United States Office of Water EPA-822-R-01-005
Environmental Protection 4304 March 2001
Agency
&EPA Streamlined
Water-Effect Ratio Procedure
for Discharges of Copper
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Streamlined
Water-Effect Ratio Procedure
for Discharges of Copper
March 2001
U.S. Environmental Protection Agency
Office of Water
Office of Science and Technology
Washington, D.C.
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Notices
This document provides guidance to states and tribes authorized to establish and
implement water quality standards under the Clean Water Act (CWA), to protect
aquatic life from acute and chronic effects of copper. Under the CWA, states and
tribes are to establish water quality criteria to protect designated uses. The CWA
and EPA regulations at 40 CFR Part 131 contain legally binding requirements. The
statutory provisions and EPA regulations described in this document contain
legally binding requirements. This document does not substitute for the CWA or
EPA's regulations; nor is it a regulation itself. Thus, it does not impose legally
binding requirements on EPA, states, tribes, or the regulated community, and may
not apply to a particular situation based upon the circumstances. State and tribal
decision makers retain the discretion to adopt approaches on a case-by-case basis
that differ from this guidance when appropriate. Therefore, interested parties are
free to raise questions and objections about the substance of this guidance and the
appropriateness of the application of this guidance to a particular situation. EPA
will, and States should, consider whether or not the recommendations or
interpretations in the guidance are appropriate in that situation. While this
guidance constitutes EPA's scientific recommendations on procedures for
obtaining site-specific values for aquatic life criteria for copper, EPA may change
this guidance in the future.
This document has been approved for publication by the Office of Science and
Technology, Office of Water, U.S. Environmental Protection Agency. Mention of
trade names or commercial products does not constitute endorsement or
recommendation for use.
Acknowledgment
This document was prepared by Charles Delos, Health and Ecological Criteria
Division, Office of Science and Technology. Appendix A was adapted and
modified from the 1994 document Interim Guidance on the Determination and
Use of Water-Effect Ratios for Metals., which had been prepared by Charles
Stephan and others. Appendix B is based primarily on a literature review by Gary
Chapman of the Great Lakes Environmental Center.
Peer review of this document was performed by Paul Jiapizian of the Maryland
Department of Environment, William Dimond of the Michigan Department of
Environmental Quality, and Cindy Roberts of U.S. EPA. This is documented in
Response to Peer Review Comments on Streamlined Water-Effect Ratio for
Discharges of Copper, available in portable document format (pdf) from the
contact below.
Please submit questions to: Charles Delos, U.S. EPA, Mail Code 4304,
Washington, DC 20460 (e-mail: delos.charles@epa.gov).
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Contents
Page
Introduction 1
Synopsis of the Streamlined Procedure 1
Discussion of Technical Approach 2
Implementation 6
Appendix A. Sampling and Testing Protocol 7
Appendix B. Species Mean Acute Values 17
Appendix C. Assessment of Streamlined Procedure 23
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Introduction
This guidance presents a Streamlined
Procedure for determining site-specific
values for a Water-Effect Ratio (WER), a
criteria adjustment factor accounting for the
effect of site-specific water characteristics on
pollutant bioavailability and toxicity to
aquatic life. This guidance is intended to
complement the 1994 Interim Guidance on
Determination and Use of Water-Effect
Ratios for Metals (EPA-823-B-94-QQI).
Whereas the 1994 Interim Procedure applies
to essentially all situations for most metals,
the Streamlined Procedure is recommended
only for situations where copper
concentrations are elevated primarily by
continuous point source effluents. Because
this is a relatively common regulatory
situation, a great deal of experience is
available to guide the development of a more
efficient procedure.
The Streamlined Procedure does not
supersede the 1994 Interim Procedure, even
for the limited situations to which it applies.
Rather, it provides an alternative approach.
In these situations the entity conducting the
study may choose between using the Interim
Procedure or using the Streamlined
Procedure.
Synopsis of the Streamlined Procedure
The Streamlined Procedure involves the
sampling of two events, spaced at least one
month apart. Flow during each event should
be stable, and water quality unaffected by
recent rainfall runoff events. Samples of
effluent and upstream water are to be taken.
These are mixed at the design low-flow
dilution, to create a simulated downstream
sample, to be used as the site-water sample
in toxicity tests spiked with various
concentrations of soluble copper salts.
In manner similar to the Interim Procedure,
the side-by-side, lab oratory-water and site-
water toxicity tests are run to obtain the 48-
hour acute EC 50 with either Ceriodaphnia
dubia or Daphnia magna. The result may be
expressed as either dissolved or total
recoverable copper. After adjusting for any
hardness differences, the WER for the
sample is the lesser of (a) the site-water
EC50 divided by the laboratory-water EC50,
or (b) the site-water EC50 divided by the
documented Species Mean Acute Value (the
mean EC50 from a large number of
published toxicity tests with laboratory
water). The geometric mean of the two (or
more) sampling event WERs is the site
WER.
The design of the Streamlined Procedure is
intended as a more efficient approach for
generating the information needed to make a
pollution control decision. The intent is to
provide a method that is both easier for the
performing organization to carry out, and
easier for the regulatory agency to review.
The Streamlined Procedure omits laboratory
or field measurements that experience with
the Interim Procedure has shown to be of
little practical value. The design is also
intended to be inherently less subject to
random sampling variability, thereby
allowing a reduction in the number of
samples while maintaining reliability.
Table 1 compares the provisions of the
Streamlined Procedure with those of the
1994 Interim Procedure.
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Table 1. Comparison of Streamlined Procedure and 1994 Interim Procedure
Characteristic
Applicability
Minimum number of
sampling events
Minimum number of WER
measurements
Minimum number of WER
measurements considered
in obtaining final site WER
Preparation of constructed
downstream water
Calculation of sample
WER
Calculation of final site
WER
1994 Interim Procedure
Universal
3
4
3
Mix effluent and upstream
samples at the dilution ratio
occurring at the time of
sampling
Site water LC + Lab water LC
Complicated scheme with
six "if... then... else" clauses and
12 possible paths
Streamlined Procedure
Copper from continuous
discharges
2
with recommended restrictions
2
2
Mix effluent and upstream
samples at the design low-flow
dilution ratio
Site water LC + The greater of
(a) Lab water LC, or
(b) SMAV
Geometric mean of the two
measurements
Discussion of Technical Approach
The key facets of the procedure are
presented below, with an explanation of their
purpose. The detailed protocol for
collecting samples, obtaining measurements,
and conducting tests is presented in
Appendix A. An analysis, through Monte
Carlo simulation, of the protectiveness of the
approach is presented in Appendix C.
1. Purpose of procedure. The procedure is
for deriving a dissolved and/or total
recoverable WER for copper from
continuous point source effluents. The
results may be used to obtain:
a. A dissolved WER used to obtain the
site-specific value of a dissolved
copper criterion.
b. A total recoverable WER used to
obtain either (i) the site-specific value
for a total recoverable criterion, or
(ii) a total recoverable effluent limit
from a dissolved criterion, merging
the functions of a dissolved WER and
a dissolved-to-total permit translator
factor.
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Commentary: If obtained from dissolved
copper ECSOs, the site WER multiplied
by the national or state's hardness-
normalized dissolved criterion becomes
the site value of the dissolved copper
criterion. Likewise, if obtained from
total recoverable measurements, the
WER is used to obtain the site value of
the total recoverable criterion. A total
recoverable WER may be applied to a
dissolved criterion by first converting the
dissolved criterion to a total recoverable
criterion, then multiplying by the WER.
In this case it eliminates the permit-
specific dissolved-total translator factor
by replacing the original dissolved
criterion with a site-specific total
recoverable criterion.
2. Recommended applicability of the
Streamlined Procedure. The procedure
is designed to apply to regulatory
situations where most of the copper is
from continuous point source effluents.
The procedure is not designed for
regulatory situations where the copper
originates primarily from wet weather or
nonpoint sources.
Commentary: The Streamlined
Procedure is intended to apply to
situations where the copper from the
regulated discharge is expected to attain
its maximum concentrations under low-
flow or low-dilution conditions.
Continuous point source discharges fit
this pattern. The most common such
situation involves municipal effluents,
which past experience and current
knowledge have generally shown to yield
low risks for copper toxicity, due to the
sequestering of copper by organic
matter. Nevertheless, the procedure is
just as suitable for non-municipal point
source effluents.
The Streamlined Procedure should not be
applied to situations where elevated
copper concentrations are the result of
wet weather runoff. In this case the
critical copper concentrations may not
occur under low-flow conditions, such
that the Streamlined Procedure's focus
on simulating low-flow conditions would
not be appropriate.
3. Collection of samples. Sampling for
WER measurements involves:
a. Two sampling events, at least one
month apart, and as recommended
below,
b. Samples of upstream and effluent
during each event.
Commentary: The Interim Procedure
calls for sampling three events. The
Streamlined Procedure, by more focused
sampling, can achieve equivalent
reliability using samples of two events, as
indicated by the Monte Carlo modeling
presented in Appendix C.
4. Plant performance during sampling
events should be as follows:
a. Average or better operating
conditions,
b. CBOD (carbonaceous biochemical
oxygen demand) and suspended
solids concentrations within permit
limits.
Commentary: Because the WER is
sensitive to the concentration of organic
matter being discharged, the plant should
be operating normally, as measured by
CBOD and suspended solids discharges.
5. Stream conditions during sampling
events should be as follows:
a. Stable flow condition, preferably
during a drier weather season
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(wherever regulatory schedules
allow).
b. Water quality conditions should be
compatible with those occurring
during time periods when nonpoint
source inputs of organic matter and
suspended solids are relatively low.
