United States

Environmental Protection

Agency

Office of Water

(4303)

EPA 821-B-00-004

July 2000

Method Guidance and

Recommendations for Whole

Effluent Toxicity (WET) Testing 

(40 CFR Part 136) 
i

Table of Contents

Executive Summary ..................................................... vi

Chapter 1 Introduction ................................................ 1-1

What is whole effluent toxicity (WET) and how is it measured? ................. 1-1

What is the regulatory background of WET testing? .......................... 1-2

What is the purpose of this document? ..................................... 1-2

What other clarification and guidance documents has EPA published on WET? .... 1-3

Chapter 2 Nominal Error Rate Adjustments ............................. 2-1

When is a nominal error rate used? ........................................ 2-1

What is a nominal error rate? ............................................ 2-1

How is the alpha level related to specific types of errors? ...................... 2-2

What alpha level is recommended in the WET method manuals? ................ 2-3

When can alpha be reduced? ............................................. 2-3

When should alpha not be reduced? ....................................... 2-4

How can adequate test sensitivity be confirmed? ............................. 2-4

What is the recommended decision process for determining the appropriate alpha 

level? ............................................................... 2-7

Chapter 3 Confidence Intervals ......................................... 3-1

When are confidence intervals not generated by point estimation techniques? ...... 3-1

Chapter 4 Concentration-Response Relationships ........................ 4-1

How will this guidance be incorporated into WET test methodology? ............ 4-1

What is the concentration-response relationship concept? ...................... 4-1

How is the concentration-response concept used in WET testing? ............... 4-2

How can the concentration-response concept be used to review WET test results? . . 4-3

What are some patterns of concentration-response relationships typically seen in WET

test data? ............................................................ 4-5

1. Ideal concentration-response relationship ............................ 4-6

2. All or nothing response ........................................... 4-6

3. Stimulatory response at low concentrations and detrimental effects at higher

concentrations .................................................. 4-7

4. Stimulation at low concentrations but no significant effect at higher

concentrations .................................................. 4-8

5. Interrupted concentration-response: significant effect bracketed by nonsignificant effects .............................................. 4-11

6. Interrupted concentration-response: non-significant effects bracketed by 

significant effects .............................................. 4-13

7. Significant effects only at highest concentration ...................... 4-14

8. Significant effects at all test concentrations but flat concentration- response

curve ........................................................ 4-15

9. Significant effects at all test concentrations with a sloped concentrationresponse curve ................................................ 4-17

10. Inverse concentration-response relationship .......................... 4-18
ii

Chapter 5 Dilution Series Selection .................................... 5-1

Do the WET method manuals specify a certain dilution series? .................. 5-1

Why is selecting an appropriate dilution series important? ..................... 5-1

How might the dilution series or dilution sequence be modified to assist in 

determining a concentration-response relationship and improving the precision of

calculated effect concentrations? ........................................ 5-2

Chapter 6 Dilution Waters ........................................... 6-1

What does EPA consider to be an acceptable dilution water? ................... 6-1

How do I choose an appropriate dilution water? ............................. 6-1

What dilution water should I use when determining absolute toxicity of an effluent? . . 6-3

What dilution water should I use when determining the toxicity of an effluent in the

receiving system? .................................................... 6-3

When and how do I use dual controls? .................................... 6-5

How might the choice of dilution waters affect WET test results? ................ 6-6

Chapter 7 References ................................................ 7-1
iii

Tables

Table 2.1. Recommended maximum MSD (minimum significant difference) criteria for

selected WET test methods and responses (adapted from Table 3-6 in USEPA,

2000). ...................................................... 2-6

Table 2.2. Number of within-treatment replicates giving equivalent MSDs (minimum

significant differences) at alpha = 0.05 and 0.01, for a test employing five

concentrations and a control. ..................................... 2-9

Table 2.3. Example results from 10 previous Ceriodaphnia dubia 3-brood reproduction

tests. ...................................................... 2-10

Table 2.4. Comparison of critical Dunnett’s values for five concentrations and a control

using alpha = 0.05 and 0.01. .................................... 2-12
iv

Figures

Figure 2.1. Possible decisions and outcomes in the hypothesis test. ................ 2-2

Figure 2.2. Recommended decision process for determining the appropriate alpha

level for WET hypothesis testing. ................................ 2-8

Figure 4.1. Classical concentration-response relationship. ....................... 4-1

Figure 4.2. Example determination of point estimates from a concentration-response 

curve. .................................................... 4-2

Figure 4.3. Ideal concentration-response relationship. .......................... 4-6

Figure 4.4. All or nothing concentration-response relationship. ................... 4-7

Figure 4.5. Stimulation at low concentrations and significant effects at high

concentrations. .............................................. 4-8

Figure 4.6. Stimulation at low concentrations but no significant effect at higher

concentrations. .............................................. 4-9

Figure 4.7. Interrupted concentration-response: significant effect bracketed by nonsignificant effects. .......................................... 4-11

Figure 4.8. Interrupted concentration-response: non-significant effects bracketed by

significant effects. .......................................... 4-13

Figure 4.9. Significant effects only at highest concentration. .................... 4-15

Figure 4.10. Significant effects at all test concentrations but flat concentration- response

curve. ................................................... 4-16

Figure 4.11. Significant effects at all test concentrations with a sloped concentrationresponse curve. ............................................ 4-18

Figure 4.12. Inverse concentration-response relationship. ....................... 4-19

Figure 6.1. Flowchart for appropriate selection and use of dilution water in WET 

testing. .................................................... 6-2
v

Disclaimer

This document, Method Guidance and Recommendations for Whole Effluent Toxicity (WET)

Testing (40 CFR Part 136), is provided to help implement national water quality-based

permitting under the National Pollutant Discharge Elimination System (NPDES) Program. 

This guidance document does not, however, substitute for the Clean Water Act (CWA) or

EPA’s regulations, nor is it a regulation itself. Thus, it cannot impose legally binding

requirements on EPA, States, Tribes, or the regulated community and may not apply to a

particular situation based upon case-specific circumstances. The material presented herein is

intended solely for guidance and does not alter any statutory or regulatory requirements, or

requirements in an NPDES permit. EPA, State, and Tribal decision makers retain the

discretion to adopt approaches on a case-by-case basis that differ from this guidance where

appropriate. EPA may change this guidance in the future.
vi

Executive Summary

I

n 1995, the U.S. Environmental Protection Agency (EPA) published a final rule

standardizing 17 whole effluent toxicity (WET) test methods for use in NPDES

(National Pollutant Discharge Elimination System) monitoring [60 FR 53529; October

16, 1995]. These WET test methods measure the aggregate acute and chronic toxicity

of an effluent using standardized freshwater, marine, and estuarine plants, invertebrates, and

vertebrates. The inclusion of WET methods in the NPDES program completes an integrated

strategy for water quality-based toxics control that fulfills the Clean Water Act’s mandate to

protect aquatic life and prohibit the discharge of toxic pollutants in toxic amounts. 

This document provides guidance and recommendations on the conduct of the approved

WET test methods and interpretation of WET test results reported under the NPDES

program. This guidance partially fulfills the obligations of a legal settlement agreement that

resolves a judicial challenge to the WET final rule. The document provides guidance on the

following issues: nominal error rate adjustments, confidence intervals, concentrationresponse relationships, dilution series, and dilution waters. A summary of the guidance and

recommendations for each issue is provided below.

C Nominal error rate adjustments - The WET method manuals (USEPA, 1993c;

USEPA, 1994a; USEPA, 1994b) recommend a nominal error rate (or alpha level) of 0.05

when using hypothesis testing to determine test results. This guidance clarifies that

alpha may be reduced to 0.01 when sublethal endpoints from Ceriodaphnia or fathead

minnow tests are reported under NPDES permit requirements, or when WET permit

limits are derived without allowing for receiving water dilution. In these situations,

however, the alpha level should be reduced only in tests that meet a set criterion for test

sensitivity, since reductions in alpha also reduce statistical power. Specifically, the

percent minimum significant difference (%MSD) calculated for the test using an alpha of

0.01 should be less than or equal to a set criterion. Increased replication may be

necessary to meet the %MSD criterion when using an alpha of 0.01. This document

provides guidance on determining the need for additional test replication, as well as the

entire decision process for reducing the alpha level in hypothesis testing. 

C Confidence intervals - Point estimation techniques described in the WET method

manuals are used to generate effect concentrations and associated 95% confidence

intervals. Software used to conduct these statistical procedures occasionally does not

provide the associated confidence intervals. This may arise when the test data are

inappropriate for the assumptions or requirements of the statistical method chosen. In

these cases, statistical flowcharts provided in the WET method manuals should guide the

analyst to more appropriate techniques. Confidence intervals also may not be generated

if the calculated point estimate is outside of the test concentration range. In this case,

confidence intervals are not applicable because exact point estimates are not reported. 

For the inhibition concentration percentage (ICp) procedure, there are additional
vii

anomalous circumstances when confidence intervals are not generated due to limitations

of the software. 

C Concentration-response relationships - The concentration-response relationship

established between the concentration of a toxicant and magnitude of the response, is a

fundamental principle of toxicology. EPA recommends the use of this concentrationresponse concept as a test review step to assist in determining the validity of WET test

results. When unexpected concentration-response relationships are encountered, a

thorough review of test performance, test conditions, and the particular concentrationresponse pattern exhibited should be conducted to determine whether the derived effect

concentrations are reliable or anomalous. This document recommends review steps for

10 different concentration-response patterns that may be encountered in WET test data. 

Based on the review, it may be determined that calculated effect concentrations are

reliable and should be reported, that calculated effect concentrations are anomalous and

should be explained, or that the test was inconclusive and the sample should be retested.

C Dilution series - This guidance clarifies that the WET method manuals do not require

the use of a specific dilution series for all WET tests. The dilution series for a specific

test should be selected to optimize the precision of calculated effect concentrations and

assist in establishing concentration-response relationships. Recommendations for

selecting an appropriate dilution series include: considering historic WET testing

information for the given effluent, using the receiving water concentration as a test

concentration, bracketing the receiving water concentration with test concentrations,

adding test concentrations within a given range of interest, and increasing the dilution

factor used to space effluent concentrations. 

C Dilution waters - This guidance clarifies that an acceptable dilution water for WET

testing is appropriate for the objectives of the test; supports adequate performance of the

test organisms with respect to survival, growth, reproduction, or other responses that may

be measured in the test (i.e., consistently meets test acceptability criteria for control

responses); is consistent in quality; and does not contain contaminants that could

produce toxicity. If the objective of the test is to determine the absolute toxicity of an

effluent, EPA recommends the use of a standard synthetic dilution water. A consistent,

high purity natural water source (e.g., uncontaminated seawater or treated well water)

also may be appropriate for determining the absolute toxicity of an effluent when

specific criteria given in this guidance are met. If the objective of the test is to determine

the toxicity of an effluent in the receiving system, a local receiving water is

recommended for use as dilution water provided that the receiving water meets specific

criteria. The receiving water should be collected as a grab sample from upstream or near

the final point of effluent discharge, have adequate year-round flow, support adequate

performance of the test organisms, be consistent in quality, be free of contaminants that

would produce toxicity, and be free from pathogens and parasites that could affect WET

test results. If the local receiving water fails to meet any of these criteria for use, a

synthetic dilution water adjusted to approximate the chemical characteristics of the

receiving water is recommended. 
1-1

1

Introduction

T

his chapter provides a brief introduction to whole effluent toxicity (WET) testing and

describes the regulatory background and context of WET testing. This chapter also

describes the purpose of this document and outlines the issues addressed in each

chapter. 

What is whole effluent toxicity (WET) and how is it measured?

Whole effluent toxicity (WET) is defined as “the aggregate toxic effect of an effluent measured

directly by an aquatic toxicity test” [54 FR 23868 at 23895; June 2, 1989]. Aquatic toxicity

test methods designed specifically for measuring WET have been codified at 40 CFR part 136

[60 FR 53529; October 16, 1995]. These WET test methods employ a suite of standardized

freshwater, marine, and estuarine plants, invertebrates, and vertebrates to estimate acute and

short-term chronic toxicity of effluents and receiving waters. Specific test procedures for

conducting the approved WET tests are included in the following three test method manuals:

C U.S. Environmental Protection Agency. 1993c. Methods for Measuring the Acute

Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms, 4th ed.,

EPA 600/4-90/027F. U.S. Environmental Protection Agency, Environmental Monitoring

Systems Laboratory, Cincinnati, OH.

C U.S. Environmental Protection Agency. 1994. Short-term Methods for Estimating the

Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms, 3rd ed.,

EPA 600/4-91/002. U.S. Environmental Protection Agency, Environmental Monitoring

Systems Laboratory, Cincinnati, OH. 

C U.S. Environmental Protection Agency. 1994. Short-term Methods for Estimating the

Chronic Toxicity of Effluents and Receiving Waters to Marine and Estuarine Organisms,

2

nd ed., EPA 600/4-91/003. U.S. Environmental Protection Agency, Environmental

Monitoring Systems Laboratory, Cincinnati, OH. 

