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

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          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

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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

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                            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

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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

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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

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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

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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

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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
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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%
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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
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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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