Commentary: Sampling of events with
elevated runoff is not desirable because
of the confounding influences of
nonpoint inputs that would not be
present under low flow conditions.
Because the upstream and effluent
samples are to be mixed at a ratio
equivalent to the low flow dilution, the
upstream water quality should not be
greatly dissimilar to the quality expected
during low flow conditions. Upstream
water quality is of greater concern if
there is a substantial fraction of upstream
water present during the design low-flow
event, and if a total recoverable WER is
to be used. Conversely, where little
upstream water is to be used in creating
the simulated low-flow downstream
sample, or if a dissolved WER is to be
applied, the quality of the sampled
upstream water is less important.
6. Merging of effluent and upstream
samples. The effluent and upstream
samples are to be combined at the
dilution corresponding to the design low-
flow condition that the permitting
authority uses in permit limit
calculations.
Commentary: Extensive data from the
State of Connecticut (discussed in the
Supplement to Appendix C) indicate no
relationship between upstream dissolved
copper WER values and streamflow.
Consequently, for continuous discharges
the critical condition, that condition
where the copper concentration can be
expected to be highest relative to the
WER, should be expected to occur at
low dilution. The most efficient way
(and often the only feasible way) to
predict conditions at low flow is to mix
the upstream and effluent samples at the
low-flow dilution ratio.
By contrast, the 1994 Interim Procedure
called for mixing the samples at the
dilution occurring during the sampling
event. The Interim Procedure was,
however, a general purpose procedure,
not restricted to the situations to which
the Streamlined Procedure applies. The
Interim Procedure thus also applies to
situations where there is little
understanding of what flow conditions
could be critical.
By restricting the use to well understood
situations, the Streamlined Procedure has
been designed to be more efficient and its
results more predictable than the Interim
Procedure. Monte Carlo simulation
indicates that the Streamlined Procedure,
under the conditions to which it applies,
is less subject to chance variation
(sampling error) than the Interim
Procedure.
7. Chemical analyses.
a. Dissolved and/or total copper
concentration
b. Hardness, alkalinity, pH, dissolved
organic carbon, and total suspended
solids
Commentary: The method may be
applied to either dissolved or total
copper. If applied to dissolved copper,
the permit authority will need to derive a
translator in order to derive a total
recoverable permit limit. If applied to
total copper, there is no need for a
translator.
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Hardness measurements are needed to
normalize the laboratory water and site-
water ECSOs to the same hardness. The
remaining measurement parameters
provide ancillary information for
understanding the chemistry influencing
the observed results and for providing a
link with the Biotic Ligand Model, which
is ultimately intended to replace the
WER toxicity test procedures for copper.
Toxicity testing.
a. Tested taxon: Either Ceriodaphnia
dubia or Daphnia magna.
b. Test: 48-hr EC50, spiking with
copper sulfate, nitrate, or chloride.
c. Side-by-side tests in laboratory
dilution water and site water.
Commentary: These stipulations are
more specific than those of the Interim
Procedure. Experience has shown that
the daphnids, which are quite sensitive to
copper, have been the most useful test
organisms for WER studies.
Furthermore, for these two species there
is a substantial amount of data on the
range of ECSOs observed in laboratory
water.
Other daphnid species, such as Daphnia
pulex, probably have similar sensitivity,
but have not been recommended here
because they have fewer data available
for estimating the appropriate value of
the Species Mean Acute Value. Other
test species, either fresh or saltwater,
could be substituted, provided that ample
data were available to determine the
appropriate SMAV. There is no
technical reason why the approach could
not be extended to saltwater copper tests
or to other metals in fresh or saltwater.
The current document's focus on
freshwater copper stems from the greater
demand for a streamlined procedure
among the numerous freshwater copper
dischargers.
The 1994 Interim Procedure
recommendation for a test with a second
species has been dropped, because the
additional test has not been found to
have value.
The simultaneously measured laboratory
water EC50, as presented in the detailed
protocol (Appendix A), is necessary
unless the state substitutes a rigorous
reference toxicant program involving
copper.
9. Analysis of data.
a. For site-water samples, if there is less
than 50 percent mortality at the
highest copper treatment
concentration, then assume that the
EC50 is the highest treatment
concentration.
b. The sample WER is the lesser of (i)
the site-water EC50 divided by the
lab-water EC50, or (ii) the site-water
EC50 divided by the Species Mean
Acute Value (from Appendix B).
c. Final site WER is the geometric mean
of the two (or more) sample WERs.
d. The acute and chronic criteria
concentrations for the site are the
national criteria concentrations (or
comparably derived state values)
multiplied by the final site WER.
Commentary: (a) As in the Interim
Procedure, it is not necessary to ascertain
the precise value of the WER, if the
actual WER is greater than the range of
values of regulatory interest, (b) The
Streamlined Procedure eliminates one
source of concern about the Interim
Procedure: the variability and apparent
non-protective bias of the lab water
ordinarily used in the side-by-side tests.
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(c) Monte Carlo modeling indicates that
use of the geometric mean of the two
samples can be expected to yield a
criterion as protective as or more
protective than intended for chemical-
specific criteria, (d) In accord with the
1994 Interim Guidance and with
common practice, the WER derived from
acute tests is applied to both acute and
chronic criteria. Because the
involvement of strong binding agents
causes the WER to increase as the effect
concentration decreases, the WER
derived from acute tests is expected to be
protective of chronic effects.
Implementation
Implementation policies for the Streamlined
Procedure are the same as for the 1994
Interim Procedure. WER-based site-specific
criteria provisions are subject to EPA review
under Section 303(c) of the Clean Water Act
and its implementing regulations at 40 CFR
Part 131. This can be structured in two
ways.
1. A state may submit each individual
determination of a WER-based site-
specific criteria value to EPA for review
and approval.
2. A state may incorporate WER
adjustment provisions into its water
quality standards, submitted to EPA for
review and approval. Once the
provisions are in place, the results of
each site-specific application of the
procedure would be subject to public
participation requirements, but would not
be submitted for further Section 303(c)
review.
In all cases, it should be noted that the WER
derivation is part of the standards setting
process. In the absence of an appropriate
specification of the site criterion, WERs are
not used for adjusting reasonable potential
calculations, wasteload allocations, or permit
limits.
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Appendix A
Sampling and Testing Protocol
for the Streamlined WER Procedure
The procedure set forth below is for deriving
a WER for copper discharged to fresh water
primarily from continuous point source
effluents, using Daphnia as the test taxon.
A. Background Information
I. Obtain information on the mean flow
and design dilution flow for the water
body segment for which the WER is
being determined. If the proposed
effluent limit was calculated using time-
variable modeling without reference to a
particular design low flow, then for
purposes of applying the Streamlined
Procedure, use the design flow that the
state customarily applies to steady-state
dilution calculations.
2. Estimate the values of the site-specific
criterion and WER corresponding to key
decision points, for example, the
minimum values needed for the
determination of "no reasonable
potential", upon which depends the need
for copper permit limits.
3. Consult the state or tribal pollution
control agency in order to assure that
the study will respond to all concerns
with respect to the peculiarities of the
specific site.
B. Acquiring and Acclimating Test
Organisms
1. Obtain, culture, hold, acclimate, feed,
and handle the test organisms as
recommended by U.S. EPA (1993)
and/or by ASTM (1999, 2000a, 2000b).
2. Acclimation to the site water is desirable
but optional.
C. Collecting and Handling Upstream
Water and Effluent
1. Obtain samples during two (or more)
sampling events, spaced at least four
weeks apart. When regulatory
schedules allow, schedule events for a
season when low streamflows are more
likely to occur.
2. For each sampling event, obtain a
representative sample of upstream
water, relatively unaffected by recent
runoff events that might elevate the total
suspended solids and organic matter
concentrations.
3. For each sampling event, obtain a
representative sample of effluent during
a period when the discharger is
operating normally, relatively unaffected
by short-term perturbations due to
rainfall inflow or slug loads. Composite
samples are preferred over grab samples.
4. Collect, transport, handle, and store
samples as recommended by U.S. EPA
(1993). Obtain a sufficient volume so
that some can be stored for additional
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testing or analyses if unusual results are
obtained. Store samples at 0 to 4°C in the
dark with no air space in the sample
container.
5. During the sampling event, measure
effluent parameters that are normally
required to be reported in the Discharge
Monitoring Report for the discharge
under study. These measurements
provide information on the
representativeness of the effluent
samples.
6. For the sampling event, obtain
streamflow data at the nearest relevant
gaging station, and rainfall data and any
other relevant meteorological
information for the preceding two
weeks.
7. Consider using chain of custody
procedures for all samples of site water
and effluent.
8. Begin toxicity tests as soon as practical,
but always within 96 hours after
collecting samples, in accord with EPA
(1993) recommendations for using site
water for the dilution water. Because
these tests are not intended for
measurement of whole effluent toxicity
(which might attenuate over time), it is
not essential to begin tests within 36
hours after the collection of the samples
(as would be recommended by EPA
(1993) for measuring the toxicity of an
effluent). This is a change from the
1994 Interim Procedure
recommendation.
9. If the site water might contain predators
of the daphnid test organisms, remove
them by filtering through a 37-60 pm
sieve or screen.
D. Laboratory Dilution Water
1. Use laboratory water that accords with
U.S. EPA (1993) or ASTM (1999,
2000a). Use ground water, surface
water, reconstituted water, diluted
mineral water, or dechlorinated tap
water that has been demonstrated to be
acceptable to aquatic organisms. If a
surface water contains predators,
remove them by filtering through a 37-
60 |jm sieve or screen. Do not use
water prepared by such treatments as
deionization or reverse osmosis unless
salts, or mineral water are added as
recommended by U.S. EPA (1993) or
ASTM (1999, 2000a).
