These figures show clear differences
in trends with pH for acute and
chronic toxicity. The differences in
these trends are especially evident in
the acute-chronic ratios also plotted
on this figure, which vary by almost
ten-fold over the pH range, in
contrast to being constant if the acute
and chronic pH relationships really
were the same.
lmoufl, Bass Acute us Ovonic Toddty
1 1W
P aEe LC5O
4 40.
fta eC nc
Ra o
S •
The figure below shows just 6 7 8 9 6 7 8 9 6 -
the chronic data from this study,
plotted with both the acute and
chronic pH relationship from the
Update, the intercepts in these relationships being adjusted to best fit this dataset. For the chronic
relationship, the residual mean square error (log scale) of the data from the line is .0 127, corresponding
to an average deviation of the line of a factor of 1.30 (about 30%), in line with reasonable uncertainties
for toxicity data. For the acute relationship, the residual mean squared error is .0695, more than five
times larger than for the chronic
relationship, and which corresponds a
deviation factor of 1.84, extremely large S m a II m 0 u t h B a s $ C h r o n i C E C 20
deviations relative to the uncertainty in 40
the data. Even if the lowest pH data is
ignored, the acute pH relationship gives a 20
much worse fit.
The contention in the comments
that there is not a significant difference
between acute and chronic pH -
relationships and that the acute pH
relationship provides an adequate fit to
the chronic data is not supported by such
analysis of this data. Using the acute pH
relationship results in much larger errors
and ignores clear differences between
chronic and acute toxicity from this
study. It should be further noted that the
lines used here are based on the average
regression across all datasets. The
differences between the acute and
6 7 8 9
pH Relationship

chronic data for smallmouth bass are actually greater than accounted for by these average relationships.
To ignore these differences would not be an appropriate use of available data. Finally, it should be
noted that the differences here between acute and chronic toxicity are not due to different endpoints.
Although the chronic toxicity EC2Os here included effects on both survival and growth, the effects
were mostly due to mortalities. Even if just mortality was used from the chronic study, the pH
dependence of chronic toxicity would still differ markedly from that of acute toxicity.
Ceriodaphnia dubia
The other dataset used in the evaluation of the pH dependence of chronic ammonia toxicity was
that from Johnson (1995) using Ceriodaphnia dubia. This dataset consists of life cycle tests conducted
at four different pHs and in waters of three different ion concentrations, with reproduction providing
the most sensitive endpoint. As reported in the Update, this data shows a similar pH dependence to
that for smallmouth bass, being significantly flatter than the acute pH relationship from the Update. It
should be noted, however, that the acute pH relationship for crustaceans also appears to be flatter at
low pH than the average acute pH relationship in the Update, so that there is not as much pH
dependence of acute-chronic ratios as observed above for smallmouth bass. But whatever the trends in
these ratios, the issue of concern here is whether the data for this organism support using a flatter pH
relationship for chronic toxicity than for acute toxicity. Although regression analysis reported in the
Update indicated this to be the case, Figure 8 of the 1998 Update did not provide good visual
presentation of these trends because the data are quite variable (reproductive endpoint) and because
data and lines from the waters of different ion concentration were superimposed.
The following figure shows the empirical trends more clearly by showing the geometric
average and range, at each tested pH, of the effect concentrations for the three different ion
concentrations. (Data are plotted at the average pH for each set of three tests.) The acute-chronic
ratios are the geometric averages for each pH and are not corrected for the small differences in pH
between acute and chronic tests. Although these ratios are variable, on average they do increase
substantially at low pH, although
the trend is not as great as for
smallmouth bass. Cedodaphnia dubia Acute rsus Chronic Todcity
The figure on the next ° CtTIXiC EC2O .ACute LC5O AC Ratio
page shows the data normalized I
and averaged to better show the 100 10
mean trends of toxicity with pH.
