United States

Environmental Protection

Agency

Office of Water

4304

EPA-822-R-01-005

March 2001

Streamlined

Water-Effect Ratio Procedure

for Discharges of Copper

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.
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).
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

1

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-94-001).

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, laboratory-water and sitewater toxicity tests are run to obtain the 48-

hour acute EC50 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. 
2

Table 1. Comparison of Streamlined Procedure and 1994 Interim Procedure

Characteristic 1994 Interim Procedure Streamlined Procedure

Applicability Universal Copper from continuous

discharges

Minimum number of

sampling events

3 2

with recommended restrictions

Minimum number of WER

measurements

4 2 

Minimum number of WER

measurements considered

in obtaining final site WER

3 2

Preparation of constructed

downstream water

Mix effluent and upstream

samples at the dilution ratio

occurring at the time of

sampling

Mix effluent and upstream

samples at the design low-flow

dilution ratio

Calculation of sample

WER

Site water LC ÷ Lab water LC Site water LC ÷ The greater of

 (a) Lab water LC, or

 (b) SMAV

Calculation of final site

WER

Complicated scheme with

six “if...then...else” clauses and

12 possible paths

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.
3

Commentary: If obtained from dissolved

copper EC50s, the site WER multiplied

by the national or state’s hardnessnormalized 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 permitspecific 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 lowflow 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
4

(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 lowflow 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.
5

Hardness measurements are needed to

normalize the laboratory water and sitewater EC50s 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. 

8. 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 EC50s 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. 
6

(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 chemicalspecific 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 sitespecific 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. 
7

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

1. 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 timevariable 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
8

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 μm

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 μm 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
9

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 metalloorganic 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-bycase 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.
10

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 uncertainty 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.1):

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
11

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 wellmixed 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.1):

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-
12

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
13

the model does not fit the data, use

computational interpolation; do not use

graphical interpolation. Use the same

computational method for each of the sideby-side tests.

3. For laboratory 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

a WER>1.

b. If no treatment affected more than

50 percent of the test organisms,

set the EC50 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:
14

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, hardnessnormalized EC50 is greater than

the hardness-normalized SMAV,

the sample WER equals the sitewater EC50 divided by the

laboratory-water EC50.

b. If the laboratory-water, hardnessnormalized 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 welldocumented 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
15

of total recoverable or dissolved

metal, TSS, and DOC.

l. 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-byside tests using laboratory dilution

water and site water.

3. Include the following information in a

summary table:

a. EC50s and hardness for each test

in a site-water, laboratory-water

comparison, not normalized for

hardness.

b. EC50s 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,

2

nd 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.
16

ASTM. 2000b. E1192-97 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/disk2/. 

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.
17

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 SMAV EC50, μg/L

At Hardness = 50 mg/L At hardness = 100 mg/L

Total Cu Dissolved Cu Total Cu Dissolved Cu

Ceriodaphnia dubia 12.49 11.51 24.00 22.11 

Daphnia magna 10.47 10.05 20.12 19.31 

The SMAV at any other hardness is obtained as follows:

The exponent, 0.9422 is the EPA’s copper criterion hardness slope. It is slightly

higher than found in those Table B-1 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.
18

Table B-1: EC50s for 59 reported results with Ceriodaphnia dubia and 55 reported

results with Daphnia magna.

Species Methoda

Hrdns

(mg/L as

CaCO3)

EC50

Total

(μg/L)

EC50

Diss.

(μg/L)

EC50 Total,

Adjusted to

Hrdns=100

(μg/L)b

EC50 Diss.,

Adjusted to

Hrdns=100

(μg/L)b

Reference

Ceriodaphnia

dubia (<24 h)

S, M 45 25.0

17.0

30.0

24.0

28.0

32.0

23.0

20.0

19.0

53.05

36.07

63.66

50.93

59.42

67.90

48.81

42.44

40.32

Belanger et al.