2. Do not use laboratory water with DOC,
TOC, or TSS >5 mg/L.
3. Use laboratory water with hardness
between 40 and 220 mg/L. Within these
ranges use laboratory water with
hardness relatively close to that of the
site water.
4. The alkalinity and pH of the laboratory
dilution water is to be appropriate for its
hardness. Values for alkalinity and pH
that are appropriate for some values of
hardness are given by U.S. EPA (1993)
and ASTM (1999, 2000a); other
corresponding values should be
determined by interpolation. If
necessary, adjust alkalinity using sodium
bicarbonate, and pH using aeration,
sodium hydroxide, and/or sulfuric acid.
E. Conducting Tests
1. Conduct the tests such that there are no
differences between the side-by-side
tests other than the composition of the
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dilution water, the concentrations of metal
tested, and possibly the water in which the
test organisms are acclimated just prior to
the beginning of the tests.
2. A single laboratory water test may be
compared with tests of multiple site
water samples conducted side by side.
3. Follow the recommendations of U.S.
EPA (1993) and/or ASTM (1999,
2000a, 2000b) regarding setting up
facilities for conducting toxicity tests
and selecting and cleaning the test
chambers.
4. Prepare a stock solution of copper
chloride 2-hydrate (CuCl2-2H2O),
copper nitrate 2.5-hydrate
(Cu(NO3)2-2.5H2O), or copper sulfate 5-
hydrate (CuSO4'5H2O).
a. Use reagent-grade material.
b. Only as necessary to get the
metal into solution, acidify the
stock solution using metal-free
nitric acid.
c. Use the same stock solution for
all tests conducted at one time.
5. In the unusual situation where the
effluent is dominated by highly stable
metallo-organic compounds, such as
copper phthalocyanine dyes, then an
exception to using the above listed
copper salts may be considered. That is,
it may be acceptable to include the
metallo-organic compounds in the stock
solution. In such case, prepare the stock
solution such that the stable metallo-
organic compounds constitute the same
percentage of total copper in the stock
solution as in the site water to be WER
tested. This exception to the usual
procedure requires case-by-case review
of its appropriateness, after
documenting the usual presence of the
compounds in the effluent, the stability
of the compounds, and the importance
of distinguishing the compounds from
other forms of copper.
6. Run the 48-hour test with Ceriodaphnia
dubia or Daphnia magna to obtain an
EC50 (U.S. EPA 1993; ASTM 1999,
2000a, 2000b), using a sufficient volume
to accommodate the needed chemical
measurements. With appropriate
modification of the protocol, other test
species may be substituted on a case-by-
case basis, only if (a) the daphnid
SMAV is either significantly below the
site criterion obtained by the
Recalculation Procedure, or daphnia is
not viable at the site water salinity; and
(b) there is sufficient data to establish an
SMAV with high reliability.
7. Static tests may be used if dissolved
oxygen remains sufficient, and if either
(a) measured dissolved copper
concentrations do not decrease more
than 50 percent by the end of a test
intending to use the dissolved copper
measurements as the basis for the WER,
or (b) total recoverable concentrations
are to be used as the basis for the WER.
8. Renew the test solutions after 24 hours
if either (a) dissolved oxygen would
decrease too much, or (b) dissolved
copper would decrease more than 50%
by the end of a test intended for a
dissolved WER, or (c) the analyst favors
renewal for any other reason.
a. If solutions in one test in a pair of
side-by-side tests are renewed,
renew solutions in the other test as
well.
b. To renew site water solutions,
prepare new test solutions from the
remaining site water sample(s)
stored at 0 to 4°C in the dark.
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9. Follow recommendations on
temperature, loading, feeding, dissolved
oxygen, aeration, disturbance, and
controls given by U.S. EPA (1993)
and/or ASTM (1999, 2000a, 2000b).
10. Use range-finding tests, of 8 to 48-hour
duration where necessary and where
they will not unduly delay the beginning
of the test.
11. Use a dilution factor of 0.6 or greater
for nominal concentrations, balancing
for the particular situation the needs for
covering the range of possible results,
reducing the uncertainly in the
calculated EC50, and curbing the cost of
the test. The value of the WER needed
for a "no reasonable potential"
determination or other decision
benchmarks should be kept in mind in
selecting the dilution series. With
regard to the risk that the dilution series
will not span the possible range of the
EC50, note that in site water (a) a "less
than value" for the EC50 would be
unusable for obtaining a WER>1, and
(b) a "greater than" value" for the EC50
would be interpreted as "equal to" in
calculating the WER. In contrast, in lab
water (c) a "greater than value" for the
EC50 would be unusable for obtaining a
WER>1, (d) a "less than value" for the
EC50 would be interpreted as "equal
to"; however, (e) the SMAV from
Appendix B would be used in place of
any lab water EC50 less that SMAV.
12. Use an unspiked dilution water control
for each test.
13. Use at least 20 organisms for each
treatment concentration (including
unspiked controls). It is desirable to use
two or more test chambers per
treatment. Assign test organisms
randomly, or at least impartially to the
side-by-side tests, and to all treatment
chambers (U.S. EPA 1993; ASTM
1999, 2000a). When assigning
impartially, do not place more than 20%
of any chamber's total number of
organisms at one time. In addition, the
test chambers should be assigned to
location in a random arrangement or in a
randomized block design.
14. Use appropriate glassware for making
serial dilutions, such as graduated
cylinders, volumetric flasks, and
volumetric pipettes.
15. For the test using site water, mix
effluent and upstream waters to accord
with the design dilution, corresponding
to the upstream design flow and effluent
flow normally used for steady state
modeling calculations. Generally use
any one of the following procedures to
prepare the test solutions for the test
chambers and the "chemistry controls",
if any (see section F.I):
a. Thoroughly mix the sample of the
effluent and place the same known
volume of the effluent in each test
chamber; add the necessary amount
of metal, which will be different for
each treatment; mix thoroughly; let
stand for 2 to 24 hours (if
overnight, keep at 0 to 4°C); add
the necessary amount of upstream
water to each test chamber; mix
thoroughly; let stand for 1 to 3
hours.
b. Add the necessary amount of metal
to a large sample of the effluent and
also maintain an unspiked sample of
the effluent; perform serial dilution
using a graduated cylinder and the
well-mixed spiked and unspiked
samples of the effluent; let stand for
10
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2 to 24 hours (if overnight, keep
at 0 to 4°C); add the necessary
amount of upstream water to
each test chamber; mix
thoroughly; let stand for 1 to 3
hours.
c. Prepare a large volume of simulated
downstream water by mixing
effluent and upstream water in the
desired ratio; place the same known
volume of the simulated
downstream water in each test
chamber; add the necessary amount
of metal, which will be different for
each treatment; mix thoroughly and
let stand for 1 to 4 hours.
d. Prepare a large volume of simulated
downstream water by mixing
effluent and upstream water in the
desired ratio; divide it into two
portions; prepare a large volume of
the highest test concentration of
metal using one portion of the
simulated downstream water;
perform serial dilution using a
graduated cylinder and the well-
mixed spiked and unspiked samples
of the simulated downstream water;
let stand for 1 to 4 hours.
Procedures "a" and "b" allow the metal
to equilibrate with the effluent before
the solution is diluted with upstream
water.
16. For the test using the laboratory dilution
water, either of the following
procedures may be used to prepare the
test solutions for the test chambers and
the "chemistry controls" (see section
F.I):
a. Place the same known volume of
the laboratory dilution water in each
test chamber; add the necessary
amount of metal, which will be
different for each treatment; mix
thoroughly; let stand for 1 to 4
hours.
b. Prepare a large volume of the
highest test concentration in the
laboratory dilution water; perform
serial dilution using a graduated
cylinder and the well-mixed spiked
and unspiked samples of the
laboratory dilution water; let stand
for 1 to 4 hours.
17. Add the test organisms, acclimated per
section B. 1, to the test chambers for the
side-by-side tests at the same time.
18. Observe the test organisms and record
the effects and symptoms as specified by
U.S. EPA (1993) and/or ASTM (1999,
2000a).
F. Chemical and Other Measurements
1. To reduce the possibility of
contamination of test solutions before or
during tests, do not place thermometers
and probes for measuring pH and
dissolved oxygen into test chambers.
Rather, perform such measurements on
"chemistry controls" that contain test
organisms or on aliquots that are
removed from the test chambers. The
other measurements may be performed
on the actual test solutions at the
beginning and/or end of the test or the
renewal.
2. Measure hardness, pH, alkalinity, TSS,
and DOC for the tested site water and
the laboratory dilution water.
3. Measure dissolved oxygen, pH, and
temperature during the test at the times
specified by U.S. EPA (1993) and/or
ASTM (1999, 2000a, 2000b), using the
same schedule for both of the side-by-
11
-------
side tests. If chemistry controls
are used, obtain measurements on
both the chemistry controls and
actual test solutions at the end of
the test.
4. For a dissolved WER calculated from
dissolved measurements, measure
dissolved copper in the appropriate test
solutions. For a total recoverable WER,
measure total recoverable copper. It is
desirable but not essential for both
dissolved and total recoverable copper
to be measured. EPA methods (U.S.