For the regression analysis
reported in the Update, the shape E
of the pH relationship was
assumed to be the same among 10
datasets and a pooled, average 2
estimate for this shape was 10
derived. Average sensitivity
differences between datasets, T i
including among the three water 2 4
types used in the C. dubia study,
1’ .. 2 . . _ .I ....,....I
6 7 8 9 6 7 8 9 6 7 8 9

were accounted for with separate . . .
estimates for the EC2O atpH=8. This Ceriodaphnia dubia Chronic Toxicity
estimate serves the role of an intercept 10 -
and this procedure is analogous to a
pooled linear regression in which the
slopes of different datasets are assumed Chronic pH Relationship
to be the same, but the Intercepts vary. o 4 -
For the data presentation here, each
EC2O was divided by the regression
estimate of the EC2O at pH=8 for its E 2 -
water type to produce normalized
EC2Os. When so normalized, the points
for all the water types can be . I -
superimposed onto the same normalized
pH relationship. Such a normalization
was used to summarize acute toxicity
data in Figures 4 and 7 of the 1998 0.4
Update, and was also used in
Appendix A of the Hall & Associates z
comments. Data so normalized still 0.2 -
show the same residual variation around
the regression curve as before
normalization. To reduce that variation, 0.1 ‘ • I • I • I
the geometric average of the three 6 7 8 9
normalized EC2Os was calculated at
each tested pH. Since each of these
data is an estimate for the same point on the relationship, such averaging is legitimate, although it is
made somewhat approximate due to pH variations among these points. This normalization and
averaging is done only to provide a more informative visual presentation of the average pH trends
present in this data. Any statistical analysis and conclusions regarding parameter values and trends is
still based on the original regression analysis.
When the data are so plotted on the above figure, it is seen that the mean pH relationship for
this dataset is similar to that for smallmouth bass presented above. As for smalimouth bass, both the
acute and chronic pH relationships from the Update are also plotted on this figure, with their intercepts
adjusted to best fit the data. For the chronic relationship, the residual mean squared error is 0.0 100,
whereas for the acute pH relationship it is -.0191, almost twice as much. This difference arises largely
from the fact that the chronic data show more flattening at lower pH, which is not accommodated for
by the acute pH relationship. Although the disparity between this data set and the acute pH
relationship is not as bad as was the case for smallmouth bass, the acute relationship still is
substantially worse than the chronic pH relationship.
All Species Aggregated
All available chronic EC2Os for all tested species are plotted against pH in Figure 12 of the
1998 Update (Figure 14 in 1999). Scatter in these data, produced by interspecies variability and
interlaboratory variability, make it difficult to infer from the plot exactly what the correct pH
relationship should be.
Acute pH Relationship

Random number simulations of the type described in Appendix I indicate that the Figure 12
EC2Os are consistent with the chronic pH relationship. The amount of scatter in the data and the
sparsity of data at low pH make it difficult, however, to conclude that the Figure 12 could not also be
consistent with the acute pH curve. Thus, EPA’s preference for a chronic pH dependence distinct from
the acute pH dependence is based on analysis of the above described Broderius et al. (1985)
smallmouth bass study and Johnson (1995) Ceriodaphnza study, and not on the aggregated data of
.Figure 12, which was presented to illustrate the general relationship between the chronic criterion and
the available EC2Os.
Appendix III - Issues Regarding Pooling of Temperature Data
Some comments were critical of the pooled analysis of temperature data used in the Update.
The concern apparently was not of the general techniques used for pooled analysis, which were
standard statistical techniques and were used in the same comments’ Appendix A. Rather, the concern
regarded the inclusion of certain data sets in the pooled analysis which had a limited temperature range
and/or a significant number of data with considerable variability. The contention appears to be that
including such data sets introduced biases or uncertainties in the analysis that was responsible for the
Update’s conclusion that there was no significant temperature dependence of acute ammonia toxicity
(in fish). The comments further suggested that temperature dependence should be based only on data
sets with at least a 20°C range, presumably so that the slope estimate from each data set has a better
reliability than those from some of the data sets used in the pooled data set.
While it is desirable for each data set to have a broad temperature range, EPA believes that it is
not correct to necessarily exclude less-than-desirable datasets. The question that should be posed is
whether the less desirable datasets make a net positive contribution to the analysis, even given their
uncertainties, and whether that contribution is appropriately weighted relative to the more desirable
datasets. The following analyses will address specific concerns raised in the comments about the
limitations of some datasets, and use statistical simulations to demonstrate that inclusion of such
datasets is appropriate. This does not preclude the possibility that a data set can contain misleading
data and skew the results, but this is true of large data sets as well as small, and of data sets with large
temperature ranges as well as small ranges. In the absence of some clear inconsistencies or
confounding influences, including a data set in any analysis should be decided on the basis of whether,
on average, it would be expected to improve the analysis or not. As the following discussion
demonstrates, the policy of the Update to include even limited data sets is not expected to obscure
underlying relationships, but would rather increase the likelihood of detecting them. Furthermore, the
analysis procedures used in the Update recognize the relative uncertainties of slope estimates from the
different datasets and weight them appropriately in developing pooled estimates for slopes, so that
“high quality” datasets will have more impact than lower quality ones.