These three method manuals (WET method manuals) were incorporated by reference into 40

CFR part 136 in the 1995 rule. As regulations, use of these methods and adherence to the

specific test procedures outlined in the WET method manuals is required when monitoring

WET under the National Pollutant Discharge Elimination System (NPDES). Of course, the

extent that such procedures are “requirements” depends on the text of the WET method

manuals themselves. Words of obligation, such as “must” or “shall” indicate a required

procedure. When WET method manuals use discretionary terms such as “may” or “should”

the manuals provide flexibility so that the laboratory analyst may optimize successful test

completion (USEPA, 1996a). 
1-2

What is the regulatory background of WET testing?

The Clean Water Act (CWA) was enacted in 1972 with the objective of “restoring the

chemical, physical, and biological integrity of the Nation’s waters.” Along with other specific

goals, CWA section 101(a)(3) states that “it is the national policy that the discharge of toxic

pollutants in toxic amounts be prohibited.” EPA has pursued this goal through the

implementation of the water quality standards program and the NPDES permitting program. 

These programs have adopted an integrated strategy of water quality-based toxics control that

includes the following approaches:

C Chemical-specific control approach

C Whole effluent toxicity (WET) control approach

C Biological criteria/bioassessment and biosurvey approach

To implement this strategy, States and Tribes are encouraged to define numeric or narrative

water quality standards that include chemical-specific criteria, criteria for whole effluent

toxicity, and biological criteria. Some states have included numeric criteria for WET, while

others have relied on narrative criteria such as, “free from toxics in toxic amounts”. These

water quality standards and criteria are maintained by controlling the discharge of pollutants

through the NPDES permitting program. When a discharge causes or has a reasonable

potential to cause or contribute to the excursion of numeric or narrative water quality

standards, a water quality-based effluent limit in the NPDES permit will be issued to control

the discharge. This includes permit limits for WET if the discharge causes, has a reasonable

potential to cause, or contributes to the excursion of water quality standards for WET,

including narrative criteria for toxicity.

Further explanation of the regulatory role and background of WET can be found in the WET

method manuals (USEPA, 1993c; USEPA, 1994a; USEPA, 1994b) and in EPA’s Technical

Support Document for Water Quality-based Toxics Control (USEPA, 1991b). 

What is the purpose of this document?

This guidance is intended to clarify the published WET method manuals on selected issues

regarding the conduct of WET tests and interpretation of WET test results. This document

provides additional guidance and recommendations to EPA Regional, State, Tribal, and local

regulatory authorities; regulated entities; and environmental laboratories on these selected

issues. Proper implementation of the guidance provided in this document should enhance

successful WET test completion, result interpretation, and the application of WET testing in

the NPDES program. 

EPA developed this guidance document as part of efforts to resolve litigation over the

rulemaking that standardized and approved the WET test methods for use in NPDES

monitoring [60 FR 53529; October 16, 1995]. In a settlement agreement, EPA agreed to
1-3

provide guidance and recommendations on five specific technical issues. Each of these issues

is addressed in a separate chapter of this guidance document.

C Nominal error rate adjustments - Chapter 2 explains the concept of a nominal error rate

(or alpha level) and the effect of alpha on false positive rates, false negative rates, and test

sensitivity. This chapter clarifies the circumstances when the alpha level for WET

hypothesis testing may be reduced from 0.05 to 0.01. This chapter also provides guidance

and recommendations for assuring that test sensitivity is not adversely affected by

reductions in alpha. This guidance includes procedures for measuring test sensitivity,

determining the need for additional test replication, and comparing test sensitivity to

recommended criteria.

C Confidence intervals - Chapter 3 clarifies the circumstances under which confidence

intervals are not generated and/or not capable of generation when using point estimation

techniques.

C Concentration-response relationships - Chapter 4 explains the concept of a

concentration-response relationship and describes how this concept may be used as a WET

test review step. This chapter identifies various forms of concentration-response

relationships encountered in WET testing and provides guidance on evaluating and

interpreting results from these concentration-response relationships.

C Dilution series selection - Chapter 5 provides guidance on selecting appropriate dilution

series for WET tests. This guidance provides recommendations for modifying the dilution

series to assist in determining the existence of a concentration-response relationship and

improving point estimate precision.

C Dilution water - Chapter 6 clarifies what EPA considers to be acceptable dilution water

for WET testing. This chapter provides guidance on selecting an appropriate dilution

water based on the objectives of the WET test and the quality and consistency of available

dilution water sources. Guidance is provided regarding when to use the following waters

for dilution: receiving water, standard synthetic water, and synthetic water adjusted to

approximate receiving water characteristics. This chapter also clarifies the use of dual

controls when dilution water differs from the water used to culture test organisms.

What other clarification and guidance documents has EPA published on WET?

The final WET methods rule [60 FR 53529; October 16, 1995] incorporated the WET method

manuals (USEPA, 1993c; USEPA, 1994a; USEPA, 1994b) by reference. EPA provided

further guidance and clarifications regarding the use of the WET test methods in a

memorandum dated April 10, 1996 from Tudor Davies, Director of the EPA Office of Water’s

Office of Science and Technology. This memorandum, titled “Clarifications Regarding

Flexibility in 40 CFR Part 136 Whole Effluent Toxicity (WET) Test Methods” (USEPA,

1996a), provided clarification on the following WET test issues: pH and ammonia control,

temperature, hardness, test dilution concentrations, and acceptance criteria for Champia

parvula. 
1-4

In January 1999, EPA published an errata sheet for the WET method manuals (USEPA,

1999). This errata sheet amended the approved WET test methods to correct typographical

errors and omissions, provide technical clarification, and establish consistency among the 1995

WET rule language and the three WET method manuals.

EPA has recently published a guidance document titled, Understanding and Accounting for

Method Variability in Whole Effluent Toxicity Applications Under the National Pollutant

Discharge Elimination System Program, (USEPA, 2000). This guidance document is

intended to provide regulatory authorities with an understanding of WET test variability and

provide guidance on accounting for and minimizing WET test variability and its effects on the

regulatory process.
2-1

2

Nominal Error Rate

Adjustments

T

he WET method manuals (USEPA, 1993c; USEPA, 1994a; USEPA, 1994b)

recommend a nominal error rate (or alpha) of 0.05 when using hypothesis testing to

determine WET test results. Under certain circumstances, it may be appropriate to

reduce alpha to 0.01. This chapter provides an explanation of the concept and use of

a nominal error rate and provides guidance on when alpha may be reduced.

When is a nominal error rate used?

A nominal error rate is used in the statistical method of hypothesis testing. According to the

WET method manuals, effect concentrations for effluent toxicity tests may be generated by

point estimation techniques or hypothesis testing techniques (see Section 9 of USEPA, 1994a;

USEPA, 1994b). Point estimation techniques are used to generate effect concentrations such

as LC50 (median lethal concentration), EC50 (median effect concentration), or IC25 (25%

inhibition concentration) values. Hypothesis testing techniques are used to generate NOEC

(No-Observed-Effect-Concentration) and LOEC (Lowest-Observed-Effect-Concentration)

values. Both statistical techniques have advantages and disadvantages (Grothe et al., 1996),

and regulatory authorities may choose to base WET permit limits on effect concentrations

generated using either technique. The WET method manuals (see Section 9 of USEPA, 1994a;

USEPA, 1994b) state that point estimation techniques are the preferred statistical methods for

calculating effect concentrations in WET tests under the NPDES permit program.

What is a nominal error rate?

The concept of hypothesis testing relies on the ability to distinguish statistically significant

differences between a control treatment and other test treatments (e.g., effluent concentrations). 

In terms of classical statistics, the hypothesis testing techniques (whether Dunnett’s Test, tTest with Bonferroni adjustment, Steel’s Many-One Rank Test, or Wilcoxon Rank Sum Test

with Bonferroni adjustment) test the null hypothesis (Hi) that there is no difference between

the control treatment and other test treatments (the effluent is not toxic). This null hypothesis

is rejected (the effluent is determined to be toxic) if the difference between the control treatment

and any other test treatment is statistically significant. In order to determine when the

difference between treatments is large enough to be statistically significant and to warrant

rejection of the null hypothesis, the statistician or analyst selects a nominal error rate. This

nominal error rate is an intended upper bound on the probability of incorrectly rejecting the

null hypothesis (determining that the effluent is toxic) when it is in fact true (the effluent is not

toxic). In selecting the nominal error rate, the analyst is deciding what level of uncertainty
2-2

he/she is comfortable with in making this type of error (determining that the effluent is toxic

when it is not). The larger the nominal error rate, the greater the probability of incorrectly

rejecting the null hypothesis (determining that the effluent is toxic when in fact it is not). In

classical statistics, the error of incorrectly rejecting the null hypothesis is termed a Type I

error, and the nominal error rate selected to place an intended upper bound on the probability

of this error is termed alpha ("). To remain consistent with statistical terminology, the nominal

error rate will be referred to as alpha in the remainder of this document. An alpha of 0.05

means a 5% probability of making a Type I error and is associated with a 95% level of

significance (i.e., on average 1 test in 20 tests could produce a Type I error). 

How is the alpha level related to specific types of errors?

Figure 2.1 describes the possible correct and erroneous decisions that can be made in

hypothesis testing. In making the decision to reject or accept the null hypothesis, two types of

error are possible. An incorrect decision can be made by determining that a sample is toxic

when in fact it is not (Type I error), or determining that a sample is not toxic when in fact it is

(Type II error). These errors also may be commonly referred to as false positive error and

false negative error, respectively. The alpha level that is selected by the statistician or analyst

in a hypothesis test represents the probability of making a Type I error (or the Type I error

rate). The probability of a Type II error (or the Type II error rate) is represented by beta ($). 

Figure 2.1. Possible decisions and outcomes in the hypothesis test.

True State of Nature

Hi is true 

(sample is not toxic)

Hi is false 

(sample is toxic)

Decision

Accept Hi

(determine that sample

is not toxic)

Correct decision

Type II error 

(false negative)

Reject Hi

(determine that sample

is toxic)

Type I error 

(false positive)

Correct decision

There are direct and indirect costs associated with both types of errors. False positives can

create undue costs and effort involved in follow-up actions such as increased testing, Toxicity

Identification Evaluation (TIE) and Toxicity Reduction Evaluation (TRE) procedures, possible

fines for permit violations, and the potential for civil lawsuits. False negatives can cause the

continuation of unchecked environmental degradation and the associated long-term cost of

reclamation or restoration. Researchers have suggested that false negatives may be more

costly than false positives because false positives may be quickly discovered by additional
2-3

testing, while false negatives may continue longer before being discovered (Thursby et al.,

1997). Since there are costs associated with each type of error, neither type of error should

be ignored, and an effort should be made to minimize both types of error. However, because

of the relationship between the Type I error rate (") and the Type II error rate ($), reductions

in one type of error generally cause an increase in the other. For instance, when test

variability and test design are held constant, reducing the alpha level of a test increases the

Type II error rate ($). This reduces the statistical power (defined as 1-$) of the test and

limits the ability of the test to detect small effects as statistically significant. Because costs

exist for both types of error, it is important to consider the impact of both types of error

before reducing alpha. 

What alpha level is recommended in the WET method manuals?

Traditionally, scientists have set alpha for biological studies at 0.01 to 0.1 (1 to 10%). The

0.01 level, at one extreme, provides a statistically conservative error rate that minimizes false

positives. The 0.1 level, at the other extreme, provides a statistically more liberal error rate

that results in increased statistical power. Zar (1984) states that a probability of 5% or less

is commonly used as a criterion for rejection of the Hi, and that when the 5% chance of an

incorrect rejection of the hypothesis is unacceptably high, then a 1% level of significance is

sometimes used. The WET test method manuals recommend an alpha of 0.05 for hypothesis

testing (see Section 9 of USEPA 1994a; USEPA 1994b). The experimental test designs of

the WET test methods (e.g., replicates, treatments, number of organisms) have limits to the

magnitude of toxic response that they are able to detect given a specific alpha level (Denton

and Norberg-King, 1996; USEPA, 2000); smaller effects will generally not be detected. If

the recommended test alpha level is reduced, the experimental test design may need

modification (e.g. increased test replication) to maintain the same level of test sensitivity.

When can alpha be reduced?

The alpha level used for hypothesis testing in WET data analysis may be reduced from 0.05

to 0.01 when:

- sublethal endpoints (reproduction or growth) from Ceriodaphnia dubia or fathead

minnow tests are reported under NPDES permit requirements, or

- the NPDES permit limit for WET was derived without allowing for receiving water

dilution due to low dilution potential in the receiving system,

provided that the WET test is able to maintain adequate test sensitivity (as demonstrated by

successfully meeting a set criterion for minimum significant differences [MSDs]) using an

alpha of 0.01.
2-4

When should alpha not be reduced?

The alpha level of a test should not be reduced unless the regulatory authority allows or

specifies an alpha of 0.01 in the NPDES permit (see “What is the recommended decision

process for determining the appropriate alpha level?”). The alpha level of a test also should

not be reduced if the test does not maintain adequate test sensitivity. This determination is

made by comparing the test MSD (calculated using the reduced alpha of 0.01) to

recommended maximum MSD levels (see “How can adequate test sensitivity be

confirmed?”). If the test MSD (calculated using the reduced alpha of 0.01) is greater than

the MSD criterion, alpha should not be reduced to 0.01, and results should be reported using

the standard alpha level of 0.05. 