EPA 1997, 1999) are recommended. In
any case use appropriate QA/QC
techniques to assure attaining the target
level of accuracy.
a. Rather than measuring the metal
in all test solutions, it is often
possible to store samples and then
analyze only those that are needed
to calculate the results of the
toxicity tests. Measure (i) all
concentrations in which some, but
not all, of the test organisms were
adversely affected, (ii) the highest
concentration that did not
adversely affect any test
organisms, (iii) the lowest
concentration that adversely
affected all of the test organisms,
and (iv) the controls.
b. For total recoverable copper,
measure once for a static test, or
twice for a renewal test (once per
day). For measurement of total
recoverable copper in the test
chamber, mix the whole solution
in the chamber before the sample
is taken for analysis. Do not
acidify the solution in the test
chamber before the sample is
taken. Rather, acidify after it is
placed in the sample container.
c. Measure dissolved metal at the
beginning and end of a static test,
or at the beginning and end of the
first day in a renewal test (that is,
just before renewal). For
measurement of dissolved metal in
a test chamber, mix the whole
solution in the test chamber before
removing a sufficient amount for
filtration. Do not acidify before
filtration. Filter the sample within
on hour after it is after it is taken,
then acidify the filtrate.
5. Perform QA/QC checks.
G. Calculating and Interpreting the
Results
1. Evaluate the acceptability of each
toxicity test individually.
a. Reject tests where deviations from
the above presented laboratory
practices are substantial,
particularly with respect to
acclimation, randomization,
temperature control, measurement
of metal, and/or disease or
disease-treatment.
b. Reject tests where more than 10
percent of the organisms in the
controls were adversely affected.
2. Calculate the EC50 using methods
described by U.S. EPA (1993) or
ASTM (1999, 2000a). If two or more
treatments affected between 0 and 100
percent in both tests in a side-by-side
pair, use probit analysis to calculate
results of both tests, unless the probit
model is rejected by the goodness of fit
test in one or both of the acute tests. If
probit analysis cannot be used, either
because fewer than two percentages are
between 0 and 100 percent or because
12
-------
the model does not fit the data, use
computational interpolation; do not use
graphical interpolation. Use the same
computational method for each of the side-
by-side tests.
3. F or 1 ab oratory water:
a. Calculate or assign the EC50 for
the lab water only if the percent of
the organisms that were adversely
affected is greater than 50 percent
in at least one treatment (although
it is preferable if at least 63
percent of organisms were
affected). That is, if there is
insufficient toxicity at all
concentrations in the laboratory
water, the side-by-side tests are
not usable for obtaining a
WER>1.
b. If no treatment other than the
control affected less than 50
percent of the test organisms, set
the EC50 equal to the lowest test
concentration (preceded by <
sign) . That is, if there is
excessive toxicity at all tested
concentrations (except the
control), the laboratory water
EC50 is known only to be less
than the lowest treatment
concentration.
c. If the hardness-normalized EC50
in laboratory water is less than the
documented SMAV for the
species, then use the SMAV in
place of the laboratory water
EC50 in the dominator of the
WER. See Appendix B for the
SMAVs for Ceriodaphnia dubia
and Daphnia magna.
4. For site water:
a. Calculate or assign the EC50 for
the site water only if the percent
of the organisms that were
adversely affected is less than 50
percent in at least one treatment
(although it is preferable if less
than 37 percent of organisms were
affected). That is, if there is
excessive toxicity at all tested
concentrations in site water, the
sample is not usable for obtaining
aWER>l.
b. If no treatment affected more than
50 percent of the test organisms,
set the EC 50 equal to the highest
test concentration (preceded by >
sign). That is, if there is
insufficient toxicity at all tested
concentrations, the site water
EC50 is known only to be greater
than the highest treatment
concentration.
5. In reporting results, highlight anything
unusual or questionable about the test
findings.
a. Report if dissolved metal
decreased by more than 50
percent from the beginning to the
end of a 48-hour static test.
b. Report if there were inversions in
the data for more than two
concentrations in the range of 20
to 80 percent mortality (or as
modified by Abbott's formula).
6. Normalize the (a) laboratory-water
EC50, (b) the site-water EC50, and (c)
the SMAV EC50 to the same hardness,
using the formula:
EC50
at Std Hdns
= EC50
at Sample Hdns ^ Samp|e
Std Hdns
13
-------
Where "Std Hdns" is any particular
standard hardness value to which all
values will be normalized, and "Sample
Hdns" is the hardness of the laboratory
water, the site water, or the SMAV.
The exponent 0.9422 is the log-log
slope for the 1984/1985 and 1995 EPA
acute criteria. If different from 0.9422,
it is appropriate to use the hardness
slope of the state criterion.
7. Calculate the sample WER from values
normalized to the same hardness.
a. If the laboratory-water, hardness-
normalized EC50 is greater than
the hardness-normalized SMAV,
the sample WER equals the site-
water EC50 divided by the
laboratory-water EC50.
b. If the laboratory-water, hardness-
normalized EC50 is less than the
hardness-normalized SMAV, the
sample WER equals the site-water
EC50 divided by the SMAV. See
Appendix B for a value of the
SMAV. Other comparably well-
documented values for the SMAV
may be used.
8. Calculate the site WER as the geometric
mean of the two (or more) sample
WERs. Compare the result against
WER values typically expected for the
type of situation under study.
Additional chemical or toxicological
data may be needed to support unusually
high WERs.
9. Calculate the site acute and chronic
criteria concentrations as the ordinary
hardness-adjusted criteria concentration
(that is, the value that would have been
applicable for a default assumption that
WER=1), multiplied by the final site
WER.
H. Reporting the Results
1. Include the following general
information in the report submitted to
the appropriate regulatory agency:
a. Identity of the investigators and
the laboratory.
b. Name, location, and description of
the discharger; description of the
effluent and the receiving water.
c. Effluent and upstream water flows
used to calculate the dilution ratio.
d. Dilution ratio used in mixing
effluent and upstream water to
prepare the site water.
e. Downstream design hardness
expected to be used for the permit
derivation.
f The values of the site-specific
criterion and WER estimated to
correspond to a determination of
no reasonable potential or to other
pollution control decision
benchmarks relevant to the
purpose of the study.
g. Identification of each sampling
station.
h. Procedures used to obtain,
transport, and store the samples of
the upstream water and the
effluent.
i. Any pretreatment, such as
filtration, of the effluent, site
water, and/or laboratory dilution
water.
j. Description of the laboratory
dilution water, including source,
and preparation.
k. Results of all chemical and
physical measurements on
upstream water, effluent, actual
and/or simulated downstream
water, and laboratory dilution
water, including hardness,
alkalinity, pH, and concentrations
14
-------
of total recoverable or dissolved
metal, TSS, and DOC.
1. Description of the experimental
design, test chambers, volume of
solution in the chambers,
photoperiod, and numbers of
organisms and chambers per
treatment.
m. Source and grade of the copper
salt, and how the stock solution
was prepared.
n. Species and source of the test
organisms, age, and holding and
acclimation procedures.
o. The average and range of the
temperature, pH, hardness,
alkalinity, and the concentration of
dissolved oxygen (mg/L) during
acclimation.
2. Include the following information for
each sample or toxicity test.
a. Date and time of sampling site
water and date of toxicity test.
b. Effluent flow during the sampling
event.
c. Upstream flow during and prior to
the sampling event, either
measured directly or estimated
from relevant neighboring gages.
d. Prior meteorological conditions
affecting flow and sampled water
quality.
e. Measurements of hardness,
alkalinity, pH, and DOC.
f The average and range of the
measured concentrations of
dissolved oxygen (in mg/L).
g. The average and range of the test
temperature.
h. A summary table of the
concentrations of copper in each
treatment, including controls, and
the number of organisms affected,
in sufficient detail to allow
independent statistical analysis of
the data.
i. The EC50 and the method used to
calculate it.
j. Anything unusual about the test,
any deviations from the
procedures described above, and
any other relevant information.
k. All differences, other than the
dilution water and the
concentrations of metal in the test
solutions, between the side-by-
side tests using laboratory dilution
water and site water.
3. Include the following information in a
summary table:
a. ECSOs and hardness for each test
in a site-water, laboratory-water
comparison, not normalized for
hardness.
b. ECSOs for each site-water,
laboratory-water (or SMAV)
comparison, after normalizing for
hardness.
c. The calculated sample WERs.
4. Present the calculated site WER and site
criterion.
References for Appendix A
ASTM. 1999. ASTM Standards on
Biological Effects and Environmental Fate,
2nd Edition. American Society for Testing
and Materials, West Conshohocken, PA.
ASTM. 2000a. E729-96 Standard Guide
for Conducting Acute Toxicity Tests on Test
Materials with Fishes, Macroinvertebrates,
and Amphibians. American Society for
Testing and Materials, West Conshohocken,
PA.
15
-------
ASTM. 2000b. El 192-97 Standard Guide
for Conducting Acute Toxicity Tests on
Aqueous Effluents with Fishes,
Macroinvertebrates, and Amphibians.
American Society for Testing and Materials,
West Conshohocken, PA.
U.S. EPA. 1993. Methods for Measuring
the Acute Toxicity of Effluents and
Receiving Waters to Freshwater and Marine
Organisms. Fourth Edition.
http ://www. epa. gov/ost/WET/di sk2/.
EPA/600/4-90/027F. National Service
Center for Environmental Publications,
Cincinnati, OH.
U.S. EPA. 1997. Trace Metals Guidance
(diskette). EPA 821/C-97-002.
http://www.epa.gov/OST/pc/analytic.html.
EPA Office of Water Resource Center,
Washington, DC.
U.S. EPA. 1999. Methods and Guidance
for the Analysis of Water, Revision 2 (on
CD-ROM). EPA 821/C-99-004.
http://www.epa.gov/OST/pc/analytic.html.
EPA Office of Water Resource Center,
Washington, DC.