A major concern raised in the comments was that some data sets had a temperature range of
10°C or less. The comments characterized this as “extrapolations” since these data sets were being
used to infer a relationship beyond there range. Such a characterization is inappropriate, because each
data set is used to provide an estimate of the slope only within its own range, and the entire relationship
is based collectively on data which do cover a broad range, so that no “extrapolations” are done, even if

the overall relationship is pieced together from relationships covering different ranges. Nonetheless,
although this characterization is wrong, the contribution of data sets with limited temperatures ranges
to the overall analysis is still a legitimate issue, because narrow temperature ranges result in more
uncertain slope estimates. However, the analysis used in the Update recognizes that these estimates
still provide legitimate information about the slope, and, if appropriately weighted, will make a positive
contribution to the overall analysis. Based on statistical simulations, the analysis here will address
whether data sets with limited temperature ranges can appropriately contribute to inferences about
relationships across a broader range.
The first simulations presented here will use two types of data sets. One data set will consist of
six points equally spaced across a 0 to 30°C range (0, 6, 12, 18, 24, 30°C), representing a single “high
quality” test series on an organism. The other data set will consist of three pairs of points, representing
three separate sets of two tests, the first set being conducted at 0 and 6°C, the second at 12 and 18°C,
and the third at 24 and 30°C. Thus, each data set has the same number of points at the same
temperatures, but the first data set will provide one slope estimate based on six points, whereas the
second data set will provide three, less certain, slope estimates based on two points each. These
smaller sets are similar to the most limited ones used in the Update, whereas the larger set is similar to
the data sets favored by the comments.
In the first set of simulations, the real temperature relationship is assumed to be:
log 1 0 (LC5O) = —0.01 (1 ëmperature(C) —15)
The log slope of -0.01 corresponds to a factor of 2 change over a 30°C range (at 15°C, the true LC5O
is 1.000, at 0°C it is 1.413, and at 30°C it is 0.707). The intercept in this equation is set to 15°C to put
it in the middle of the data, rather than the extremes. Data points were randomly created based on this
relationship, with a standard deviation of 0.1 for the log LC5Os (the average standard deviation
observed in the temperature data sets in the Update). A total of 10,000 samples were created for each
type of data set and subject to linear regression of log LC5O versus temperature. For the second data
set type (with three sets of data pairs), the regression produced separate intercepts for each data pair,
but a pooled estimate of slope. The mean and variability of the regression estimates for slopes and
intercepts are summarized in the following table:
Data Set Type
Mean of Param Est
SD of Param Est
One Set of Six
-0.0 100
0.04 1
Three Sets of Two
; )
Pooled Slope
Intercept (0,6°C Pair)
Intercept (12,18°C Pair)
Intercept (24,30°C Pair)
As expected, the single data set of six points produced much more precise estimates of the parameters,
but the threes sets of data pairs still produced unbiased estimates for the true slope (-0.0 100) and
intercepts (0.000). These simulations also tracked how often the regressions produced a slope

(a) greater than -0.0050 (i.e., less than half as steep) and (b) positive rather than negative. For the
single data set of six, the slope was >0.005 10% of the time and positive 0.5% of the time. For the
three sets of data pairs, the slope was >0.005 36% of the time and positive 23% of the time. Clearly,
just three sets of paired data like this would produce imprecise estimates, and reliance shouldn’t be put
on just on such a limited data set. But just because such a data set by itself produces imprecise
estimates of slopes does not mean that it cannot make a worthwhile contribution as part of a larger
pooled analysis, so the next simulations were run to demonstrate this point.