How can adequate test sensitivity be confirmed?

As described above, alpha may be reduced only when the test maintains adequate test

sensitivity. Adequate test sensitivity is determined by calculating the MSD for a given test

and comparing this value to maximum MSD criteria. This procedure is described below.

C Calculate test MSD - To measure the sensitivity of the test, the minimum significant

difference or MSD is calculated. The MSD is defined as the smallest difference between

the control and another test treatment that can be determined as statistically significant in

a given test. The MSD is a measure of statistical sensitivity that is dependent upon the

within test variability, the alpha level selected for the test, and the test design (i.e.,

number of replicates and treatments). The MSD decreases (i.e., statistical sensitivity

increases) with decreasing test variability, increased test replication, and increased alpha. 

According to the WET method manuals (USEPA, 1994a; USEPA, 1994b), the MSD may

be calculated for Dunnett’s multiple comparison test using the following equation:

MSD d s

n n

w

c

= × +

1 1

0

where:

d = Dunnett’s t for the selected " and N - (k+1) degrees of freedom

sw = square root of the error mean square from analysis of variance

(ANOVA)

n0 = number of replicates in the control

nc = number of replicates for each effluent concentration

N = total number of replicates in the ANOVA

k = number of non-control treatments being compared to the control

The pooled variance estimate, sw, is obtained from an analysis of variance (ANOVA). 

Test concentrations that exhibit 0% survival are excluded from the ANOVA for survival

endpoints, and test concentrations greater than the NOEC for survival are excluded from

the ANOVA for sublethal endpoints. 
2-5

When the number of replicates is not the same for all test treatments, but variances are

expected to be the same, the t-test with Bonferroni’s adjustment is used for hypothesis tests

(USEPA, 1994a; USEPA, 1994b). Under these circumstances, the MSD is calculated

using the formula shown above, except that “d”is replaced by the standard t-statistic for a

one-sided test at level 1-"/k, where k is the number of treatments being compared to the

control. Further details and a table of critical values for t are provided in Appendix D of

the WET method manuals (USEPA, 1994a; USEPA, 1994b).

The above equation (with the slight modification for unequal replicates, if needed) may be

used to calculate the MSD for all tests in which results are derived from hypothesis testing,

regardless of the hypothesis testing technique used (e.g., Dunnett’s Test, t-test with

Bonferroni adjustment, Steel’s Many-One Rank Test, or Wilcoxon Rank Sum Test with

Bonferroni adjustment). When a given data set does not meet the assumptions (e.g.,

normal distribution or homogeneous variance) necessary for the use of parametric

hypothesis testing procedures (i.e., Dunnett’s test or t-test with Bonferroni adjustment), the

MSD still may be derived as described above for use as an approximate indicator of test

sensitivity. However, when there are significant differences in variances among

treatments, the best approach is to identify a variance-stabilizing transformation

(preferably one which applies generally and not to just one test) and which leaves the

treatment means approximately normal. 

To facilitate the comparison of MSD values among tests and with established criteria, the

MSD is generally expressed as a percentage of the mean control value for the given test. 

This transformation is conducted using the following equation:

% MSD

MSD

= ×

Control mean

100%

Other measures of test sensitivity, such as test power (1- $) also can be used to determine

the statistical sensitivity of a test. However, the MSD is recommended in this guidance for

determining the appropriateness of reducing alpha levels in hypothesis testing. The MSD

is easily calculated and is generated by most statistical software packages used in WET

test data analysis. In addition, the Pellston Workshop on Whole Effluent Toxicity

(Chapman et al., 1996; Denton and Norberg-King, 1996) and other researchers (Thursby

et al., 1997; Warren-Hicks et al., 1999) recommend the use of MSDs to assure that

acceptable statistical sensitivity is achieved. The MSD is currently used to access the

acceptability of test sensitivity in the West Coast WET methods (USEPA, 1995), and

criteria for acceptable MSD levels have been recommended for most of the approved WET

test methods in a newly published EPA guidance document titled, Understanding and

Accounting for Method Variability in Whole Effluent Toxicity Applications Under the

National Pollutant Discharge Elimination System Program (USEPA, 2000).
2-6

C Compare test MSD to maximum MSD criteria - In EPA’s recently published guidance

document on WET method variability (USEPA, 2000), EPA recommends criteria for

maximum MSD values in an effort to reduce method variability. EPA compiled a national

database of WET reference toxicant test data from 75 laboratories and 23 test methods

conducted over a 10-year period. EPA used these data to make inferences about WET test

method variability and to evaluate recommendations for reducing variability. From an

analysis of MSD values from these tests, it was determined that placing upper and lower

bounds on MSDs improved test precision. Based on this finding, EPA recommended

setting upper and lower limits for MSDs at the 10th and 90th percentiles of the MSD

distribution compiled from this national database. Table 2.1 shows the recommended

upper bounds on WET test MSDs for given test methods. 

EPA recommends that these maximum MSD criteria be met for all tests (USEPA, 2000),

regardless of the alpha value used in hypothesis testing. Therefore, EPA recommends that

alpha be decreased from 0.05 to 0.01 only when the test MSD (expressed as %MSD)

calculated with the new, lower alpha (0.01) meets the criteria recommended in Table 2.1

(i.e., calculated test %MSD should be less than or equal to the value in Table 2.1 for the

given method). If the calculated test %MSD is greater than the maximum criterion stated

in Table 2.1, the test results should be reported using an alpha of 0.05. In order to meet

these MSD criteria using an alpha of 0.01, additional test replication may be required (see

Step 2 under “What is the recommended decision process for determining the appropriate

alpha level?”).

Table 2.1. Recommended maximum MSD (minimum significant difference) criteria for

selected WET test methods and responses (adapted from Table 3-6 in USEPA, 2000).

WET test method

Biological

Response

Maximum MSD

Criterion (%MSD)

1000.0- Fathead Minnow, Pimephales promelas,

Larval Survival and Growth Test

Growth 35

1002.0- Daphnid, Ceriodaphnia dubia, Survival and

Reproduction Test

Reproduction 37

1003.0- Green Alga, Selenastrum capricornutum,

Growth Test

Growth 23

1004.0- Sheepshead Minnow, Cyprinodon

variegatus, Larval Survival and Growth Test

Growth 23

1006.0- Inland Silverside, Menidia beryllina, Larval

Survival and Growth Test

Growth 35

1007.0- Mysid, Mysidopsis bahia, Survival, Growth,

and Fecundity Test

Growth 32
2-7

What is the recommended decision process for determining the appropriate

alpha level?

Figure 2.2 summarizes the recommended decision process for determining the appropriate

alpha level for use in hypothesis testing. This figure is provided to assist regulatory

authorities, permittees, and laboratories in this decision-making process. The recommended

three-step decision process is described below.

C Step 1 - In step one, the regulatory authority determines the target alpha level that will be

specified in the permit. If either of the following circumstances apply, the regulatory

authority may allow a target alpha of 0.01:

- sublethal endpoints (reproduction or growth) from Ceriodaphnia dubia or fathead

minnow tests are reported under NPDES permit requirements, or

- the NPDES permit limit for WET was derived without allowing for receiving water

dilution due to low dilution potential in the receiving system.

The target alpha level is the alpha level that the analyst will attempt to use in the statistical

analysis of test data for all samples of the given effluent. While a target alpha level may

be specified for all tests, each test should be evaluated independently to determine if the

target alpha level is appropriate (see Step 3). The regulatory authority should specify (as

a permit condition) that when a target alpha level of 0.01 is allowed, the test MSD should

not exceed the recommended MSD criterion for test sensitivity (Table 2.1). If the test fails

to meet the MSD criterion using the target alpha level, results should be reported using the

standard alpha of 0.05. 

C Step 2 - After the regulatory authority has determined that a target alpha level of 0.01 is

allowable, the permittee should consult with the testing laboratory to determine if increased

test replication is needed to meet the MSD criterion using the target alpha level. Since the

MSD is a function of alpha, test variability, and test design (i.e., number of replicates and

test treatments), an increase in the MSD caused by reducing alpha can be offset by an

increase in test replication. Table 2.2 shows the increase in test replication needed to

completely offset a reduction in alpha from 0.05 to 0.01. For instance, replication in the

fathead minnow chronic test would need to be increased from four to seven replicates to

maintain the same MSD level when alpha is decreased from 0.05 to 0.01 (assuming that

variability remains constant).
2-8

No

Regulatory authority may allow alpha of 0.01 independently for each

test, provided that the MSD criteria is met in the test. Otherwise, an

alpha of 0.05 is specified.

Regulatory

authority

specifies

alpha of 0.05

Step 1:

Regulatory authority

determines the target

alpha level

No

Yes Yes

Step 2:

Permittee in

consultation with

testing laboratory

determines the need

for increased

replication

Evaluate the test sensitivity (MSD) of the previous 10 - 12 tests

using an alpha of 0.05 and 0.01.

Would all tests

 have passed the MSD

criterion using an alpha

 of 0.01?

Evaluate the extent

of increased test

replication needed

No

Step3:

Permittee tests each

sample and reports

results using the

appropriate alpha

level

Perform each

subsequent test using

increased replication

Perform each subsequent test

using traditional replication

Does the test

meet the MSD criteria

using an alpha

of 0.01?

Report test results using

an alpha of 0.05

Report test results using

an alpha of 0.01

Yes

No

Yes

Start

Are

sublethal endpoints

 for Ceriodaphnia or

Fathead minnow

reported?

Is the

permit limit derived

without allowing for

receiving water

dilution?

Figure 2.2. Recommended decision process for determining the appropriate alpha level for WET

hypothesis testing.
2-9

To determine the need for increased test replication, the permittee and testing laboratory

should evaluate the laboratory’s recent performance on tests with the given effluent. 

Laboratories that consistently conduct tests with low variability and high sensitivity (low

MSDs) will require smaller increases in test replication than laboratories with high

variability and low sensitivity (high MSDs). Laboratories should calculate MSDs for the

previous 10 - 12 tests of the given effluent using an alpha of 0.05 and 0.01. While results

from these tests already will have been reported using an alpha of 0.05, this exercise will

provide the permittee with an idea of how often the laboratory might fail to meet the

MSD criterion using the new, reduced alpha of 0.01. It is important that this evaluation

is made using a single laboratory’s performance (i.e., the laboratory that will perform

testing with the new, reduced alpha) for the single effluent of interest. If all of the tests

evaluated would have passed the MSD criterion using a reduced alpha of 0.01, then no

increase in test replication will be necessary. If some of the tests evaluated would have

failed the MSD criterion using a reduced alpha of 0.01, then increased test replication is

needed.

Table 2.2. Number of within-treatment replicates giving equivalent MSDs (minimum

significant differences) at alpha = 0.05 and 0.01, for a test employing five

concentrations and a control.

Number of replicates 

for alpha = 0.05

Number of replicates

for alpha = 0.01

3 5

4 7

5 8

6 10

7 11

8 13

9 15

10 16

If increased test replication is needed, the extent of the increase should be determined by

calculating the replication needed to pass the MSD criterion in the least sensitive of the

10 previous tests evaluated. This level of within-treatment replication will be sufficient

to meet the MSD criterion in approximately 90% of tests conducted. The following

steps and calculations should be followed to determine the needed increase in test

replication across all treatments. A hypothetical example using Ceriodaphnia dubia 3-

brood reproduction test data from 10 tests (Table 2.3) illustrates this determination. 

When unequal replication among treatments is desired (e.g., more replicates in the
2-10

control treatment than in other treatments), consult Dunnett (1964) for optimizing the

allocation of replicates between the control and other treatments.

1. Determine the least sensitive of the previous 10 tests - Tabulate the results

from the previous 10 tests conducted on the effluent of interest by a single laboratory

(Table 2.3). For each test, include the mean control response, the error mean square

(EMS) from the ANOVA, and MSDs calculated using an alpha of 0.05 and 0.01. 

The test with the highest MSD calculated using an alpha of 0.01 should be

considered the least sensitive test of those evaluated. If replication varied among the

tests evaluated, the least sensitive test should be identified as the test with the largest

ratio of EMS to control mean. In the example given (Table 2.3), 2 of the 10 tests

(tests 7 and 9) failed to meet the MSD criterion of 37% (Table 2.1) when using an

alpha of 0.01. Test 9 should be determined to be the least sensitive test since the

MSD of 43.81% is the largest observed in the previous 10 tests. The following

calculations will determine the additional replication that would be needed for this

test to pass the MSD criterion. 

Table 2.3. Example results from 10 previous Ceriodaphnia dubia 3-brood reproduction

tests.