16
-------
Appendix B
Species Mean Acute Values
for Ceriodaphnia dubia and Daphnia magna
Species Mean Acute Values, SMAVs, for Ceriodaphnia dubia and Daphnia
magna were obtained by tabulating available toxicity data, normalizing for
hardness differences using the 1985 and 1995 EPA hardness slope for copper, and
calculating the geometric mean of all EC50 results for each species.
Normalized to a hardness of 50 and 100 mg/L as CaCO3, the results are as
follows:
Species
Ceriodaphnia dubia
Daphnia magna
SMAV EC50, ug/L
At Hardness = 50 mg/L
Total Cu
12.49
10.47
Dissolved Cu
11.51
10.05
At hardness = 100 mg/L
Total Cu
24.00
20.12
Dissolved Cu
22.11
19.31
The SMAV at any other hardness is obtained as follows:
Site Hdns\ o.9422
SMAVatSiteHdns = SMAVatHdns=100
100 mg/L
The exponent, 0.9422 is the EPA's copper criterion hardness slope. It is slightly
higher than found in those Table B-l studies that investigated the effect of
hardness: Belanger et al. 1989, Belanger and Cherry 1990, Chapman et al.
manuscript, and Borgmann and Charlton 1984. Other well documented slopes
may also be suitable for normalizing the data.
17
-------
Table B-1: ECSOs for 59 reported results with Ceriodaphnia dubia and 55 reported
results with Daphnia magna.
Species
Ceriodaphnia
dubia (<24 h)
Ceriodaphnia
dubia (<24 h)
Ceriodaphnia
dubia (<24 h)
Ceriodaphnia
dubia (<24 h)
Ceriodaphnia
dubia (<24 h)
Ceriodaphnia
dubia (<24 h)
Ceriodaphnia
dubia (^2 h)
Ceriodaphnia
dubia
Ceriodaphnia
dubia
Ceriodaphnia
dubia (<24 h)
Hrdns
(mg/L as
Method3 CaCO3)
S, M 45
S, M 94.1
S, M 179
S, M 97.6
S, M 113.6
S, M 182.0
S, M 90
S, M 87.5
80.8
80.8
60
30
S, U 188
204
428
410
494
440
S, M 80
EC50
Total
(M9/L)
14
28
31
52
76
91
56
84
93
13.4
11.25
13.17
25.25
11.25
4.5
36.6
19.1
36.4
11.7
12.3
12.0
6.98
EC50
Diss.
(M9/L)
25.0
17.0
30.0
24.0
28.0
32.0
23.0
20.0
19.0
26.0
21.0
27.0
37.0
34.0
66.0
63.0
71.0
67.0
38.0
78.0
81.0
EC50 Total,
Adjusted to
Hrdns=100
(M9/L)b
14.32
28.65
31.72
46.11
67.40
80.70
31.85
47.78
52.90
14.80
12.76
16.10
30.87
18.20
13.99
20.19
9.76
9.25
3.10
2.73
2.97
8.61
EC50 Diss.,
Adjusted to
Hrdns=100
(M9/L)b
53.05
36.07
63.66
50.93
59.42
67.90
48.81
42.44
40.32
27.53
22.24
28.59
39.18
36.00
69.89
66.72
75.19
38.71
21.96
45.07
46.80
Reference
Belanger et al.
1989
Belanger et al.
1989
Belanger et al.
1989
Belanger and
Cherry 1990
Belanger and
Cherry 1990
Belanger and
Cherry 1990
Orisetal. 1991
Neserke 1994
Bright 1995
Diamond et al.
1997b
18
-------
Species Method
Ceriodaphnia S, M
dubia
Ceriodaphnia S, M
dubia
Daphnia magna S, U
(<24 h)
Daphnia magna S, U
(1d)
Daphnia magna S, U
Daphnia magna S, M
Daphnia magna S, U
(<24 h)
Daphnia magna S, U
(<24 h)
Daphnia magna S, M
(<4h)
Daphnia magna S, M
(1d)
(2d)
(3d)
(4d)
(5d)
(6d)
Daphnia magna S, U
Daphnia magna S, U
Hrdns
(mg/L as
CaCO3)
99
70
74
72
148
148
142
144
148
200
193
198
194
78
90
90
45.3
99
45
100
45
240
54
80
240
10
EC50
Total
(M9/L)
10.1
14.65
6.72
6.59
23.3
24.99
18.91
73.5
18.48
31.77
58.82
31.53
39.38
13.1
8.88
10.3
9.8
85
50
10
31.8
54
41
7
10
6
14
7
10
18
93
21.5
EC50
Diss.
(M9/L)
10.65
13.34
6.53
4.55
17.74
15.34
14.7
49
8.24
20.99
32.39
21.14
18.25
EC50 Total,
Adjusted to
Hrdns=100
(M9/L)b
10.20
20.50
8.92
8.98
16.10
17.27
13.59
52.13
12.77
16.53
31.66
16.57
21.09
16.56
9.81
11.37
20.67
85.81
50.48
21.22
31.80
114.59
17.97
12.51
12.34
7.40
17.28
8.64
12.34
22.21
40.76
188.21
EC50 Diss.,
Adjusted to
Hrdns=100
(M9/L)b
10.75
18.67
8.67
6.20
12.26
10.60
10.56
34.75
5.70
10.92
17.43
11.11
9.77
Reference
Tetra Tech
1998
Dimond 2000
Biesinger &
Christensen
1972
Adema &
Deg root-van
Zijl 1972
Cairns et al.
1978
Borgmann &
Ralph 1983
Mount &
Norberg 1984
Elnabarawy et
al. 1986
Nebeker et al.
1986
Nebeker et al.
1986
Khangarot and
Ray 1989
Mickey and
Vickers 1992
19
-------
Species Method3
Daphnia magna S, U
Daphnia magna S, U
Daphnia magna S, M
Daphnia magna S, M
Daphnia magna S, M
(<24 h)
Daphnia magna S, M
(<24 h)
Daphnia magna S, M
(<24 h)
Hrdns
(mg/L as
CaCO3)
33.8
50
52
105
106
207
170
100
170
72
76
80
72
84
84
44
44
36
76
88
EC50
Total
(M9/L)
11.5
7
26
30
38
69
41.2
10.5
20.6
17.3
70.7
31.3
7.1
18.7
18.9
31
38
35
58
37
51
39
50
52
31
30
46
63
4.8
6.24
6.17
6.62
10.2
10.8
4.6
7.8
3
10.4
16.9
EC50 EC50 Total,
Diss. Adjusted to
(|jg/L) Hrdns=100
(M9/L)b
31.96
13.45
48.15
28.65
35.97
34.76
24.99
6.37
12.50
10.49
42.88
18.99
7.10
18.70
18.90
18.80
23.05
21.23
35.18
22.44
30.93
23.66
30.33
31.54
18.80
18.20
27.90
38.21
6.54
8.08
7.61
9.02
12.02
12.73
9.97
16.91
7.86
13.47
19.06
EC50 Diss.,
Adjusted to
Hrdns=100 Reference
(M9/L)b
Koivisto et al.
1992
Oikari et al.
1992
Chapman et al.
Manuscript
Baird et al.
1991
Meador 1991
Lazorchak and
Waller 1993
Dimond 2000
a Methods: S = Static, M = Measured, U = Unmeasured
b Adjusted EC50 at hardness 100 mg/L is obtained from the test measured dissolved or total EC50 as follows:
Adjusted EC50 = Test EC50
TestHdns'
Where only the test total EC50 is known, the test dissolved EC50 is estimated from EC50D = EC50T • 0.96.
Where only the test dissolved EC50 is known, the test total EC50 is estimated from EC50T = EC50D/0.96.
20
-------
References for Appendix B
Adema, D.M.M. and A.M. Degroot-van Zijl.
1972. The influence of copper on the water
fieaDaphnia magna. TNO Nieuws 27:474.
Baird, D.J., I. Barber, M. Bradley,
A.M.V.M. Scares and P. Calow. 1991. A
comparative study of genotype sensitivity to
acute toxic stress using clones ofDaphnia
magna Straus. Ecotoxicol. Environ. Saf.
21(3):257-265.
Belanger, S.E., J.L. Farris andD.S. Cherry.
1989. Effects of diet, water hardness, and
population source on acute and chronic
copper toxicity to Ceriodaphnia dubia.
Arch. Environ. Contam. Toxicol. 18(4):601-
11.
Belanger, S.E. andD.S. Cherry. 1990.
Interacting effects of pH acclimation, pH,
and heavy metals of acute and chronic
toxicity to Ceriodaphnia dubia
(Cladoceran). J. Crustacean Biol. 10(2):225-
235.
Biesinger, K.E. and G.M. Christensen. 1972.
Effects of various metals on survival,
growth, reproduction, and metabolism of
Daphnia magna. Jour. Fish Res. Board Can.
29:1691.
Borgmann, U. andK.M. Ralph. 1983.
Complexation and toxicity of copper and the
free metal bioassay technique. Water Res.
17:1697.
Bright, G.R. 1995. Variability of the "water
effect ratio" for copper toxicity - a case
study. Proc. Toxic. Subst. Water Environ.
5/23-5/30. Water Environment Federation,
Alexandria, VA.
Cairns, J., Jr. et al. 1978. Effects of
temperature on aquatic organism sensitivity
to selected chemicals. Bulletin 106. Virginia
Water Resources Research Center,
Blacksburg, VA.
Chapman, G.A. et al. Manuscript. Effects of
water hardness on the toxicity of metals to
Daphnia magna. U.S. EPA, Corvallis, OR.
Diamond, J.M., D.E. Koplish, J. McMahon,
III and R. Rost. 1997b. Evaluation of the
water-effect ratio procedure for metals in a
riverine system. Environ. Toxicol. Chem.