Two simulations were run, each with a data set with a total of twelve points. In the first
simulation, the data set consisted of two of the “high quality” sets of six evenly spaced points. In the
second simulation, the data set consisted of one of the “high quality” sets and three of the sets of data
pairs. The results of the simulation are presented below:
Data Set Type
Mean of Estimates
SD of Estimates
Two Sets of Six
Pooled Slope
Intercept (Set 1)
0.04 1
Intercept (Set 2)
0.04 1
One Set of Six and
Three Sets of Two
Pooled Slope
Intercept (0,6°C Pair)
Intercept (12,18°C Pair)
Intercept (24,30°C Pair)
Intercept (Set of Six)
The first simulation produced the expected results. Using two sets of six increased the precision
(reduced the standard deviation) of the slope estimate by a factor equal to the square root of two
(=1.41), but had no effect on the precision of the intercept estimates since such centered intercepts rely
just on the mean of the six points within each set. In the second simulation, using some “low quality”
data along with one “high quality” data set also improved the precision of the slope estimate, but only
slightly because the slope estimates from these small, narrow-range sets are uncertain. The precision
of the intercept estimates from the low temperature and high temperature pairs improved markedly
because the intercept is outside their temperature ranges and using the pooled slope to extrapolate to
the intercept improves the estimation.
The point to emphasize here is that including this “low quality” data does result in net
improvements to the overall slope estimate. The fact that this data is uncertain and does not cover the
whole temperature range does not mean it will make the analysis worse; rather, it will on average
improve it, although its impact will be relatively small because of its uncertainty. This type of pooled
analysis recognizes the uncertainty in each data set, and weights the slopes from each data set in
proportion to the relative uncertainties.
But in the simulations so far, each data set had the same “true” underlying slope. It is in fact
likely that different species of organisms have different temperature relationships. In the absence of
adequate data to define relationships for individual species, the Update evaluated whether there was

any evidence for an overall trend in the available data for all fish species. If some of the species had
different “true” slopes, how would the analysis respond to this? In fact, it would use whatever slope
was present in each individual data set, and weight each according to its uncertainty to develop a
pooled slope estimate, so that “high quality” data sets would affect the slope more than “low quality”
ones. The following simulation results demonstrates this point. This repeats the previous two
simulations, except that the “true” slope used to generate the data for the second half of the data was set
to 0.00, rather than -0.01:
Data Set Type
Mean of Estimates
SD of Estimates
One Set of Six w/
Slope=-0.01 and
One Set of Six w/
Pooled Slope
Intercept (Set 1)
0.04 1
Intercept (Set 2)
One Set of Six w/
Slope-0.01 and
Three Sets of Two
Pooled Slope
Intercept (0,6°C Pair)
Intercept (12,18°C Pair)
Intercept (24,30°C Pair)
0.08 5
Intercept (Set of Six)
In the first simulation, when both sets of data are of equal quality, the resulting pooled slope analysis is
the average of the two, as is appropriate. In the second simulation, the slope is heavily weighted
toward the slope of the more certain data. Thus, this pooled analysis technique does not allow data sets
which are inherently uncertain to unduly influence the analysis, as the comments imply.
Another concern raised in the comments was that some data sets not only had a limited range
of temperatures, but also had a large number of data points, so that the analysis was unduly skewed to
whatever slope that data set happened to have. Additionally, it was noted that some of these data sets,
such as that of Thurston et aL, had substantial variability and that it would be inappropriate for such
variable data to have a heavy influence on the pooled slope estimate. The comments further suggested
that the large variability in some of the data sets suggested problems with the data that would also
argue against it being given substantial weight in the analysis. The concerns of the comments were not
justified on several counts:
(1) Part of the apparent variability of the Thurston et al. data is due to the large size of the data
sets, which are more likely to show data in the tails of the distribution. In fact, the standard
deviation of the Thurston et al. data in 1998 Update Figure 3 is less than that of the Reinbold
and Pescitelli bluegill gill data and only slightly greater than some other data sets. It is also less
than the fathead minnow, walleye, and rainbow trout data of Arthur et al., which the comments
use as their preferred data sets.
(2) The variability noted in some of these data sets is not necessarily unusually large for toxicity
data when tests are repeated enough times to actually produce good estimates of their
variability. Actually, the variability around the regression line for some the data sets is

more deserving of attention. The residual error around several of the regression lines in 1998
Update Figure 2 and 3 is less than the uncertainty of LC5O estimates, indicating that, by
random chance, the relationships look better than they really are.