Test

%MSD with

alpha = 0.05

%MSD with

alpha = 0.01

Error Mean

Square (EMS)

Control mean

1 20.78 26.82 24.98 24.6

2 16.50 21.29 16.14 24.9

3 20.12 26.273 28.97 26.6

4 23.82 30.75 19.18 18.8

5 23.94 30.90 31.57 24.0

6 26.32 34.94 26.53 18.7

7 29.53 38.11 29.78 18.9

8 17.75 22.90 18.52 24.8

9 33.94 43.81 68.31 24.9

10 18.38 23.73 15.07 22.2

2. Transform %MSD criterion to MSD - The MSD criterion that should be met

for all tests (Table 2.1) is expressed as a %MSD. This %MSD should be

transformed to a MSD using the control mean performance in the least sensitive of

the previous 10 tests that are being evaluated. Perform this transformation using the

following equation:

MSD

MSD

max

%

=

× Control mean

100%
2-11

where:

MSDmax = the MSD that should have been met in the least sensitive of

the previous 10 tests

%MSD = the %MSD criterion (Table 2.1)

Control mean = the mean control response in the least sensitive of the

previous 10 tests

For the example given, the control mean for test nine should be used in conjunction

with the MSD criterion for the Ceriodaphnia dubia chronic test method (Table 2.1) to

calculate the MSDmax as: 

MSDmax

.

=

37% × 24 9

100%

 MSDmax = 9.213

3. Calculate the square root of the error mean square (sw) - The error mean square

(EMS) is a measure of test variability that is obtained from an ANOVA of test data. 

To evaluate increased replication needs, use the EMS calculated in the least sensitive

of the previous 10 tests. Calculate the square root of this EMS to obtain the variable

sw that is used in the calculation of test MSDs. In the example given, the EMS from

test nine should be used to calculate sw as:

sw = EMS

sw= 68.31

sw = 8.265

4. Calculate the MSD using an increase in test replication - Using the equation

below and Table 2.4, calculate the MSD with an alpha of 0.01 and assuming one

additional replicate per treatment.

MSD d s

n n

w

c

= × +

1 1

0

where:

d = Dunnett’s t obtained from Table 2.4 using an alpha of 0.01 and

the increased number of replicates

sw = square root of the error mean square from the least sensitive of

the previous 10 tests

n0 = increased number of replicates in the control

nc

 = increased number of replicates for each effluent concentration
2-12

For the example given, the MSD first should be calculated with one additional

replicate (10 original replicates + 1 additional replicate = 11 replicates) to obtain:

MSD = 2 940 × 8 265 +

1

11

1

11 . .

MSD = 10.36

Table 2.4. Comparison of critical Dunnett’s values for five concentrations and a control

using alpha = 0.05 and 0.01.1

Number of replicates Degrees of freedom alpha = 0.05 alpha = 0.01

3 12 2.502 3.420

4 18 2.407 3.206

5 24 2.362 3.107

6 30 2.335 3.049

7 36 2.318 3.012

8 42 2.305 2.986

9 48 2.296 2.967

10 54 2.289 2.952

11 60 2.284 2.940

12 66 2.279 2.931

13 72 2.275 2.923

14 78 2.272 2.916

15 84 2.269 2.910

16 90 2.267 2.905

17 96 2.265 2.901

18 102 2.263 2.897

19 108 2.261 2.894

20 114 2.260 2.891

1

 Critical values were calculated using the Dunnett’s procedure in SAS (SAS Institute, 1990). Critical values were determined using

equal replication in five test concentrations and a control. Degrees of freedom were determined as N - (k+1), where, N = total

number of replicates in the experiment, and k = number of non-control treatments.

5. Determine if the increased replication meets the MSD criterion - If the MSD

calculated in the above step is less than or equal to the MSDmax calculated in step 2,

then the number of replicates used in this calculation is the appropriate replication that

should be used in future testing. If the MSD calculated in the above step is greater
2-13

than the MSDmax, then repeat step 4 using one additional replicate. Continue to repeat

step 4, each time with an additional replicate, until the MSD is less than or equal to the

MSDmax calculated in step 2. 

For the example given, the MSD calculated with 11replicates (10.36) was larger than

the MSDmax (9.213) calculated in step 2, so additional replicates are needed. The

above equation is repeated using one additional replicate until the calculated MSD

meets the criterion. For this example, the criterion is first met at a level of 14

replicates: 

MSD = 2 916× 8 265 +

1

14

1

14

. .

MSD = 9.109

Based on the above calculations for this example, the laboratory should use 14 test

replicates per treatment in future testing using an alpha of 0.01.

C Step 3 - After a target alpha level of 0.01 has been specified (Step 1) and a decision has

been made regarding the need for increased test replication (Step 2), testing may begin

using the target alpha level (0.01) and the revised test design (i.e., replication). For each

test that is performed, the MSD should be calculated and compared to the MSD criterion

(Table 2.1). If the test meets the MSD criterion, the results may be reported using the

target alpha level (0.01). If the test does not meet the MSD criterion, the results should be

reported using the traditional alpha of 0.05. If more than 1 in 10 tests fail to meet the

criterion, the permittee should reconsider the need and extent of increased replication.
3-1

3

Confidence Intervals

T

he WET method manuals (USEPA, 1993c; USEPA, 1994a; USEPA, 1994b) provide

specific directions for the derivation of effect concentrations from WET tests. Effect

concentrations recommended for reporting results from WET tests are either based on

hypothesis testing (NOEC, LOEC) or point estimation (LC50, EC50, IC25). Multiple

effect concentrations are possible for each WET method. For example, the potential endpoints

reported for the fathead minnow larval survival and growth chronic test include an IC25 for

growth, NOEC for growth, LC50 for survival, and a NOEC for survival. For each type of

endpoint, flowcharts in the WET method manuals guide the analyst to the proper choice of

statistical methods based on assumptions and determinations that can be made from the data. The

proper statistical method can then be performed using EPA or commercially available software to

derive the desired effect concentration. For point estimation techniques (LC50, EC50, IC25) the

statistical methods generally produce an effect concentration with associated 95% confidence

intervals. However, under certain circumstances confidence intervals are not produced or they are

unreliable. This chapter provides clarification and guidance on the circumstances under which

confidence intervals are not generated or are not suitable. Currently, confidence intervals are not

reported in the permit compliance system but may be used in interpreting results of WET tests. 

Statements in this method guidance document regarding software refer to current versions of

software available from USEPA at the following web site address:

http://www.epa.gov/nerleerd/stat2.htm.

When are confidence intervals not generated by point estimation techniques?

Point estimation techniques may fail to generate confidence intervals if: 

C Test data do not meet specific assumptions required by the statistical methods - Under

these circumstances, an alternate statistical method should be used as indicated in the

flowcharts for statistical analysis provided in the WET method manuals. These flowcharts

guide the analyst to the proper statistical technique based on the appropriateness of data

assumptions. In order to obtain reliable point estimates and confidence intervals from the

Probit method, it is required that the data contain at least two partial mortalities (i.e., percent

mortalities between 0 and 100%) and that the slope differ significantly from zero. If the

assumption of two partial mortalities is not met, the software will provide a warning and

neither point estimates nor confidence intervals will be generated. If the slope does not differ

significantly from zero, point estimates will be generated without confidence intervals. In either

of two situations (less than two partial mortalities or a significant Chi-square test indicating

lack of fit to the model), the analyst should resort to use of the Spearman-Karber or Trimmed

Spearman-Karber methods as indicated by the flowcharts in the WET method manuals. The
3-2

Spearman-Karber and Trimmed Spearman-Karber methods require at least one partial

mortality to calculate an effect concentration and associated confidence intervals. If this

assumption is not met by the data, EPA’s Trimmed Spearman Karber software will

automatically default to the use of the Graphical Method for determining point estimates. 

Since the Graphical Method does not estimate confidence intervals, EPA’s Trimmed Spearman

Karber software will produce a point estimate without confidence intervals and state that 95%

confidence limits are not calculated. For sublethal effects, the inhibition concentration

percentage (ICp) procedure is recommended for determining effect concentrations. Data

assumptions for the ICp method are not tested by the ICp software. Thus, failure of test data

to meet assumptions of the ICp method does not result in a failure to generate point estimates

or confidence intervals.

C Point estimates are outside of the test concentration range - The Probit method may not

produce confidence intervals if the generated point estimate is greater than the highest test

concentration. In this case, the software will provide a warning that the slope is not

significantly different from zero. The Spearman-Karber and Trimmed Spearman-Karber

methods will produce neither point estimates nor confidence intervals if the point estimate is

outside of the test concentration range. In this case, the software will produce an error

message stating that the required trim is too large. The ICp method will not generate

confidence intervals if a point estimate is above the test concentration range. The software will

produce a warning that none of the group response means were less than 75% of the control

mean. Whenever a point estimate lies above the test concentration range, the test result should

be reported as greater than the highest test concentration (e.g., IC25 >100% or LC50 >100%). 

Whenever a point estimate lies below the test concentration range, the test result should be

reported as less than the lowest test concentration (e.g., IC25 <6.25% or LC50 <6.25%).

Under these circumstances, confidence intervals are not applicable since exact point estimates

are not reported. 

C Specific limitations imposed by the software are encountered - The ICp software may fail

to generate confidence intervals if the number of random resamplings of the data used in the

bootstrapping technique is not a multiple of 40. This may occur when the analyst selects a

number of resamplings that is not a multiple of 40, or it may occur if one or more of the

random resamples is automatically removed from the analysis. The ICp software will

automatically remove random resamples that produce effect concentrations above the highest

test concentration. If this occurs, the software will produce an error message that states that

the number of resamplings was not a multiple of 40. The occurrence of this error increases

with increasing test variability, increases as the point estimate approaches the highest test

concentration, and increases with an increasing number of random resamples selected. This

anomaly is due to a limitation of the ICp software and not necessarily an inherent limitation of

statistical bootstrapping techniques upon which the software is based. For this reason, EPA

recommends that confidence intervals for the ICp method not be reported or used in WET

testing until the ICp software has been thoroughly reviewed by experts and possibly modified. 

This recommendation should not affect NPDES reporting in the interim since confidence

intervals are not currently reported in the permit compliance system.
3-3

In summary, the choice of statistical methods, the choice of software for analysis, and the

appropriateness of test data for those methods and software is important in generating reliable

results. Computer programs for WET data analysis, modifications to those programs, data

appropriateness for the programs, and user decision points within the programs should be

evaluated by a statistician to verify that use of the programs is consistent with the WET method

manuals and current statistical science. Laboratory analysts and regulatory authorities should also

recognize that confidence intervals from statistical programs should always be considered

approximate. Confidence intervals may not provide the exact coverage intended because of

deviations from method assumptions. Lastly, investigators should keep informed of additional and

improved techniques and software for WET data analysis that may become available.

 
4-1

4

ConcentrationResponse

Relationships

T

his chapter is designed to explain the concept of a concentration-response relationship. 

This chapter also identifies common patterns of WET test data and provides guidance on

using the concentration-response concept to review WET test results.

How will this guidance be incorporated into WET test methodology?

EPA plans to incorporate the guidance presented in this chapter into the WET method manuals

(USEPA, 1993c; USEPA, 1994a; USEPA, 1994b). A proposal to amend the manuals is expected

to appear in the Federal Register by March 2001.

What is the concentration-response relationship concept?

The concept of a concentration-response, or more classically, a dose-response relationship is “the

most fundamental and pervasive one in toxicology” (Casarett and Doull, 1975). This concept

assumes that there is a causal relationship between the dose of a toxicant (or concentration for

toxicants in solution) and a measured response. A response may be any measurable biochemical or

biological parameter that is correlated with exposure to the toxicant. The classical concentrationresponse relationship is depicted as a sigmoidal shaped curve (Figure 4.1), however, the particular

shape of the concentration-response curve may differ for each coupled toxicant and response pair. 
4-2

Concentration

0

50

100

LC50

Figure 4.2. Example determination of point estimates from a concentrationresponse curve.

In general, more severe responses (such as acute effects) occur at higher concentrations of the

toxicant, and less severe responses (such as chronic effects) occur at lower concentrations (Figure

4.1). A single toxicant also may produce multiple responses, each characterized by a

concentration-response relationship. 

In classical toxicology, concentration-response curves are generally displayed such that responses

increase with increasing concentration (Figure 4.1). This is accomplished by defining responses in

terms of adverse effects (e.g., mortality, reduction in growth, reduction in reproduction). The

WET method manuals do not follow this convention; rather, responses are displayed in terms of

survival, growth, and reproduction such that concentration-response curves for toxicants decrease

with increasing concentration. This guidance will remain consistent with the convention

established in the WET method manuals and will display concentration-response relationships for

WET data such that responses decrease with increasing concentration. 

How is the concentration-response concept used in WET testing?

The concentration-response concept is the basis for the determination of point estimates (LC50,

EC50, IC25, etc.) in WET testing. A biological response (mortality, growth inhibition,

reproductive inhibition, etc.) is measured at a range of effluent concentrations to develop a

concentration-response curve. This curve, which is typically sigmoidal, is then linearized by

various transformations of the data (e.g., probit transform) to assist in drawing conclusions from

the relationship. From the resulting linearized concentration-response curve, a point estimate effect

concentration can be calculated (Figure 4.2). The effect concentration is an estimate of the

concentration of effluent that will produce a specific level of response (e.g., 50% mortality). In

WET testing, effect concentrations such as the LC50, EC50, IC25 and IC50 are commonly used to

report WET test results. 
4-3

How can the concentration-response concept be used to review WET test results?