16(3):509-520.
Dimond, W.F. 2000. Letter to Charles
Delos: peer review of Streamlined Water-
Effect Ratio Procedure. July 21, 2000.
State of Michigan, Department of
Environmental Quality. Lansing, MI.
Elnabarawy, M.T., A.N. Welter and R.R.
Robideau. 1986. Relative sensitivity of three
daphnid species to selected organic and
inorganic chemicals. Environ. Toxicol.
Chem. 5(4):393-8.
Hickey, C.W. andM.L. Vickers. 1992.
Comparison of the sensitivity to heavy
metals and pentachlorophenol of the mayflies
Deleatidium spp. and the cladoceran
Daphnia magna. N.Z.J. Mar. Freshwater
Res. 26(l):87-93.
Khangarot, B.S. and P.K. Ray. 1989.
Investigation of correlation between
physicochemical properties of metals and
their toxicity to the water flea Daphnia
21
-------
magna Straus. Ecotoxicol. Environ. Saf.
18(2): 109-20.
Koivisto, S., M. Ketola and M. Walls. 1992.
Comparison of five cladoceran species in
short- and long-term copper exposure.
Hydrobiol. 248(2): 125-36.
Lazorchak, J.M. and W.T. Waller. 1993. The
relationship of total copper 48-H LCSOs to
Daphnia magna dry weight. Environ.
Toxicol. Chem. 12(5):903-11.
Meador, J.P. 1991. The interaction of pH,
dissolved organic carbon, and total copper in
the determination of ionic copper and
toxicity. Aquat. Toxicol. 19(1): 13-31.
Mount, D.I. and T.J. Norberg. 1984. A
seven-day life-cycle cladoceran toxicity test.
Environ. Toxicol. Chem. 3:425.
Neserke, G. 1994. Written testimony to
Colorado Department of Health Water
Quality Control Commission on behalf of the
Coors Brewing Company.
Oikari, A., J. Kukkonen and V. Virtanen.
1992. Acute toxicity of chemicals to
Daphnia magna in humic waters. Sci. Total
Environ. 117-188:367-77.
Oris, J.T., R.W. Winner and M.V. Moore.
1991. A four-day survival and reproduction
toxicity test for Ceriodaphnia dubia.
Environ. Toxicol. Chem. 10(2):217-24.
Tetra Tech. 1998. Determination of Copper
LCSOs for Calculation of Water Effect
Ratios and Effluent Limits for POTWs in
Southeastern Pennsylvania. Tetra Tech,
Owings Mills, MD.
Nebeker, A.V., M.A. Cairns, S.T. Onjukka
and R.H. Titus. 1986. Effect of age on
sensitivity of Daphnia magna to cadmium,
copper and cyanazine. Environ. Toxicol.
Chem. 5(6):527-30.
22
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Appendix C
Assessment of the Streamlined Water-Effect Ratio Procedure
Abstract
The protectiveness of the Streamlined
Procedure for obtaining a copper water-
effect ratio (WER) for streams affected by
point source discharges has been assessed
using Monte Carlo probabilistic modeling.
The Streamlined Procedure uses two WER
samples to set the site WER. The
probabilistic modeling considered that the
two WER samples were collected from a
situation where the flow, toxicant
concentration, and WER vary over time.
This analysis evaluated the suitability of
calculating each site's WER as the geometric
mean of the two WER measurements for the
site, when the effluent and upstream samples
are mixed at the design low-flow dilution.
Comparison was made against the predicted
unbiased value for the WER: that is, the
value that the WER would have if the site-
specific criterion were to have the same level
of protection as intended for the national
criterion.
Overall the results of this work indicated that
the Streamlined Procedure tends to yield a
site WER slightly more restrictive than the
unbiased site WER. In 50 percent of the
Monte Carlo trials, the calculated WER was
less than 0.84 times the unbiased site WER.
Within the range of conditions investigated,
the design downstream dilution had no
significant effect on the level of protection
provided.
In addition, in the Supplement to this
Appendix, an estimate was made of the
effect, relative to the 1994 Interim
Procedure, of having the Streamlined
Procedure restrict the lab water EC50 to a
value not less than the EPA SMAV. Sixteen
lab water ECSOs from three WER studies
were evaluated. By including the difference
in the way the two procedures set the lab
water EC50, the Streamlined Procedure and
the Interim Procedure could be appropriately
compared through the Monte Carlo
simulation. Results indicated that the two
procedures yielded similar results.
Introduction
The purpose of this assessment is to
determine, through modeling, whether the
Streamlined Procedure provides the degree
of protection intended for aquatic life
criteria. The Supplement to this Appendix
also deals with the following issues: (a) the
purpose of streamlining the copper WER
procedure, (b) the reason for preparing
samples by mixing at design dilution, (c) the
need for the simultaneous laboratory water
test, (d) the potential for reducing the
number of samples, and (e) a comparison
with the 1994 Interim Procedure.
Assessment Strategy
The water-effect ratio (WER) reflects the
effect that local site water constituents have
on increasing or reducing the pollutant
bioavailability and toxicity. The
23
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concentrations of the site water constituents
that control the WER can be expected to
vary over time, as do all water quality
parameters. A site's WER would thus also
vary over time. Consequently, the number of
samples in any reasonably feasible sampling
scheme cannot fully characterize the site.
The question investigated here is whether it
is appropriate to calculate the site WER as
the geometric mean of the two samples,
mixed at the low-flow dilution. This issue
can be addressed by asking: How does the
level of protection provided by such a WER
derivation compare with the level of
protection intended for aquatic life criteria?
If one continually knew what the actual
WER was during each increment of time,
then one could continually reset the site
criterion such that it would always correctly
reflect the actual conditions. Thus, if one
made continual WER measurements and
continual adjustments to the site criterion,
then there would be no "final WER" and no
issue about its method of calculation.
Assuming that the WER measurements were
correct, the continual adjustments of the site
criterion would yield a level of protection
exactly equal to what was intended for the
criterion.
It is of course infeasible to continually
measure the WER over time. However,
since we know something about the
statistical properties of water quality
parameters, it is feasible to set up a
mathematical modeling experiment to mimic
continual measurements, using realistic-
looking hypothetical distributions of WER
values and other site parameters. In this way
it is possible to examine the behavior of
various sampling and calculation alternatives.
That is, given an assumed distribution of
time-variable WERs, one can compare (a)
the level of protection provided by any
alternative for obtaining the final WER (and
site criterion), to (b) the neutral or unbiased
level of protection provided by continually
resetting the site criterion using the WER
specific to each event.
The basic strategy for performing this
analysis (modified from Delos 1998, which in
turn evolved from Delos 1994) is to set up
one or more scenarios of hypothetical site
applications by specifying the statistical
properties of the facility WERs, flows, and
toxicant concentrations. Reflecting the time
variability of a site, a very large number of
such WERs, with accompanying flow and
toxicant concentration values, can then be
randomly generated. One may then compare
the behavior of (a) the Streamlined
Procedure for deriving a site WER, with (b)
the unbiased technique of continually
resetting the WER and site criterion.
For a situation where the WER is varying
over time, and the site criterion is continually
being re-set in accordance with the WER,
the time variations in the toxicant
concentration will yield some frequency of
criteria violations. Assuming that this
frequency of criteria violations is acceptable,
one can find, by trial and error, a single,
fixed (time-invariant) value for the WER,
and likewise a single, fixed site criterion, that
yields exactly the same frequency of
violations as the time-variable WER and
time-variable site criterion. This single, fixed
value for the WER, yielding the same
violation frequency as the time-variable
WER, may be termed the unbiased final
WER.
This fixed unbiased WER represents the
"correct" value for site's final WER.
However, its value could only be ascertained
if one had a long record of measured WERs
and toxicant concentrations for the site,
something that can be obtained for a model
24
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simulation site but not a real-world site. In a
real-world site only a small sampling is
obtained for the values that the WER may
take over time. The purpose of the Monte
Carlo modeling is to compare the final WER
values derived from small samples of the of
the site's distribution of WERs to the correct
value represented by the unbiased final
WER.
The Monte Carlo analysis considers that a
site has time-variable values for streamflow,
toxicant concentration, and WER. It also
considers that the sites to which the
Streamlined Procedure applies differ
somewhat in their design flow dilution ratio.
Modeling Procedure
It is not necessary to read this section in
order to understand and use the modeling
results presented later. Readers not
interested in the procedural details may skip
to Results, the next section.
The Monte Carlo model is set up as follows.
For any particular discharge situation, the
following sources of variability are taken into
account:
• Time variable dilution ratio of upstream
flow to effluent flow
• Upstream time-variable toxicant
concentration
• Upstream time-variable WER
• Effluent time-variable toxicant
concentration
• Effluent time-variable WER
That is, the modeling work assumes that the
dilution flow, toxicant concentration, and
WER vary over time. In addition to time
variability within a site, the analysis
recognizes that between various sites,
discharges differ somewhat in their design-
flow dilution.
The steps for setting up and evaluating a site
scenario are set forth below. The
computations were set up as a Quattro Pro
spreadsheet.
1. Consider an effluent discharged to a
stream. The effluent and upstream
waters are each characterized by three
parameters: WER, toxicant
concentration, and flow. The
downstream toxicant concentration and
WER are calculated by dilution. Such a
calculation for the WER assumes that
the WER is controlled by and directly
related to the underlying concentrations
of water quality parameters (such as
dissolved organic carbon) that are
appropriately calculated by the dilution
formula.