(3) While the variability in the Thurston et at. studies may be higher than average, the
comments present no basis for suggesting that this data does not contain legitimate information
about the temperature relationship. Even if other factors contribute to this variability, only if
these factors are confounded with temperature so as to obscure or enhance the temperature
relationship should this data not be considered. Except for pH, which was accounted for in the
Update analysis, there is no such indication that such confounding is of concern, and there is no
apparent reason to think that this data set is more likely to be confounded by other variables
than is any other data set.
(4) The greater number of points in the Thurston et aL data sets will increase its weight in the
analysis, but this is appropriate, standard statistical procedure, since more data provide greater
certainty in the slope estimate, other factors being equal. However, this effect is not
proportional to the number of data, but rather to the square root of the number of data (or, for
some regression parameters, less than the square root). Thus, if the Thurston et at. data set has
four times as much data as another data set, it will not have four times the influence on the
slope estimate, but rather it will have at most two times the influence.
(5) Finally, the limited temperature range and large variability in the Thurston Ct al. data sets
make the slope estimate from these data sets less certain, and thus decreases their weight in the
development of the pooled slope estimate. Again, these data sets do not have as much
influence on the slopes as the comments suggest.
To suggest that variability per se should preclude the use of this data is contrary to basic statistical
estimation procedures. The procedures used in the Update consider the temperature range of the data,
the number of data, and the variability of the data and suitably weight them in the analysis.
In order to further evaluate this issue of whether data sets with limited temperature range and
with a large number of variable points should be included in the analysis, a series of simulations were
run to demonstrate that such data sets provide an appropriate contribution to the pooled analysis. This
involved the use of a data set with a temperature range from 10 to 20°C, with a size of either 6 (to
match earlier sets) or 28 (the size of the Thurston fathead minnows dataset), and with a log variability
of either 0.10 or 0.15. The first set of simulations looked just at using such a data set as shown at the
top of the next page.
These simulations demonstrate how a restricted range will greatly increase the uncertainty of
the slope estimate. The “high quality” data set with six points over a 30°C range had a standard
deviation of 0.0040 for the slope estimates. Reducing the temperature range here by a factor of three
increased the standard deviation by a factor of three, to 0.0 120. Increasing the number of points to 28
reduced the uncertainty, but only by about a factor of 2, so that even with 28 data points the 10°C
range data set here is less certain than the 30°C range data set with just 6 points. The above table also
shows, as expected, that increasing the variability of the data points proportionately increases the
uncertainty in the parameter estimates.

Data Set Type
Mean of Param Est
SD of Param Est
6 Pts 10-20°C
Std Dev = 0.1
0.04 1
28 Pts 10-20°C
6 Pts 10-20°C
Std Dcv = 0.15
0.06 1
28 Pts 10-20°C
Std Dcv = 0.15
Simulations were then run in which the data sets included both a small “high quality” set (6
points, 30°C range, 0.1 log standard deviation) and a large “low quality” set (28 points, 10°C range,
0.15 log standard deviation). Furthermore, the high quality data was generated assuming a -0.0! slope
and the low quality with a 0.00 slope, so that the resultant pooled slope estimate will more clearly
indicate the relative weight given to the different data.
Data Set Type
Mean of Param Est
SD of Param Est
6 Pts 0-30°C
Pooled Slope
Slope = 0.01
Std Dev 0.1
28 Pts 10-20°C
Slope = 0.00
Intercept (Set of 6 Pta)
Intercept (Set of 28 Pts)
Std Dcv = 0.15
The resultant slope is much closer to that used to generate the smaller groups of points, because the
analysis procedure recognized that, despite the larger number of points in the second group, the smaller
temperature range and greater variability made its slope estimate less certain. But the second group
still provides useful information on the slope, so some influence is apparent in the pooled slope.
Other simulations and analyses could provide further information on the utility of different
types of data sets, but will not change the basic conclusion -- that data sets with limited temperature
range, whether it be with little or many data and with small or large variability, are useful in this kind of
analysis. Such data sets will not unduly influence the analysis so that relationships from “quality” data
sets are obscured; rather, including all the data will improve the chances of detecting any true mean
trends in the data as a whole. Consequently, the data analysis should not be restricted to a few selected
data sets, especially since the data sets that the comments recommend suffer from various problems
and do not themselves demonstrate statistically significant trends. As mentioned earlier, further
collection of appropriate data may well demonstrate temperature dependence of ammonia for some
fish, but the most appropriate analysis of all the currently available data do not support the use of any
temperature dependence.