A corollary of the concentration-response concept is that every toxicant should exhibit a

concentration-response relationship, given that the appropriate response is measured and given that

the concentration range evaluated is appropriate. Use of this concept can be helpful in determining

whether an effluent possesses toxicity and in identifying anomalous test results. An evaluation of

the concentration-response relationship generated for each sample is an important part of the data

review process that should not be overlooked. This chapter provides guidance on identifying valid

concentration-response relationships and interpreting results from unexpected concentrationresponse patterns. This guidance on reviewing concentration-response 

relationships should be viewed as a component of a broader quality assurance and data review and

reporting process that includes: 

C Review of test conditions - The WET method manuals provide a summarized method-specific

list of test conditions that should be followed in all WET test (e.g., test temperatures, number

of replicates, test chamber sizes and volumes, lighting, feeding regimes, etc.). The conduct of

each test should be reviewed to ensure that these conditions were met within the flexibility

provided by the method manuals. The test conditions used in the test and any deviation from

WET method manual requirements should be clearly reported. Daily measurements should be

reviewed to ensure that values are within the acceptable ranges. Calibration of equipment

should be verified and noted.

C Review of test acceptability criteria - The WET method manuals provide method-specific

minimum criteria for the acceptability of tests (e.g., minimum control survival, reproduction,

growth, or variability). These criteria are requirements of the methods, and any test not

meeting the minimum test acceptability criteria should be considered invalid. All invalid tests

should be repeated with a newly collected sample. While permit compliance should not be

based on an invalid test, EPA’s promulgation of the methods requires the results of all tests to

be reported (valid or invalid).

C Review of reference toxicant testing - Reference toxicant testing is an important quality

control practice that is required in the WET method manuals. Reference toxicant testing

should be conducted on at least a monthly basis for each test method routinely conducted in a

laboratory. WET test review should include evaluation of the most recent reference toxicant

test and the reference toxicant cusum chart maintained by the laboratory. All reference

toxicant tests should be conducted similarly (e.g., test duration, test conditions, test endpoint)

to effluent tests being conducted. For instance, acute reference toxicant testing should be

conducted to accompany acute testing of effluents, and short-term chronic reference toxicant

testing should be conducted to accompany short-term chronic testing of effluents. 

C Review of organism culture health and performance - EPA recommends that laboratories

monitor and record the health and performance of organism cultures from which test organisms

are obtained. For instance, the survival and reproduction of Ceriodaphnia dubia brood stock

should be monitored and recorded during routine culture maintenance (i.e., water changes). 

This can be accomplished with a subset of 10 to 20 brood culture animals in individual culture

vessels. This monitoring and documentation allows a laboratory to assess the current condition
4-4

of organism cultures prior to initiating a test and can allow the laboratory to postpone testing if

organism cultures are unhealthy. This can potentially reduce the incidence of invalid tests and

the cost associated with retesting. In the test review step, the documentation of culture health

and performance can be useful in either identifying or eliminating poor culture health as a

cause for marginal control performance in a test. Laboratories should maintain culture control

charts (cusum charts) for survival, reproduction, growth, or other parameters for the

appropriate species.

C Review of test variability - EPA recommends that the variability of each WET test, measured

as a minimum significant difference (MSD) or percent MSD, be calculated and reported with

all test results. EPA also recommends that laboratories maintain control charts for percent

MSDs (USEPA, 2000). These control charts will allow laboratories to assess individual test

variability in the context of typical variability within the laboratory. High test variability can

result in insensitive tests or unexpected concentration-response relationships. Consult USEPA

(2000) for additional guidance on WET test method variability. 

C Review of concentration-response relationships - The guidance provided in this chapter may

be used to assist in evaluating the concentration-response relationship as a part of the data

review and reporting process. The succeeding section (“What are some patterns of

concentration-response relationships typically seen in WET test data?”) provides examples of

common patterns in WET test data, discusses possible causes and solutions for unexpected

patterns, and provides guidance on when to accept or reject test data based on the

concentration-response concept. Some states have already developed similar guidance

(Washington State Department of Ecology, 1997). It should be noted that the determination of

a valid concentration-response relationship is not always clear cut. Data from some tests may

suggest consultation with professional toxicologists and/or regulatory officials. Tests that

exhibit unexpected concentration-response relationships also may indicate a need for further

investigation and possible retesting. In general, when unexpected or apparently anomalous

concentration-response relationships are encountered, EPA recommends the following:

- attempt to determine a cause for the response - The above mentioned test review steps

and specific guidance for individual concentration-response relationships (see “What are

some patterns of concentration-response relationships typically seen in WET test data?”)

may assist in determining a cause for unexpected concentration-response relationships. 

Unexpected concentration-response relationships could be valid response patterns or

anomalies resulting from Type I test error, high test variability, or other causes. If a given

effluent consistently produces a specific, unexpected concentration-response relationship,

there is likely a physical, chemical or biological cause. In situations where difficult-tointerpret concentration-response relationships are produced consistently by a given

effluent, consultation with professional toxicologists is recommended. Toxicity

identification evaluation (TIE) procedures (USEPA, 1991a; USEPA, 1992; USEPA,

1993a; USEPA, 1993b; USEPA, 1996b) also provide guidance that may be useful in

determining a cause for such concentration-response relationships. 

- follow guidance for specific concentration-response patterns - The succeeding section

(“What are some patterns of concentration-response relationships typically seen in WET
4-5

test data?”) provides examples of 10 concentration-response patterns that may be exhibited

by WET test data. This section provides guidance in interpreting each concentrationresponse pattern using a step-by-step review process. Based on this review, the guidance

may recommend acceptance of the calculated results (e.g., NOEC or IC25) as valid and

reliable, explanation of the calculated results as anomalous, or retesting. 

- increase testing frequency - EPA recommends a testing frequency increase after any

anomalous, questionable, or failing test result, with the number of tests and duration of

testing to be determined by the regulatory authority. 

- coordinate with regulatory authorities, permittees, and testing laboratory - EPA

recommends that regulatory authorities, permittees, and testing laboratory personnel work

together to resolve difficult-to-interpret WET test data. EPA also recommends that

discussions be initiated as soon as possible when questions arise regarding WET test

results.

This chapter provides additional guidance on reviewing test data; it is not the intent of this chapter

to recommend the frequent disqualification and repetition of WET tests. Several warnings and

safeguards should be considered when implementing the guidance in this chapter. First,

unexpected concentration-response relationships should not occur with any regular frequency. 

Second, it is not recommended to screen only those tests in which toxicity is found at or below the

receiving water concentration (RWC). If screening is to be done for unexpected concentrationresponse relationships, all tests should be screened in a similar manner. Third, all testing results

should be reported to the regulatory authority, and the regulatory authorities should review all tests

(including those disqualified and repeated). Regulatory authorities should be alert to patterns such

as a high or increasing test rejection rate or a tendency for disqualified tests to show toxicity more

often than tests accepted without qualification.

What are some patterns of concentration-response relationships typically seen in

WET test data?

Ten concentration-response patterns that may appear in WET testing are individually described

and illustrated below using hypothetical test data. This section provides guidance in interpreting

each concentration-response pattern. The guidance focuses on determining a cause for unexpected

concentration-response patterns by recommending a step-by-step review process. Based on this

review, the guidance may recommend acceptance of the calculated results (e.g., NOEC or IC25) as

valid and reliable, explanation of the calculated results as anomalous, or retesting. When retesting

is recommended, this generally means beginning a new test on a newly collected sample since

sample holding times are typically expired by the time results are obtained from the original test. 

Test results should be reported for all tests conducted, even if retesting is recommended.
4-6

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Control 6.25 12.5 25 50 100

Percent Effluent

Mean 7-day Survival

1. Ideal concentration-response relationship

This response pattern (Figure 4.3) shows a clear concentration-response relationship, with multiple

effluent concentrations identified as significantly different from the control. This pattern also

shows a monotonic decrease in response, meaning that the response steadily decreases for each

higher effluent concentration. This pattern is indicative of a well designed test with appropriately

chosen concentrations that bracket the effluent’s range of toxicity. Under these circumstances, the

hypothesis testing and point estimation techniques recommended in the WET method manuals

provide reliable results. 

Figure 4.3. Ideal concentration-response relationship. 1

1

 Solid squares indicate data points that are statistically significantly different from the control, and hollow squares indicate data

points that were not significantly different from the control. The dotted line shows the control mean minus the minimum significant

difference (MSD); any test treatment response mean less than this value is considered to differ significantly from the control mean.

2. All or nothing response 

The “all or nothing” response pattern is very common in WET test data. This response pattern

(Figure 4.4) is characterized by a transition from no significant effect at one effluent concentration

to a complete effect (100% mortality) at the next higher concentration. While not ideal, this

pattern also represents a valid concentration-response relationship, and both hypothesis testing and

point estimation techniques recommended in the WET method manuals will provide reliable

results. This pattern of response is indicative of a steep concentration-response curve for the given

effluent, and under these circumstances, the precision of the estimate may be improved by closer

spacing of effluent concentrations (increased dilution factor) or the addition of intermediate effluent

concentrations in future testing.
4-7

0

0.2

0.4

0.6

0.8

1

1.2

Control 6.25 12.5 25 50 100

Percent Effluent

Mean 7-day Survival

Figure 4.4. All or nothing concentration-response relationship. 1

1

 Solid squares indicate data points that are statistically significantly different from the control, and hollow squares indicate data

points that were not significantly different from the control. The dotted line shows the control mean minus the minimum significant

difference (MSD); any test treatment response mean less than this value is considered to differ significantly from the control mean.

3. Stimulatory response at low concentrations and detrimental effects at higher

concentrations 

A stimulatory response is a nonmonotonic concentration-response relationship characterized by a

measured increase in the response (stimulation) at low concentrations. This stimulation at low

concentrations can be followed by a detrimental effect at higher concentrations (Figure 4.5) or by

no effect at higher concentrations (see Section 4 following). Davis and Svendsgaard (1993) found

that such nonmonotonic concentration-response relationships occurred in 12-24% of the

toxicological studies surveyed. The stimulatory response pattern characterized in Figure 4.5 is

typically found with sublethal endpoints such as reproduction, growth, fertilization, or larval

development. For instance, test organism reproduction may increase (relative to the control) at low

concentrations of an effluent and decrease relative to the control at higher concentrations. This

concentration-response pattern, while nonmonotonic, is still a valid concentration-response

relationship, and both hypothesis testing and point estimation techniques recommended in the WET

method manuals will provide reliable results.
4-8

0

5

10

15

20

25

30

35

40

Control 6.25 12.5 25 50 100

Percent Effluent

Mean Number of Young

Figure 4.5. Stimulation at low concentrations and significant effects at high concentrations. 1

1 Solid squares indicate data points that are statistically significantly different from the control, and hollow squares indicate data

points that were not significantly different from the control. The dotted line shows the control mean minus the minimum significant

difference (MSD); any test treatment response mean less than this value is considered to differ significantly from the control mean.

4. Stimulation at low concentrations but no significant effect at higher concentrations

This concentration-response relationship is similar to the previous example in that stimulation is

observed at lower concentrations, but in this case, higher concentrations do not produce significant

effects (Figure 4.6). In this situation, hypothesis testing techniques should produce reliable results,

assuming that adequate test sensitivity is achieved. Results from point estimation techniques

should be interpreted carefully when this response pattern is encountered, because the inhibition

concentration percentage (ICp) procedure may produce effect concentrations (particularly IC25s)

that indicate toxicity at effluent concentrations where the response is comparable to the control

response. The ICp procedure assumes that responses: (1) are from a random, independent, and

representative sample of test data; (2) follow a piecewise linear response function; and (3) are

monotonically non-increasing, meaning that the mean response for each higher concentration is less

than or equal to the mean response for the previous concentration. If the data are not

monotonically non-increasing, the ICp procedure adjusts the response means using a “smoothing”

technique that averages adjacent means (see Appendix M of USEPA, 1994a). This technique

averages response means (including that of the control) with those of the next highest test

concentration until responses are monotonically non-increasing. In cases where the responses at

the low effluent concentrations are much higher than in the control, the smoothing process may

result in a large upward adjustment in the control mean. This can lead to an IC25 result that is less

than the highest test concentration, even though the highest test concentration was not statistically

different from the control treatment and even if a percent difference of less than 25% was observed

between the control response and the response at the highest test concentration. 
4-9

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5

10

15

20

25

30

35

40

Control 6.25 12.5 25 50 100

Percent Effluent

Mean Number of Young 

Figure 4.6. Stimulation at low concentrations but no significant effect at higher

concentrations. 1

1

 Solid squares indicate data points that are statistically significantly different from the control, and hollow squares indicate data

points that were not significantly different from the control. The dotted line shows the control mean minus the minimum

significant difference (MSD); any test treatment response mean less than this value is considered to differ significantly from the

control mean.