2. Assume that effluent and upstream
WERs, toxicant concentrations, and
flows are log normally distributed, each
with particular values for its log mean
and log standard deviation. Time
variable effluent and upstream flows
were made to fit one of three categories:
(a) design IWC of 91%, (b) design IWC
of 50%, and (c) design IWC of 33%.
Flow parameters were selected such that
downstream flows less than the design
low flow occurred 3% of the time.
Because the copper discharged by
sewage treatment facilities (by far the
most common copper discharge) is
physico-chemically associated with the
discharged organic matter, effluent
toxicant concentration and effluent
WER were modeled to be 40-50%
correlated with each other. Such degree
of correlation is directly supported by
State of Connecticut data (Dunbar
1997c), by wastewater treatment
25
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process modeling, and indirectly by
Pennsylvania municipal discharger
data (Hall and Associates 1998).
3. Using random number generators that
produce log normal values having the
proper mean and standard deviation,
generate random values for five
parameters: effluent and upstream
WERs and toxicant concentrations, and
upstream flow. This constitutes one
event.
4. Using the dilution formula, calculate the
downstream WER and toxicant
concentration for the Step 3 event.
Because the streamlined approach mixes
effluent and upstream water at the
design IWC, irrespective of the actual
IWC occurring during the sampling
event, this modeling analysis must keep
track of two separate downstream
WERs for the event: (a) the downstream
WER calculated to occur for the actual
event IWC, and (b) the downstream
WER that would be measured if effluent
and upstream waters were mixed at the
design IWC.
5. In like manner, generate a large number
of events. For this work, 1000 events
were used for each of the three dilution
categories (33%, 50%, and 91% design
IWC). Each event is characterized by
the upstream and effluent values for
WERs, concentrations, and flows, and
by the calculated downstream values for
these parameters.
6. Specify an appropriate relative value for
the national criterion for the toxicant.
Then for each of the 1000 events
calculate a site criterion as the product
of the national criterion times the event's
downstream WER (at the actual event
dilution). For each event determine
whether the toxicant concentration
exceeded the event-specific site
criterion. Count the number of
excursions of the event-specific criterion
in all 1000 events.
Toxicant concentration and WER mean
values and relative national criterion values
in Steps 2 and 6 were selected such that
using reasonable values for log variances, the
site criterion was violated 1% of the time
overall.
7. Arbitrarily pick some value for the
overall final WER, to remain a fixed
constant across all events. Calculate a
fixed value for the site criterion, as the
product of the national criterion times
the fixed final WER. For each event
determine whether the toxicant
concentration exceeded the fixed value
for the site criterion. Count the number
of excursions of the event-specific
criterion for all 1000 events. Compare
this excursion frequency with that
obtained in Step 6.
8. Repeat Step 7, adjusting the value by
trial and error, until the fixed final WER
yields the same excursion frequency as
did the variable criterion from Step 6.
The value so obtained may be called the
unbiased final WER. It is the value the
final WER should have if it were to
produce the level of protection intended
for the criterion.
The remaining steps consider the possible
outcomes if (a) sample WERs were obtained
for two events, in accord with the
Streamlined Procedure, and (b) the final
WER is set equal to the geometric mean the
two sample WERs.
9. For each of 999 pairs of WER
measurements in each of the three
26
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10.
dilution scenarios (33%, 50%, and
91% IWC), calculate a site WER
equal to the geometric mean of each
pair of measurements. (Form pairs
from the first and second events,
second and third events, and so on
down the list, thereby yielding 999
pairs.)
For each of the three dilution scenarios,
examine the distribution of 999 site
WERs obtained by Step 9. Determine
the WERs corresponding to various
percentiles in the distribution (such as 50th,
85th, and 95th percentiles). Compare these
with the unbiased WER obtained from Step
8, in order to ascertain how often the site
WER would be under-protective, and how
often it would be over-protective.
Values for key parameters are shown in
Table C-l. The log standard deviations are
based on past experience with selected water
quality measurements from well conducted
studies.
Table C-1. Values for key input parameters and selected intermediate outputs.
0.5
0.5
1.0
0.5
0.5
0.0
0.5
0.43
4.0
33%
51%
91%
8%
14%
63%
3%
1%
Standard deviation of log upstream toxicant concentration
Standard deviation of log upstream WER
Standard deviation of log upstream flow
Standard deviation of log effluent concentration
Standard deviation of log effluent WER
Standard deviation of log effluent flow
Correlation (r2) between log effluent toxicant concentration and log effluent WER
Correlation (r2) between effluent toxicant concentration and effluent WER
Ratio, effluent mean WER : upstream mean WER
Design IWC for higher dilution facility in scenario
Design IWC for medium dilution facility in scenario
Design IWC for low dilution facility in scenario
Overall geometric mean IWC for higher dilution facilities in group
Overall geometric mean IWC for medium dilution facilities in group
Overall geometric mean IWC for low dilution facilities in group
Frequency of flows below design flow
Frequency of site-specific criteria violations
27
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Results
The results were used to address the
question: How much protection is provided
if the site WER is set equal to the geometric
mean of two sample WERs?
The WER established as such a geometric
mean will be here termed the procedure
WER. Table C-2 shows the ratio of the
procedure WER to the unbiased WER for
the typical or 50th percentile situation and
worst case 95th percentile situation among
the 999 final WERs obtained from the Monte
Carlo analysis of each of the three dilution
scenarios.
The level of protection provided by the
Streamlined Procedure does not vary
significantly among the dilution scenarios,
relative to other uncertainties and random
influences. Irrespective of design IWC, the
Table C-2 Monte Carlo results indicate that
the substantial majority of cases, a
streamlined Procedure WER will be below
the site's unbiased WER. The probability of
obtaining a procedure WER greater than the
site's unbiased WER averaged 29% among
sites.
Data from Dunbar (1997a, 1997b, 1997c)
indicate that over time, the measured WERs
at a site are less variable than assumed in this
Monte Carlo analysis. Consequently, these
results probably represent a conservative
worst case portrayal of the performance of
the Streamlined Procedure for the type of
scenarios considered.
The possibility that a site could be assigned a
criterion concentration somewhat greater
than ideal is an inherent risk associated with
both national and site-specific criteria. If
most dischargers are to be assigned a WER
not too far below what they deserve, the luck
of the draw during sample collection will
yield some site WERs somewhat higher.
However, for the criterion in question, there
is no reason to expect aquatic communities
to be sensitive to minor errors or
uncertainties in criteria setting. Application
of any criterion will always involve some
potential for inaccuracy, whether adjusted
using the Streamlined Procedure, the 1994
Interim Procedure, the Biotic Ligand Model,
an empirical hardness relationship, or
whether not adjusted at all for site water
quality.
The performance of the Streamlined
Procedure was also compared with the 1994
Interim Procedure. This comparison is
discussed in the Supplement to Appendix C.
Table C-2. Monte Carlo prediction of relationship between the procedure WER and the
unbiased WER.
Design IWC
91% Design IWC
50% Design IWC
33% Design IWC
Mean of scenarios
Ratio of Procedure WER : Unbiased WER
50th Percentile
0.82
0.81
0.90
.84
95th Percentile
1.49
1.38
1.42
1.43
Probability of
exceeding the
Unbiased WER
30%
25%
33%
29%
28
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Conclusion
Analysis of the behavior of the Streamlined
Procedure using Monte Carlo modeling
techniques has indicated that the procedure
provides a level of protection close to that
intended for the criteria.
References for Appendix C
Biological Monitoring, Inc. 1992. Site-
Specific Study for Computing Metal
Standards for the Lehigh River and City of
Allentown, Pennsylvania. BMI, Blacksburg,
VA.
Delos, C. 1994. Probabilistic analysis of the
level of protection provided by the interim
guidance on determining water-effect ratios.
Draft. Office of Water, U.S. EPA,
Washington, DC.
Delos, C. 1998. Assessment of WER Study
for Aggregated Southeast Pennsylvania
Facilities. Health and Ecological Criteria
Division, U.S. EPA, Washington, DC 20460.
Dunbar, L.E. 1997a. Effect of streamflow
on the ability of ambient waters to assimilate
acute copper toxicity. Connecticut
Department of Environmental Protection.
Dunbar, L.E. 1997b. Derivation of a site-
specific dissolved copper criteria for selected
freshwater streams in Connecticut.
Connecticut Department of Environmental
Protection.
Dunbar, L.E. 1997c. Lotus spreadsheet
(data).
Hall & Associates and Environmental
Engineering & Management Associates.
1998. Evaluation of copper toxicity and
water effect ratio of treated municipal
wastewater. Prepared for Pennsylvania
Copper Group.
MacKnight, E.S. 1997. September 17 letter
to James Newbold. U.S. EPA, Region 3.
Neserke, G. 1994. Written testimony of
George Neserke on behalf of the Coors
Brewing Company. Colorado Department of
Health, Water Quality Control Commission.
U.S. EPA. 1994. Interim guidance on
determination and use of water-effect ratios
for metals. EPA-823-B-94-001.
29
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30
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Supplement to Appendix C
Discussion of Issues Pertaining to the Streamlined Procedure
This supplement discusses several issues
associated with the Streamlined Procedure.
Why was a streamlined WER procedure
developed?
The national recommended criterion for
copper has now taken on the role of a
screening tool. Violation of that criterion
does not usually trigger immediate pollution
control, but rather triggers WER studies to
derive a site-specific value for the criterion.
It is common for sewage treatment facilities
to exceed the national recommended copper
criterion. However, when evaluated by
TIEs, actual toxicity due to copper from
such facilities is rare, apparently because of
the excess of complexing organics.