1989

Ceriodaphnia

dubia (<24 h)

S, M 94.1 26.0

21.0

27.0

37.0

34.0

66.0

63.0

71.0

27.53

22.24

28.59

39.18

36.00

69.89

66.72

75.19

Belanger et al.

1989

Ceriodaphnia

dubia (<24 h)

S, M 179 67.0

38.0

78.0

81.0

38.71

21.96

45.07

46.80

Belanger et al.

1989

Ceriodaphnia

dubia (<24 h)

S, M 97.6 14

28

31

14.32 

28.65 

31.72 

Belanger and

Cherry 1990

Ceriodaphnia

dubia (<24 h)

S, M 113.6 52

76

91

46.11 

67.40 

80.70 

Belanger and

Cherry 1990

Ceriodaphnia

dubia (<24 h)

S, M 182.0 56

84

93

31.85 

47.78 

52.90 

Belanger and

Cherry 1990

Ceriodaphnia

dubia (<12 h)

S, M 90 13.4 14.80 Oris et al. 1991

Ceriodaphnia

dubia

S, M 87.5

80.8

80.8

60

30

11.25

13.17

25.25

11.25

4.5

12.76

16.10

30.87

18.20

13.99

Neserke 1994

Ceriodaphnia

dubia

S, U 188

204

428

410

494

440

36.6

19.1

36.4

11.7

12.3

12.0

20.19 

9.76 

9.25 

3.10 

2.73 

2.97 

Bright 1995

Ceriodaphnia

dubia (<24 h)

S, M 80 6.98 8.61 Diamond et al.

1997b
Species Methoda

Hrdns

(mg/L as

CaCO3)

EC50

Total

(μg/L)

EC50

Diss.

(μg/L)

EC50 Total,

Adjusted to

Hrdns=100

(μg/L)b

EC50 Diss.,

Adjusted to

Hrdns=100

(μg/L)b

Reference

19

Ceriodaphnia

dubia

S, M 99

70

74

72

148

148

142

144

148

200

193

198

194

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

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

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

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

Tetra Tech

1998

Ceriodaphnia

dubia

S, M 78

90

90

13.1

8.88

10.3

16.56

9.81

11.37

Dimond 2000

Daphnia magna

(<24 h)

S, U 45.3 9.8 20.67 Biesinger &

Christensen

1972

Daphnia magna

(1 d)

S, U 99 85

50

85.81 

50.48 

Adema &

Degroot-van

Zijl 1972

Daphnia magna S, U 45 10 21.22 Cairns et al.

1978

Daphnia magna S, M 100 31.8 31.80 Borgmann &

Ralph 1983

Daphnia magna

(<24 h)

S, U 45 54 114.59 Mount &

Norberg 1984

Daphnia magna

(<24 h)

S, U 240 41 17.97 Elnabarawy et

al. 1986

Daphnia magna

(<4 h)

S, M 54 7 12.51 Nebeker et al.

1986

Daphnia magna

 (1 d)

 (2 d)

 (3 d)

 (4 d)

 (5 d)

 (6 d)

S, M 80

10

6

14

7

10

18

12.34 

7.40 

17.28 

8.64 

12.34 

22.21 

Nebeker et al.

1986

Daphnia magna S, U 240 93 40.76 Khangarot and

Ray 1989

Daphnia magna S, U 10 21.5 188.21 Hickey and

Vickers 1992
Species Methoda

Hrdns

(mg/L as

CaCO3)

EC50

Total

(μg/L)

EC50

Diss.

(μg/L)

EC50 Total,

Adjusted to

Hrdns=100

(μg/L)b

EC50 Diss.,

Adjusted to

Hrdns=100

(μg/L)b

Reference

20

Daphnia magna S, U 33.8 11.5 31.96 Koivisto et al.

1992

Daphnia magna S, U 50 7 13.45 Oikari et al.

1992

Daphnia magna S, M 52

105

106

207

26

30

38

69

48.15 

28.65 

35.97 

34.76 

Chapman et al.