If the response pattern depicted in Figure 4.6 (stimulation at low concentrations but no

significant effect at higher concentrations) is encountered, the following review steps should be

taken in addition to standard test review procedures:

C Evaluate the concentration range - If the highest concentration used in the test was less

than 100% effluent (or the highest achievable effluent concentration for marine tests), the

effluent should be retested using higher test concentrations to establish if a valid

concentration-response relationship exists. This may not be necessary if the permit limit is

set at much lower than 100% effluent and test results indicate no toxicity at the permit limit

level and at least one concentration above the permit limit.

C Compare hypothesis testing results and point estimates - If there is agreement between

the NOEC and the IC25 for tests producing the concentration-response pattern depicted in

Figure 4.6 (i.e., neither value indicates toxicity at or below the permitted RWC, or both

values indicate toxicity at or below the RWC) the test results should be reported and

considered valid. If, however, the NOEC indicates no toxicity at the RWC (i.e., NOEC

greater than or equal to RWC) but the IC25 is calculated as less than the RWC, the remaining

recommended actions should be taken.

C Evaluate control response - It is possible that the response pattern depicted in Figure 4.6

could result from poor performance in the controls rather than stimulation at the lower test

concentrations. This poor control performance could cause a toxic effect at higher test

concentrations not to be detected. To evaluate this possibility, compare the control response
4-10

to the normal control performance for the laboratory. If (1) a particular test exhibits the

response pattern depicted in Figure 4.6, (2) there is disagreement between NOEC and IC25

estimates, and (3) the mean control response is well below the laboratory’s normal range of

control performance; retesting of the effluent is recommended even if the minimum test

acceptability criteria have been met. For example, if a laboratory consistently achieves a

control mean of 25-30 neonates for the Ceriodaphnia dubia 3-brood chronic test, a control

mean of 15-18 neonates (in conjunction with a non-ideal concentration-response curve and

disagreement between the NOEC and IC25) would warrant retesting. In this situation,

suppressed control performance could be considered as the cause for this response pattern

rather than stimulation. A review of control performance should also investigate the

possibility of poor performance in a single replicate substantially reducing the mean control

response. In this case, retesting is also recommended.

C Evaluate the test sensitivity - Discrepancies between IC25 and NOEC values could be due

to low test sensitivity. To determine if this is the case, evaluate the sensitivity of the test by

comparing the test MSD to MSD criteria for the given test method (see Chapter 2 of this

guidance and USEPA, 2000) and to the laboratory’s historical test sensitivity performance. 

Laboratories are encouraged to track test sensitivity (as %MSDs) for tests conducted over

time. If a test exhibits the response pattern depicted in Figure 4.6 and the test MSD is above

maximum recommended criteria for the method or above the laboratory’s typical range, the

sample should be retested. 

C Evaluate the ICp calculation - If a test exhibits the response pattern depicted in Figure 4.6

and it has been determined from the above actions that the pattern is not due to poor control

performance or low test sensitivity, then discrepancies between the NOEC and IC25 may be

due to bias from the ICp smoothing technique. To determine if this is the case, calculate the

observed percent difference between the response at the RWC and the control as: 

(m m )

m

c RWC

c

−

× 100%

where:

m = mean control response

c

m RWC = mean response at the receiving water concentration (RWC)

If the observed percent difference between the response at the RWC and the control is less

than 25% and the response at the RWC is not statistically significantly different from the

control response, then a calculated IC25 of less than the RWC should be noted as anomalous

and the effluent determined to be non-toxic at the RWC. If the observed percent difference

is equal to or greater than 25%, then the calculated IC25 should be considered valid. 
4-11

0

0.2

0.4

0.6

0.8

1

1.2

Control 6.25 12.5 25 50 100

Percent Effluent

Mean 7-day Survival

5. Interrupted concentration-response: significant effect bracketed by non-significant effects

This response pattern is characterized by a single test concentration showing a significant

difference from the control while adjacent higher and lower test concentrations do not differ

significantly from the control (Figure 4.7). When this response pattern is encountered, point

estimation techniques generally will yield reliable results, but hypothesis testing results should be

interpreted carefully. The method manual definitions of NOEC (the highest concentration of

toxicant in which the values for the observed responses are not statistically significantly different

from the controls) and LOEC (the lowest concentration of toxicant in which the values for the

observed responses are statistically significantly different from the controls) were intended for

situations where the concentration-response relationship is monotonically non-increasing. Under

these circumstance, the NOEC and LOEC are always adjacent values with the NOEC being the

test concentration just below the LOEC. In circumstances where the concentration-response

relationship is non-monotonic (as in Figure 4.7), the identification of NOEC and LOEC values is

severely compromised (Chapman et al., 1996). For this response pattern, the following review

actions should be taken in addition to standard test review procedures to determine the validity of

results obtained by hypothesis testing:

Figure 4.7. Interrupted concentration-response: significant effect bracketed by nonsignificant effects. 1

1 Solid squares indicate data points that are statistically significantly different from the control, and hollow squares indicate data

points that were not significantly different from the control. The dotted line shows the control mean minus the minimum significant

difference (MSD); any test treatment response mean less than this value is considered to differ significantly from the control mean.

C Check for test condition or procedural errors - The concentration-response relationship

depicted in Figure 4.7 could result from test conditions errors (such as pH, DO, salinity, or

temperature excursions) occurring in isolated test replicates. This concentration-response
4-12

pattern also could be due to procedural errors such as failure to properly randomize test

organisms or test chamber placement. The laboratory should verify that all test conditions

were within ranges required by the WET method manuals for the given test method. The

laboratory should verify that the assignment of test organisms to individual treatments was

properly randomized (Davis et al., 1998). This can be complete randomization or block

randomization (as with the Ceriodaphnia dubia 3-brood reproduction test). The laboratory

also should verify that the positions of test chambers within the experiment were properly

randomized. If test condition or procedural errors are identified, the sample should be retested.

C Evaluate within-treatment variability - It is possible for poor performance in a single

replicate to bias the mean response for a given test concentration and cause that concentration

to differ significantly from the control. For this reason, the within-treatment variability should

be evaluated for the significantly different treatment. If the variability (standard deviation or

CV) for that treatment is considerably greater than for other treatments, then responses of

individual replicates should be investigated. This investigation may show that a single outlier

replicate has biased the treatment mean. If this is the case and the responses from all but the

single outlier replicate are consistent with the control response, then the sample should be

retested.

C Evaluate test sensitivity - When the response pattern depicted in Figure 4.7 is encountered, it

is important to evaluate test sensitivity. If test sensitivity is low (e.g. high MSD values), large

effects at higher test concentrations may not be detected as statistically significant. To evaluate

test sensitivity, compare the MSD for the test to benchmark criteria for the given test method

(see Chapter 2 of this guidance and USEPA, 2000) and to the laboratory’s historical test

sensitivity performance. As previously mentioned, laboratories are encouraged to track test

sensitivity (as %MSDs) for tests conducted over time. If test sensitivity is low (i.e., MSDs are

above maximum recommended criteria or typical laboratory performance), then the sample

should be retested. Consult Section 6.4 in USEPA (2000) for additional guidance on

implementing upper and lower bounds on test sensitivity. 

If test sensitivity is moderate to high (i.e., MSDs below the maximum recommended criteria

and within the laboratory’s typical performance range) and none of the preceding evaluations

have determined a cause for this response pattern, it is likely that the significantly different

treatment is the result of a Type I error. A Type I error is the error of incorrectly rejecting the

null hypothesis (assuming that the treatment is significantly different from the control) when in

fact the null hypothesis is true (the treatment is not significantly different from the control). In

this situation, due to the absence of a valid concentration-response relationship, the

intermediate concentration that was determined by hypothesis testing to be statistically

different from the control should be considered anomalous, and the NOEC should be

determined as the highest concentration that was not significantly different from the control. 

Using Figure 4.7 to illustrate, the 25% concentration would be considered anomalous, the

reported NOEC would be 100%, and the reported LOEC would be >100%. Under these

circumstances, test results should still note that the 25% concentration was statistically
4-13

0

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0.6

0.8

1

1.2

Control 6.25 12.5 25 50 100

Percent Effluent

Mean 7-day Survival

different from the control but was considered anomalous due to analysis of the concentrationresponse curve and the above review steps.

6. Interrupted concentration-response: non-significant effects bracketed by significant effects

This response pattern is similar to the previous response pattern in that the concentration-response

curve is nonmonotonic (or interrupted), however, this response pattern is characterized by two or

more test concentrations showing a significant difference from the control while an intermediate

test concentration does not differ significantly from the control (Figure 4.8). When this response

pattern is encountered, point estimation techniques will generally yield reliable results, but

hypothesis testing results should be interpreted carefully. As mentioned for the previous

concentration-response pattern, the identification of NOEC and LOEC values is severely

compromised (Chapman et al., 1996) when the concentration-response relationship is nonmonotonic (as in Figure 4.8). For this response pattern, the test sensitivity should be evaluated as

described below in addition to standard test review procedures to determine the validity of results

determined by hypothesis testing.

Figure 4.8. Interrupted concentration-response: non-significant effects bracketed by

significant effects. 1

1

 Solid squares indicate data points that are statistically significantly different from the control, and hollow squares indicate data

points that were not significantly different from the control. The dotted line shows the control mean minus the minimum significant

difference (MSD); any test treatment response mean less than this value is considered to differ significantly from the control mean.

C Evaluate test sensitivity - When the response pattern depicted in Figure 4.8 is encountered, it

is important to evaluate test sensitivity by comparing test MSDs to minimum and maximum

MSD criteria recommended by EPA (USEPA, 2000). If the test MSD is lower than the

minimum MSD criterion, only effects larger than the minimum MSD criterion should be
4-14

considered significant. For example, if the minimum MSD criterion for a method is 15% and

the calculated test MSD is 10%, only effects greater than 15% difference compared to the

control should be considered significant. If test sensitivity is low (i.e., test MSD is above

maximum MSD criterion), the sample should be retested. If test sensitivity is moderate (i.e.,

test MSD is within minimum and maximum MSD criterion), the test results should be

considered valid and the NOEC should be reported as the concentration below the LOEC. For

the case depicted in Figure 4.8, a NOEC of 12.5% should be reported. Consult Section 6.4 in

USEPA (2000) for additional guidance on implementing upper and lower bounds on test

sensitivity. 

7. Significant effects only at highest concentration

This response pattern is characterized by only the highest test concentration producing a

significantly different response from the control (Figure 4.9). This response pattern should be

considered to be a valid concentration-response relationship and results determined by point

estimation should be assumed to be reliable. Hypothesis testing results are also assumed to be

reliable following the evaluation of test sensitivity as described below. If the response pattern

depicted in Figure 4.9 (significant effects only at highest concentration) is encountered, the

following review steps should be taken in addition to standard test review procedures:

C Evaluate the concentration range - When this response pattern occurs, the concentrations

used for testing should be evaluated in future tests using this effluent. If the highest effluent

concentration used in the test was less than 100% (or the highest achievable effluent

concentration for marine tests), future testing using this sample should include at least one

higher test concentration to confirm the presence of a concentration-response relationship. If

the test used a 100% effluent concentration treatment, it is difficult to confirm a concentrationresponse relationship through retesting because concentrations are constrained to less than or

equal to 100% in whole effluent testing. If this response pattern occurs commonly with a given

effluent, future testing of the effluent should use a dilution factor of >0.5 such that test

concentrations closer to the 100% effluent concentration are used (i.e., a dilution factor of 0.65

would provide a test concentration series of 18%, 27%, 42%, 65%, and 100%). This would

provide a better opportunity to confirm a concentration-response relationship that may exist at

the upper end of the concentration range. This approach should be used only if historical

testing of the effluent indicates consistency and the effect concentration is not likely to fall

below the adjusted test concentration series.

C Evaluate test sensitivity - Evaluate test sensitivity by comparing test MSDs to minimum and

maximum MSD criteria recommended by EPA (USEPA, 2000). If the test MSD is lower than

the minimum MSD criterion, only effects larger than the minimum MSD criterion should be

considered significant. For example, if the minimum MSD criterion for a method is 15% and

the calculated test MSD is 10%, only effects greater than 15% difference compared to the

control should be considered significant. If test sensitivity is low (i.e., test MSD is above

maximum MSD criterion), the sample should be retested. If test sensitivity is moderate (i.e.,

test MSD is within minimum and maximum MSD criterion), the test results should be
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0.6

0.8

1

1.2

Control 6.25 12.5 25 50 100

Percent Effluent

Mean 7-day Survival

considered valid and the NOEC should be reported as the concentration below the LOEC. For

the example given in Figure 4.9, a NOEC of 50% effluent should be reported. Consult Section

6.4 in USEPA (2000) for additional guidance on implementing upper and lower bounds on test

sensitivity. 

Figure 4.9. Significant effects only at highest concentration. 1

1

 Solid squares indicate data points that are statistically significantly different from the control, and hollow squares indicate data

points that were not significantly different from the control. The dotted line shows the control mean minus the minimum significant

difference (MSD); any test treatment response mean less than this value is considered to differ significantly from the control mean.