Screening tools can be judged by their
sensitivity and efficiency. EPA's approach
for copper appeared to have few problems
with sensitivity, but its efficiency (actual
problems divided by the sum of false
positives and actual problems) appeared to
be very low, and when applied to sewage
treatment facilities, possibly approaching
zero. Because the cost and complication of
the needless WER studies divert federal,
state, and local government resources away
from more productive efforts, the criteria
program has been subjected to criticism both
from within EPA and from outside EPA.
Recognizing the need for improving the
efficiency of the copper WER approach,
EPA developed a more streamlined
approach, simpler to perform, simpler to
review, but fully protective.
What is involved in recommendations
about the mixing of effluent and upstream
waters in preparation for the toxicity
tests?
The Streamlined Procedure mixes effluent
and upstream waters at the design low-flow
dilution ratio (the design IWC). The 1994
Interim Procedure mixes effluent and
upstream waters at the dilution actually
occurring during the sampling event.
The thinking behind the 1994 Interim
Procedure recommendation, which was
designed primarily for application to total
recoverable metals, was that the suspended
solids concentration tends to increase with
increasing flow, such that merging the high-
flow, high-solids event and the low-dilution,
high-organic-matter sample mixture might
yield an unjustifiably optimistic scenario for a
total recoverable WER. However, for
dissolved metals, particulate matter is not
relevant, and should have little effect on the
dissolved WER. Furthermore, for copper,
since total recoverable is generally not much
greater than dissolved, and since the method
is restricted to copper problems associated
with continuous point sources, it is only
necessary to assure that the event sampled is
not substantially influenced by rainfall runoff.
State of Connecticut (Dunbar 1997a) data
support this: in upstream waters., there is no
apparent relationship between the WER and
streamflow. Absent such a relationship in
upstream waters, there is no particular
reason not to measure the WER using the
IWC in which one is most interested: the
design flow condition.
31
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The State of Connecticut data comparing the
WER with the IWC also suggest that the
WER does not increase as rapidly as the
IWC. In this case, the IWC of greatest
concern would be the design low-flow IWC.
At this time the discharged metal would be
expected to be at its highest level compared
to the WER and the sample-specific
criterion.
The modeling analysis in the main part of
Appendix C nevertheless recognizes and
accounts for the potential difference between
the a sampling-event WER measured for the
actual event IWC, and the sampling-event
WER measured when the sample is prepared
at the low-flow design IWC. Consequently,
the assessed protectiveness of the
Streamlined Procedure is not an artifact of
any assumption that the low flow condition
is the critical condition with respect to
criteria excursions. The modeling analysis
considered all criteria excursions no matter
when they occurred.
Mixing upstream and effluent samples at a
specified dilution substantially reduces the
unpredictability of the results, and allows the
needed sampling work to proceed in a timely
manner (an essential feature for an efficient
approach), the only stipulation being that
rainfall runoff impacted events be avoided.
How does the protectiveness of the
Streamlined Procedure compare with that
of the 1994 Interim Procedure?
The features of the Streamlined Procedure
and Interim Procedures were compared in
Table 1 in the main body of this document.
There are two key differences that affect the
relative protectiveness of the two
procedures. (1) The Interim Procedure
mixes effluent and upstream water at the
dilution occurring at the time of sampling;
the Streamlined Procedure mixes effluent
and upstream at the low-flow dilution. (2)
The Interim Procedure uses the laboratory
water EC50 in the WER denominator; the
Streamlined Procedure uses the SMAV if it
is greater than the laboratory water EC50.
The influence these differences have on the
protectiveness of the procedures appear
largely to balance each other. The two
procedures are expected to be equally
protective.
Mixing effluent and upstream water:
Because flow at the time of sampling is such
a random parameter, effluent dilution and
downstream organic carbon concentration
are thus random uncontrolled factors in the
Interim Procedure. In contrast, flow
occurring at the time of sampling is not
relevant in conducting the test under the
Streamlined Procedure. In general, it can be
expected that organic carbon concentrations
in the tested site water will be higher and less
variable when tested per the Streamlined
Procedure than when tested per the Interim
Procedure. This tends to slightly elevate the
WER under the Streamlined Procedure.
SMA V versus laboratory water EC50:
Experience with the 1994 Interim Procedure
has caused some concern about the values of
the lab-water EC50 used for the side-by-side
comparison. There is a perception among
many WER study reviewers that these lab-
water ECSOs, while within the range of
reasonable values, are usually less than the
SMAV for the test species, thus yielding a
slight bias toward increasing the WER. The
comparatively low calcium-magnesium ratio
of the laboratory water commonly used in
WER tests may account for their relatively
low ECSOs.
Data for 30 separate WER measurements
from studies by four different laboratories
indicated that the laboratory water EC50 for
32
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Ceriodaphnia dubia and Daphnia magna,
when measured for WER purposes, averaged
less than 67% of the SMAVs shown in
Appendix B. There was relatively little
difference among the four laboratories in this
regard. This potential for variability and bias
has been eliminated from the Streamlined
Procedure by stipulating that the
denominator of the WER is the greater of the
lab water EC50 or the SMAV. This
stipulation tends to slightly depress the WER
under the Streamlined Procedure.
The net result is that the differences between
the two procedures appear to essentially
balance each other. As shown in Table C-3,
the Streamlined Procedure yields results that
are comparable to the 1994 Interim
Procedure, analyzed by the Monte Carlo
technique in a similar scenario, while
accounting for the differences between the
two procedures.
Relative to the Streamlined Procedure, the
Interim Procedure may be more variable and
uncertain than Table C-3 would suggest,
because this analysis accounts only for the
typical bias of the lab-water EC50, and not
for its imprecision or variability. Overall the
Streamlined Procedure is expected to yield
somewhat more stable results than the
Interim Procedure.
Could a single WER measurement ever be
used to derive a site WER?
The modeling analysis in the main part of
Appendix C easily lends itself to evaluating
the suitability of using a single WER
measurement. Table C-4, in format similar
to the Table C-2, presents the results. The
results did not vary significantly with design
IWC, so the table shows the combined
results for all three dilution scenarios (33%-
91% IWC).
Table C-3. Comparison of Protectiveness of Streamlined Procedure and 1994 Interim
Procedure.
Streamlined Procedure
Interim Procedure
Ratio of Procedure WER : Unbiased WER
50th Percentile
0.84
0.83
95th Percentile
1.43
1.51
Probability of
exceeding the
Unbiased WER
29%
30%
Table C-4. Monte Carlo predictions comparing the two-sample Streamlined Procedure
with a single-sample approach, both relative to the true unbiased WER.
Two-sample Procedure
Single-sample Approach
Ratio of Procedure WER : Unbiased WER
50th Percentile
0.84
0.84
95th Percentile
1.43
1.72
Probability of
exceeding the
Unbiased WER
29%
35%
33
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Using a single sample rather than the
geometric mean of two samples has no effect
on the median result. However, the luck of
the draw has more influence on a single
sample than the mean of two samples. Of
greatest concern is large difference between
the median and the 95th percentile in the
single-sample approach when compared to
the two sample approach.
Nevertheless, it is recognized that in many
municipal discharge situations, there is a
wide margin of safety between value of the
WER needed to conclude that there is no
reasonable potential for impairment and the
value of the measured values of the site
WER. In a tiered testing approach the
criterion would be more stringent where less
data are available. For a single sample,
obtained during stable flow events, not
influenced by rainfall runoff, with treatment
plant operating well, if the measured WER is
more than a factor of two greater than WER
needed to conclude that there is no
reasonable potential, the above modeling
results do not suggest any particular need for
an additional sample before concluding the
decision making process.
If a single sample is obtained for purposes of
reconfirmation of a previously established
site-specific criterion, then a single
reconfirmation sample should be compared
with the original sample results, without
reference to the above factor of two. In all
cases, occasional reconfirmation testing is
desirable, perhaps on concert with the permit
issuance cycle, even though site-specific
criteria do not generally incorporate any
legally recognized expiration date.
Is simultaneous side-by-side laboratory
water and site water testing needed? Can
the laboratory water EC50 be dispensed
with and replaced by the SMAV in
conjunction with reference toxicant
testing?
Over the years this question has remained
with the WER approach. Insistence on
simultaneous side-by-side testing implies that
the relative positions of the laboratory water
and site water ECSOs are more reliable than
the absolute values. Nevertheless, the
Agency clearly puts a substantial amount of
trust in the absolute values: they are the basis
for the national criterion and the basis for
determining compliance with WET limits.
The advantage of having the simultaneous
laboratory water test is that it accounts for
any variations in the organism culture or test
condition that might affect sensitivity to
copper, even though such variations toward
non-optimal test conditions might often be
expected to depress rather than increase the
EC50. The disadvantage of the laboratory
water test is that it is an additional source of
variation and uncertainty in the WER, and
may be subject to unjustified manipulation.
It also adds somewhat to the study cost.
Nevertheless, without some type of
laboratory water EC50 check, there is
nothing to discourage use of a daphnid
culture that had been bred in water having
high concentrations of copper. This might or
might not mimic genetic adaptation
processes actually occurring at the site. In
any case, it is considered undesirable for the
national criteria program to depend on such
adaptation processes to assure aquatic life
protection.
If the simultaneous laboratory water EC50
were to be eliminated, a state or tribe would
need to substitute in its place a reference
toxicant testing program involving copper.
This would be technically necessary to assure
that the organism culture used for testing
was not insensitive to copper toxicity. The
34
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state or tribe would be responsible for
assuring the adequacy of such an approach.
References for the Supplement
Dunbar, L.E. 1997a. Effect of streamflow
on the ability of ambient waters to assimilate
acute copper toxicity. Connecticut
Department of Environmental Protection
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