Manuscript

Daphnia magna S, M 170 41.2

10.5

20.6

17.3

70.7

31.3

24.99 

6.37 

12.50 

10.49 

42.88 

18.99 

Baird et al.

1991

Daphnia magna

(<24 h)

S, M 100 7.1

18.7

18.9

7.10 

18.70 

18.90 

Meador 1991

Daphnia magna

(<24 h)

S, M 170 31

38

35

58

37

51

39

50

52

31

30

46

63

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 

Lazorchak and

Waller 1993

Daphnia magna

(<24 h)

S, M 72

76

80

72

84

84

44

44

36

76

88

4.8

6.24

6.17

6.62

10.2

10.8

4.6

7.8

3

10.4

16.9

6.54 

8.08 

7.61 

9.02 

12.02 

12.73 

9.97 

16.91 

7.86 

13.47 

19.06

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:

 

 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.
21

References for Appendix B

Adema, D.M.M. and A.M. Degroot-van Zijl.

1972. The influence of copper on the water

flea Daphnia magna. TNO Nieuws 27:474.

Baird, D.J., I. Barber, M. Bradley,

A.M.V.M. Soares and P. Calow. 1991. A

comparative study of genotype sensitivity to

acute toxic stress using clones of Daphnia

magna straus. Ecotoxicol. Environ. Saf.

21(3):257-265.

Belanger, S.E., J.L. Farris and D.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. and D.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. and K.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 WaterEffect 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. and M.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(1):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
22

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 LC50s 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.

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.

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

LC50s for Calculation of Water Effect

Ratios and Effluent Limits for POTWs in

Southeastern Pennsylvania. Tetra Tech,

Owings Mills, MD.
23

Appendix C

Assessment of the Streamlined Water-Effect Ratio Procedure

Abstract

The protectiveness of the Streamlined

Procedure for obtaining a copper watereffect 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 sitespecific 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 EC50s 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
24

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 realisticlooking 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
25

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 designflow 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
26

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
27

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.)

10. 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-1. 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 Standard deviation of log upstream toxicant concentration

0.5 Standard deviation of log upstream WER

1.0 Standard deviation of log upstream flow

0.5 Standard deviation of log effluent concentration

0.5 Standard deviation of log effluent WER

0.0 Standard deviation of log effluent flow

0.5 Correlation (r2) between log effluent toxicant concentration and log effluent WER

0.43 Correlation (r2) between effluent toxicant concentration and effluent WER

4.0 Ratio, effluent mean WER : upstream mean WER

33% Design IWC for higher dilution facility in scenario

51% Design IWC for medium dilution facility in scenario

91% Design IWC for low dilution facility in scenario 

8% Overall geometric mean IWC for higher dilution facilities in group

14% Overall geometric mean IWC for medium dilution facilities in group

63% Overall geometric mean IWC for low dilution facilities in group

3% Frequency of flows below design flow

1% Frequency of site-specific criteria violations
28

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 Ratio of Procedure WER : Unbiased WER Probability of

exceeding the

50 Unbiased WER th Percentile 95th Percentile 

91% Design IWC 0.82 1.49 30%

50% Design IWC 0.81 1.38 25%

33% Design IWC 0.90 1.42 33%

Mean of scenarios .84 1.43 29%
29

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. SiteSpecific 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 sitespecific 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.
30
31

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 highflow, 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.
32

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. 

SMAV 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 labwater EC50s, 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 EC50s. 

Data for 30 separate WER measurements

from studies by four different laboratories

indicated that the laboratory water EC50 for
33

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.

Ratio of Procedure WER : Unbiased WER Probability of

exceeding the

50th Percentile 95th Percentile Unbiased WER

Streamlined Procedure 0.84 1.43 29%

Interim Procedure 0.83 1.51 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.

Ratio of Procedure WER : Unbiased WER Probability of

exceeding the

50th Percentile 95th Percentile Unbiased WER

Two-sample Procedure 0.84 1.43 29%

Single-sample Approach 0.84 1.72 35%
34

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 EC50s 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
35

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