8. Significant effects at all test concentrations but flat concentration-response curve

This response pattern is demonstrated in Figure 4.10. All of the test concentrations produce a

response that is significantly different from the control response, but a clear concentration-response

relationship cannot be determined. This response pattern could be due to: (1) extremely low

variability in the control, (2) an unusually high control response, (3) an inappropriate dilution

water and improper use of dilution water controls, (4) inappropriate test dilution series, (5)

potential pathogen effects in the effluent, (6) an unusual effluent-dilution water interaction. The

following review actions should be taken to determine a cause for this concentration-response

pattern and to subsequently determine the validity of calculated results.

C Evaluate test sensitivity - The response pattern depicted in Figure 4.10 may be an artifact of

the data resulting from extremely precise control results and extremely high test sensitivity. 

Investigate this possibility by comparing test MSDs to minimum MSD criteria recommended

by EPA (USEPA, 2000). If the test MSD is lower than the minimum MSD criterion, only

effects larger than the minimum MSD criterion should be considered significant. For example,

if the minimum MSD criterion for a method is 15% and the calculated test MSD is 10%, only

effects greater than 15% difference compared to the control should be considered significant. 
4-16

0

5

10

15

20

25

30

Control 6.25 12.5 25 50 100

Percent Effluent

Mean Number of Young 

If test sensitivity is low (i.e., test MSD is above maximum MSD criterion), the sample should

be retested. Consult Section 6.4 in USEPA (2000) for additional guidance on implementing

upper and lower bounds on test sensitivity. 

Figure 4.10. Significant effects at all test concentrations but flat concentration-response

curve. 1

1

 Solid squares indicate data points that are statistically significantly different from the control, and hollow squares indicate data

points that were not significantly different from the control. The dotted line shows the control mean minus the minimum significant

difference (MSD); any test treatment response mean less than this value is considered to differ significantly from the control mean.

C Evaluate control response - The concentration-response pattern depicted in Figure 4.10 could

result from an unusually high response in the control treatment. Laboratories are encouraged

to track the performance of controls in tests conducted over time. When the response pattern

depicted in Figure 4.10 is exhibited, the control response for the test should be compared to

historic control performance in the laboratory using the given dilution water. If the mean

control response is above the normal range for that laboratory and dilution water, the sample

should be retested.

C Evaluate dilution water - The improper use of dilution waters and dilution water controls

could cause the concentration-response pattern depicted in Figure 4.10. It should be confirmed

that test treatment concentrations were compared to the dilution water control and not a culture

water control. A statistical comparison of the dilution water control and the culture water

control should also be made if they are from different sources. If the dilution water control

shows a statistically significant difference from the culture water control, alternate dilution

waters should be considered and the sample retested (see Chapter 6 of this guidance).

C Evaluate test concentrations - If all test concentrations produce a complete effect (e.g., 100%

mortality, zero reproduction, etc.), a flat concentration-response relationship will result. This

concentration-response relationship should be considered valid, and it indicates high toxicity in
4-17

the sample. Assuming that the concentration range used in the test brackets the permitted

RWC, it is not necessary to retest the sample, since the test results clearly indicate toxicity. If

all test concentrations were significantly different from the control but did not produce

complete effects (as in Figure 4.10), the dilution series should be investigated. It is possible

that the test concentration range used for the test was too narrow to distinguish a shallow

sloped concentration-response curve. Test concentrations may not have been low enough to

produce no significant effect and may not have been high enough to produce severe effects. If

this situation is suspected, the sample should be retested using an expanded dilution series

range. Effluent concentrations that are lower than those used in the previous test should be

added. Effluent concentrations that are higher than those used in the previous test also should

be added (if possible) to assist in determining a concentration-response relationship.

C Consider pathogen effect - The concentration-response pattern depicted in Figure 4.10 could

also be due to the presence of pathogens in the effluent. The most common identifier of

pathogen effects are sporadic mortalities and extremely high variability between replicates. 

The pathogen effect is more common in tests using fish species than in invertebrate testing. 

This pathogen effect also may be evident only in chronic tests and not in acute tests. Pathogen

effects also may be seasonal in occurrence. If within-treatment CVs for survival are >40% for

effluent concentrations and relatively small for control replicates in standard synthetic water,

pathogen effect should be considered. If pathogen effects are suspected in the effluent, this

may be confirmed in subsequent side-by-side testing using the effluent and the effluent treated

by brief exposure to UV light or the addition of antibiotics, or increasing the number of

replicates and using less test organisms in each replicate. If pathogen effects in the effluent are

confirmed, the sample should be retested and the regulatory authority should be consulted prior

to changing testing procedures.

C Continued testing - If all of the above scenarios have been investigated and have not revealed

the cause of the response pattern, the results should be considered valid; however, continued

testing should be initiated in an effort to identify the cause of the response pattern. If an

effluent consistently exhibits this response pattern, additional investigations could include

chemical analysis or initiation of TIE procedures.

9. Significant effects at all test concentrations with a sloped concentration-response curve 

This concentration-response pattern is similar to the pattern identified in item #8 above except a

concentration-response curve can be identified at the higher effluent concentrations (Figure 4.11). 

This pattern is considered to be a valid concentration-response relationship, and point estimation

techniques will generally yield reliable results. Results determined by hypothesis testing techniques

should be interpreted carefully, and the cause for significantly different effects at low

concentrations should be investigated as described for the response pattern described in item #8. 
4-18

0

5

10

15

20

25

30

Control 6.25 12.5 25 50 100

Percent Effluent

Mean Number of Young 

Figure 4.11. Significant effects at all test concentrations with a sloped concentration-response

curve. 1

1

 Solid squares indicate data points that are statistically significantly different from the control, and hollow squares indicate data

points that were not significantly different from the control. The dotted line shows the control mean minus the minimum significant

difference (MSD); any test treatment response mean less than this value is considered to differ significantly from the control mean.

10. Inverse concentration-response relationship

This response pattern is characterized by a relationship in which adverse effects decrease with

increasing effluent concentration (Figure 4.12). This situation is most often encountered in algal

growth tests, and is typically caused by excess nutrients in the effluent. While a valid

concentration-response relationship is demonstrated in this circumstance, the effluent should be

considered nontoxic since the direction of the concentration-response relationship indicates

decreasing adverse effects. It should be noted that while the effluent is considered non-toxic, the

presence of excess nutrients still may pose a potential risk to the environment due to nutrient

enrichment and oxygen depletion. 

An inverse concentration-response pattern also may occur in tests other than algal growth assays

when the dilution water used is a receiving water or synthetic water adjusted to approximate the

receiving water characteristics. In such situations, the inverse concentration-response pattern can

result from toxicity in the receiving water or the limitation of necessary components (i.e., hardness)

in the receiving water or adjusted synthetic water. Under such circumstances, the objective of the

toxicity test should be evaluated (see Chapter 6 of this guidance). If the objective of the test is to

determine the toxicity of the effluent in the natural receiving water, then the results indicate no

toxicity in the sample. If the objective of the toxicity test is to determine the absolute presence of

toxicity in the effluent, the sample should be retested using a standard synthetic dilution water. 

Toxicity or limiting components in the receiving water or adjusted synthetic water may mask the
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0

0.5

1

1.5

2

2.5

3

3.5

4

Control 6.25 12.5 25 50 100

Percent Effluent

Mean Algal Growth 

 (10^6 cells/ml)

presence of low level toxicity in the effluent, making the absolute determination of toxicity in the

effluent difficult. 

Figure 4.12. Inverse concentration-response relationship. 1

1

 Solid squares indicate data points that are statistically significantly different from the control, and hollow squares indicate data

points that were not significantly different from the control. The dotted line shows the control mean minus the minimum significant

difference (MSD); any test treatment response mean less than this value is considered to differ significantly from the control mean.
5-1

5

Dilution Series

Selection

T

his chapter provides guidance on the selection of an appropriate dilution series for a WET

test. 

Do the WET method manuals specify a certain dilution series?

The WET method manuals (USEPA, 1993c; USEPA, 1994a; USEPA, 1994b) suggest, but do not

require, a dilution series of 6.25%, 12.5%, 25%, 50%, and 100% effluent for most effluents. This

dilution series should be used as a default when little information is known about the effluent being

tested and when initial range finding indicates that the effect concentration of interest is within the

6.25% to 100% effluent range. In many situations, a more appropriate dilution series can be

selected based on experience from repeated testing of a given effluent. The WET method manuals

do recommend a dilution factor of 0.5 for preparing test concentrations. This recommendation

does not fix the dilution factor, but is provided to establish a lower limit on the dilution factor. The

use of dilution factors greater than 0.5 is encouraged when historical testing indicates that an

effluent is relatively consistent and effect concentrations generally fall within a given range. 

Why is selecting an appropriate dilution series important?

The selection of a dilution series (number and spacing of test concentrations) for WET tests is

extremely important in producing reliable and precise results. This is most obvious for effect

concentrations such as NOEC and LOEC values generated by hypothesis testing. These values are

by definition limited to one of the effluent concentrations selected for the test. The precision of

these values also is determined by the distance from the NOEC or LOEC to the next highest or

lowest effluent concentration. For instance, using a standard dilution series of 6.25%, 12.5%,

25%, 50%, and 100% effluent, a measured NOEC value of 50% indicates that the transition from

no observable effects to observable effects occurs somewhere between 50% and 100% effluent

concentration (the NOEC-LOEC interval). If an alternative dilution series of 12.5%, 25%, 50%,

75%, and 100% were used for this test, then a NOEC of 50% would be a more precise estimate. 

In this test, the point of transition from no observable effect to observable effects is now known to

lie between 50% and 75%.

The appropriate selection of a dilution series also is important for accurately identifying

concentration-response relationships and increasing the precision of effect concentrations estimated

from those relationships. For example, toxicants or effluents with steep concentration-response

curves, often produce “all or nothing” results when using a standard dilution series of 6.25%,
5-2

12.5%, 25%, 50%, and 100% effluent. An “all or nothing” response means that one effluent

concentration produces no effect and the next highest concentration produces a complete (e.g.,

100% mortality) effect. Under these circumstances, the effect concentration is graphically

determined between the no effect and complete effect concentrations. The effect concentration

derived in this situation is less precise than when multiple concentrations with partial effects occur. 

The proper selection and spacing of dilutions can increase the opportunity of obtaining an ideal

concentration-response relationship (see Chapter 4 of this guidance) that exhibits smooth

transitions from no effect to partial effect to complete effect. 

How might the dilution series or dilution sequence be modified to assist in

determining a concentration-response relationship and improving the precision of

calculated effect concentrations?

The preceding chapter identified and discussed 10 concentration-response patterns typically

observed in WET testing. When applicable, recommendations for modifying the dilution series or

dilution sequence were provided in the discussion of individual response patterns. In general, the

following considerations and recommendations should improve the identification of concentrationresponse relationships and the precision of calculated effect concentrations.

C Consider historic WET testing information for the given effluent - Due to the importance

of dilution series selection, this decision should be based on knowledge of the effluent from

historical testing and permit information rather than simply on standard laboratory practice. 

Historic testing information on a given effluent will provide a typical range of effects that can

characterize the consistency of the effluent’s toxicity. This information is valuable and should

not be overlooked. If historical testing shows toxicity consistently within a specified range of

concentrations, the test dilution series for future tests can be selected to focus on that range. 

For example, if the LC50 for a given effluent is consistently between 50% and 100% effluent,

it may be needless to continue testing concentrations as low as 6.25% effluent. A larger

dilution factor, such as 0.75 could be used to provide a dilution series of 31.6%, 42.2%,

56.3%, 75%, and 100%. The analyst should be cautious not to narrow the range of

concentrations too much, to avoid causing the effect concentration to fall outside the test

concentration range when an unusually toxic sample is encountered.

C Use the receiving water concentration as a test concentration - As previously mentioned, a

limitation of hypothesis testing is that NOEC and LOEC values are constrained only to

effluent concentrations used in a test. Due to this limitation, hypothesis testing should be used

only in situations where the toxicity of a particular effluent concentration of interest is to be

evaluated (i.e., the receiving water concentration or RWC). In addition, the effluent

concentration of interest, usually the RWC, should be included as one of the concentrations in

the dilution series. Even if point estimation techniques are to be used for calculating effect

concentrations, it is good practice to include the RWC as a test concentration in the dilution

series. 

C Bracket the receiving water concentration with test concentrations - Test concentrations

selected should not only include the RWC, but also should bracket the RWC (unless the RWC
5-3

is 100%). This will allow the most precise determination of effect concentrations around the

RWC and will aid in the determination of a valid concentration-response relationship.

C Consider adding test concentrations within a given range of interest - For better test

resolution and more precise effect concentration estimates, additional test concentrations can

be added within a given range of interest. This may be most beneficial when testing an effluent

or toxicant that possesses a steep concentration-response relationship. Additional test

concentrations placed between concentrations of no effect and complete effect may allow for

partial effects to be measured and improve the precision of calculated effect concentrations. 

For instance, if no effect was observed at 100% effluent concentration and a complete effect

was observed at 50% effluent concentration, an additional test concentration of 75% could be

added to improve the precision of calculated effect concentrations. If historical testing

information for this effluent indicates that effect concentrations are consistently between 50%

and 100%, it may be possible to add the 75% concentration in place of the 6.25%

concentration (i.e., 12.5%, 25%, 50%, 75%, and 100%). The addition of test concentrations

also may be beneficial when very shallow concentration-response relationships are

encountered. In this case, additional test concentrations should be added to extend the

concentration range tested (e.g., 3.125%, 6.25%, 12.5%, 25%, 50%, and 100%).

C Consider increasing the dilution factor used to space effluent concentrations - Increasing

the dilution factor for a test (i.e., reducing the space between concentrations) is encouraged if

historic testing of the given effluent indicates relative consistency, and the given effect

concentration is not expected to lie outside of the concentration range. Similar to adding test

concentrations, increasing the dilution factor has the effect of narrowing the test focus on a

concentration range of interest. This effect is accomplished while maintaining a logarithmic

spacing of test concentrations, which is standard practice in toxicity testing. A possible

disadvantage of increasing the dilution factor is that all of the test concentrations are typically

changed when the dilution factor is altered; this may limit the comparability of results with

previous testing, if test results are determined exclusively by hypothesis testing techniques. 

The comparability of point estimates should not be affected by alterations in the dilution

factor. 
6-1

6

Dilution Waters

T

his chapter provides guidance for selecting a dilution water that is appropriate for the

objective of the WET test.

What does EPA consider to be an acceptable dilution water?

An acceptable dilution water for WET testing:

C is appropriate for the objectives of the test;

C supports adequate performance of the test organisms with respect to survival, growth,

reproduction, or other responses that may be measured in the test (i.e., consistently meets test

acceptability criteria for control responses); 

C is consistent in quality; and 

C does not contain contaminants that could produce toxicity.

In the WET method manuals (USEPA, 1993c; USEPA, 1994a; USEPA 1994b), Section 7

describes the types of dilution water that may be used for WET testing depending upon the

objectives of the test. This section provides procedures for preparing synthetic dilution waters and

procedures for the collection and handling of receiving waters or natural dilution waters. The

selection of the appropriate dilution water type should be made independently for each effluent

based upon the objectives of the test, the condition and quality of ambient receiving water, instream dilution potential, and recommendations or requirements from local regulatory authorities.

How do I choose an appropriate dilution water?

Figure 6.1 is provided to assist in selecting an appropriate dilution water for WET testing. First,

the choice of dilution waters should be consistent with the objectives of the WET test, thus the

objective of testing should be clearly defined by the regulatory authority. Tests can be conducted

in the standard reconstituted dilution water to assess the absolute toxicity of the effluent. The

WET method manuals (USEPA, 1993c; USEPA, 1994a; USEPA 1994b) describe this as the

primary objective of NPDES permit-related toxicity testing. To determine the toxicity of the

effluent in the receiving system, tests can be conducted using receiving water for dilution or

synthetic dilution water adjusted to approximate receiving water characteristics (USEPA, 1993c;

USEPA, 1994a; USEPA 1994b; USEPA, 1996a). EPA’s Technical Support Document discusses

this objective in context of EPA’s water quality based toxics control program (USEPA, 1991b). 
6-2

What is the objective of

the WET test?

Determine the

absolute toxicity of

the effluent

Determine the toxicity of

the effluent in the

receiving system

Use a standard

synthetic or

acceptable natural

dilution water that

matches the

organism culture

water

Does the receiving water

possess ambient toxicity

or fail to meet other

criteria for use as dilution

water?

Calculate test results

according to WET

method manual

procedures using

control data from

standard synthetic

(or acceptable

natural) dilution

water control

treatment

Use the local

receiving water as

the dilution water

Is the objective of

the test to determine

the additive or

mitigating effects of

the effluent on

contaminated

receiving water?

Use the receiving

water as the dilution

water

Use two sets of

controls:

1. culture water

2. receiving water

Compare the two sets

of controls

Is the receiving water

toxic?

Calculate test results

according to WET

method manual

procedures using

control data from the

receiving water

control treatment

For the dilution

water, use a synthetic

water adjusted to

approximate

receiving water

Use two sets of

controls:

1. culture water

2. adjusted synthetic

water

Are the two controls

significantly

different?

Calculate test results

according to WET

method manual

procedures using

control data from the

adjusted synthetic

water control

treatment

Calculate test results

according to WET

method manual

procedures using

control data from the

receiving water

control treatment

Consider using

organisms cultured

in or acclimated to

the adjusted

synthetic dilution

water

No

Yes

No

Yes

Yes

No

No

Yes

Unknown

Figure 6.1. Flowchart for appropriate selection and use of dilution water in WET testing.
6-3

What dilution water should I use when determining absolute toxicity of an

effluent?

If the objective of the WET test is to determine the absolute toxicity of the effluent, then a

standardized synthetic water is recommended for use as dilution water. A standardized synthetic

dilution water has the following advantages: proven success in maintaining organism health, known

chemical composition, reduced potential for effluent/dilution water interactions that may affect

toxicity, and better test reproducibility and repeatability. Under some circumstances, a consistent,

high purity natural water source (e.g., uncontaminated seawater or treated well water) may be used

in lieu of a synthetic water to determine the absolute toxicity of an effluent. Such waters may be

used if:

C the water is similar in physical and chemical composition to the standardized synthetic water

(i.e., hardness, alkalinity, pH, salinity);

C the water is used consistently and successfully by the testing laboratory for culturing the test

organisms; and

C survival and reproduction records demonstrating the successful use of the water for culturing

are provided and approved by the local regulatory authority.

What dilution water should I use when determining the toxicity of an effluent in the

receiving system?

If the objective of the WET test is to determine the toxicity of the effluent in the receiving system,

the local receiving water may be the most appropriate choice of dilution water. The use of

receiving water increases the environmental relevance of WET testing by simulating

effluent/receiving water interactions in the test. This also improves the capacity of the WET test to

predict in-stream effects. Despite these benefits, the local receiving water should first be evaluated

to determine its appropriateness for use as dilution water. To be acceptable for use as dilution

water, a receiving water should meet all of the following requirements: 

C The receiving water should be collected as a grab sample from upstream or near the final

point of discharge for the effluent of interest. The receiving water sample should be

collected from as close to the point of discharge as possible while remaining outside of the

influence of the discharge. This determination may be made by physical or chemical

measurements or by preliminary testing. Once an appropriate collection site has been located,

the location should be fully described and established as the standardized receiving water

collection location for the effluent discharge of interest.

C The receiving system should have adequate flow year round at the established receiving

water collection location. For instance, where the receiving water is classified as an

intermittent stream or where zero flow conditions exist, the use of receiving water for dilution

is inappropriate. Under these circumstances, a synthetic water adjusted to approximate the

characteristics (pH, hardness, alkalinity) of the closest downstream perennial water should be

used. 
6-4

C The receiving water should support adequate performance of the test organisms with

respect to survival, growth, reproduction, or other responses that may be measured in the

test. This is a primary requirement for all dilution waters (see question, “What does EPA

consider to be an acceptable dilution water?”). This means that the 100% receiving water

concentration used as a dilution water control should consistently meet test acceptability

criteria for control responses.

C The receiving water should be consistent in quality and not contain contaminants that

could produce toxicity. This is a primary requirement for all dilution waters (see question,

“What does EPA consider to be an acceptable dilution water?”). In the case of receiving

waters, this requirement is evaluated by the use of dual controls. For each test using receiving

water for dilution, a 100% receiving water control and a 100% culture water control should be

run concurrently in the test and compared to determine the presence of toxicity in the receiving

water (for more information on the use of dual controls, see the following question, “When and

how do I use dual controls?”). If and when toxicity is identified in the receiving water, the use

of receiving water for dilution should be discontinued. While it is recognized that receiving

water characteristics are dynamic, the receiving water should consistently display no ambient

toxicity. The presence of ambient toxicity may cause many receiving systems to be

inappropriate for use as a dilution water source. In many circumstances the receiving system

may be impacted by many other point and non-point sources of pollution. Use of receiving

water that possesses consistent or intermittent ambient toxicity is discouraged in most cases. 

Test results are difficult to interpret, and low to moderate toxicity in the effluent is difficult to

detect in the presence of contaminated dilution water. Receiving water that possesses ambient

toxicity is recommended for use as dilution water only if the objective of the test is specifically

to determine the additive or mitigating effects of the effluent on the contaminated receiving

water.

C The receiving water should be free from pathogens and parasites that could affect WET

test results. The presence of pathogens or parasites in the dilution water can cause sporadic

mortalities in the test that are unrelated to effluent toxicity. Due to these sporadic mortalities,

tests may fail to meet test acceptability criteria or anomalous concentration-response patterns

may be produced. Receiving water that is confirmed or suspected to contain pathogens or

parasites should not be used as dilution water.

If the local receiving water is inappropriate for use as dilution water due to failure to meet one of

the above requirements, a synthetic dilution water adjusted to approximate the chemical

characteristics (pH, hardness, alkalinity, salinity) of the receiving water should be used. The

adjustment of synthetic dilution waters should be within the bounds of the test method and

organism tolerances and should be conducted only for the purpose of matching dilution water to

receiving water conditions. For most freshwaters in the U.S., a reasonable match can be obtained

by adjusting the amounts of standard synthetic freshwater reagents (as described in Table 6 of

Section 7 in the WET method manuals) to produce the desired hardness (from very soft to very

hard). Mineral water also may be diluted appropriately (as described in Table 7 of Section 7 in the

WET method manuals) to achieve the desired hardness. These standard preparations span the
6-5

range of hardness, pH, and alkalinity that is commonly found in U.S. waters. When the receiving

water possesses an ionic balance that is atypical, the amounts of individual ion constituents in the

synthetic freshwater preparation may be further adjusted to approximate the ionic balance of the

receiving water. This may occur in coastal or arid regions, where the ionic composition may be

more dominated by sodium and chloride ions than calcium and bicarbonate ions. For marine and

estuarine testing, receiving water composition generally can be matched by preparing synthetic

seawater at the appropriate salinity or adjusting the salinity of a natural seawater using deionized

water, artificial sea salts, or hypersaline brine. 

In the case of freshwater and marine testing, the preparation of synthetic dilution water can be

adjusted to approximate the chemical characteristics of the receiving water; however, the dilution

water should not be adjusted to match the properties of the effluent. High concentrations of

common ions and ion imbalance in the effluent can be a source of toxicity (McCulloch et al., 1993;

Goodfellow et al., 2000), and therefore should be included in the analysis of toxicity and not

adjusted for in the test. 

If an adjusted synthetic water is used for dilution and this water differs from the water used for

culturing the organisms, dual controls are required by the WET method manuals as described

below.

When and how do I use dual controls?

When the dilution water used in a test differs from the water used to culture, hold, and maintain the

test organisms, an additional set of dilution water controls should be evaluated in the WET test. 

This is generally the case when a natural receiving water or an adjusted synthetic water is used for

dilution, but additional controls also may be necessary for standard synthetic dilution waters if

organisms are cultured in an alternative water. A culture water control should consist of 100%

culture water, and a dilution water control should consist of 100% of the dilution water used in the

test. These two controls should be run concurrently in the test and undergo the same test

conditions.

Prior to the analysis of test treatment data, the two controls (dilution water control and culture

water control) should be compared to determine if statistically significant differences exist. This

comparison should be made using a t-test as described in Appendix H of the freshwater method

manual (USEPA, 1994a) and Appendix G of the marine method manual (USEPA, 1994b). If there

is no statistically significant difference between the two controls, the dilution water control should

be used for further analysis and comparisons with the treatment groups. If a receiving water

control is significantly different from the culture control, this may indicate ambient toxicity in the

receiving water. In this case, the use of a synthetic dilution water adjusted to approximate the

receiving water may be more appropriate. If an adjusted synthetic dilution water shows a

significant difference from the culture control, this generally indicates that either the chemical

adjustments of the dilution water were outside of the tolerance range of the test organism or
6-6

acclimation of the test organisms to the dilution water is necessary. In this situation, the analyst

should consider using organisms cultured in water more similar to the dilution water or consider

acclimating the test organisms to the adjusted dilution water prior to the test. These options,

however, may increase test cost and may be impractical for laboratories that test effluents from

numerous dischargers, each with specific dilution water requirements. For this reason, local

regulatory authorities may wish to reevaluate test objectives for this effluent and consider the use

of a standardized synthetic water.

How might the choice of dilution waters affect WET test results?

The selection of dilution waters can have significant impact on the results of a WET test. The

physical and chemical properties of the dilution water can interact with contaminants in the sample

to increase or reduce toxic effect. The presence of acid volatile sulfides (Di Toro et al., 1992),

hardness (Belanger et al., 1989), and acidity (Schubauer-Berigan et al., 1993) are all known to

significantly affect the bioavailability (and hence the toxicity) of metals. Organic and other

hydrophobic contaminants may bind or adsorb to colloids or organic matter in natural waters

(Larson and Weber, 1994). These reactions could potentially decrease toxicity by reducing the

free concentration of the contaminant, or increase toxicity for filter feeding, sediment dwelling, or

sediment ingesting organisms through increased exposure and uptake of the contaminant from food

sources. For these reasons, the selection of dilution water for WET testing should be carefully

considered.
7-1

7

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