REGION VIII NPDES WHOLE EFFLUENT TOXICS CONTROL PROGRAM
AUGUST 1997
INTRODUCTION
This document is the 1997 revision to the original Region
VIII Whole Effluent Toxicity (WET) Policy. The contents and
procedures supplement and, in the case of conflicts, supersede
EPA national guidance and other non-regulatory EPA national
documents on this subject. Requirements in this document should
be considered minimum, and can be expanded at any time.
PURPOSE
This program provides significant controls over the
discharge of toxicants into waters of the United States in Region
VIII. These controls are called for by:
1.	Section 101(a)(3) of the Clean Water Act;
2.	State Water Quality Standards which uniformly
prohibit the discharge of toxicants or require
state waters to be free from toxicants;
3.	EPA policy dated March 4, 1984 (5);
4.	epa's Basic Permitting Principles for Whole
Effluent Toxicity, dated January 25, 1989;
5.	Surface Water Toxic Control Regulations published
on June 2, 1989;
6.	Whole Effluent Toxicity (WET) Control Policy, July
1994.
GENERAL IMPLEMENTATION PROCEDURE AND SUMMARY OF MODIFICATIONS
I. Whole Effluent Controls:
The primary impetus for the inclusion of WET limits is
40 CFR 122.44 (d) which states that if a discharger "..causes,
has the reasonable potential to cause, or contributes to an in-
stream excursion above a narrative criterion within an applicable
State water quality standard...", the discharger's permit must
contain enforceable WET limits. These regulations also state
that numerical limits can be substituted for WET limits if the
"...chemical-specific limits for the effluent are sufficient to
attain and maintain applicable numeric and narrative State water
quality standards." In the case of ammonia and chlorine
toxicity, as noted later in this Policy, if the WET is due to
these substances, the wasteload allocation process allows high
levels of chlorine and ammonia, and numeric limits exist in the
WQS for them, the WET requirements may be appropriately modified
as long as the numeric and narrative WQS are satisfied.
As in the previous revision, chronic limits may still be
required if it is concluded that a discharge has the reasonable
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potential to violate water quality standards exclusively because
of chronic toxicity. Under these circumstances, the permit must
contain appropriate WET limits to assure correction of the
chronic toxicity problem. Thus, as examples, facilities with
measured in-stream chronic toxicity, site specific chronic
aquatic toxicity information, and environmentally sensitive or
pristine receiving waters may need chronic toxicity limits,
and/or the acute toxicity limits may have to be less than 50%
mortality. Chronic lethality is no longer allowed as a test
endpoint. Chronic toxicity is the combined effect on the
organisms growth and survival, or reproduction and survival.
The 50% mortality acute toxicity definition is continued.
The 50% acute limit applies at any test dilution and is
applicable at the end of the, pipe. An exemption to this can be
granted if the discharger can show conclusively that
instantaneous or complete mixing occurs naturally below the
discharge, or if a properly designed diffuser is in place. If
this is done, dilution can be allowed, and the 50% mortality
would be applicable at a dilution concentration commensurate with
the applicable instream waste concentration (IWC). In a complete
mix situation, the 50% mortality for acute toxicity should be
applied at the instream waste concentration based on acute low
flows. Region VIII has developed a mixing zone policy which
defines a complete mix as. 1) no more than a 10% difference from
bank to bank in constituents in a downward distance of two stream
widths, or 2) a greater than 1:1 effluent to receiving water
volume. The actual amount of dilution allowed should be in
conformance with this policy.
Selenastrum capricornuturn can be used as a chronic test
organism. This plant can be rotated on an alternating basis with
the vertebrate or the invertebrate, or used as an'additional test
species. This green alga is the same one used as a food source
in Ceriodaphnia culturing and testing. The testing protocol is
in Appendix B.
WET test methods have been codified in 40 CFR part 136.
This results in the following changes in Region VIII WET Control
Program Policy:
1)	Chronic test endpoints must be based on survival and
growth, or survival and reproduction.
2)	Acute test duration for Pimephales promelas may be
shortened to 48 hours. Where testing has demonstrated
greater than 20% mortality at 9 6 hours and not at 48
hours, the test duration should remain at 96 hours.
3)	On chronic tests, five dilutions and a control are
required.
4)	First priority for diluent should be the receiving
water. If that is unsuitable then moderately hard (80
to 100, expressed as mg. CaC03/L) reconstituted water
should be used for diluent.
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5)	Acute test temperature tolerance is ^C.
6)	Pimephales promelas acute test is conducted with 200
mis. test solution in a 250 mis. test container. The
chronic test is conducted with 250 mis. test solution
in a 500 mis. test container.
7)	Age of Pimephales promelas larvae for the acute test
should be 1 to 14 days old and born within 24 hours of
each other.
8)	Replace the statistical NOEC hypothesis testing with
the linear interpolation technique (IC25 )as the test
statistic on chronic tests.
9)	Use of zeolite for routine compliance testing is
prohibited.
The reporting guidance form has been changed to reflect the
following:
1)	Test temperatures and test dissolved oxygen readings do
not have to be reported.
2)	Initial mean weight on Pimephales chronic test do not
have to be reported.
3)	Line space has been added for recording the temperature
of samples upon arrival at the testing laboratory.
The use of Toxic Units (TU) in the permit program is being
introduced in this revision. Its incorporation in the permit
development phase and the compliance reporting phase is
recommended. Toxic units are a standard mechanism for
quantifying whole effluent toxicity. The TU increases as toxicity
increases. Acute Toxic Unit (Tua) is 100/LC5o. As an example, if
a sample produced a LC50 of 100% then Tua= 100/100 or 1. Chronic
Toxic Unit (Tuc) is 100/IC25 or NOEC. If a sample produced an IC25
or a NOEC of 80%, then Tuc = 100/80 or 1.25.
In changing over to the terms above, the permit and
reporting requirements for WET should be consistent. If acute
WET is limited at 100% effluent the permit limit should be stated
as less than 1 Tua. For chronic toxicity, if the IWC is 50%,
then the limit would be expressed a Tuc less than 2 (100/50) .
Discharge Monitoring Reports will have to be coded appropriately.
The basic approach is contained in the block diagrams which
vary slightly from state to state and reflect practices in the
individual Region VIII states. It is expected that permits for
all major and all significant minor dischargers in each state
will contain the essence of the relevant diagram. As noted in
footnote (1) of the diagrams, exceptions can be made for those
facilities where there is no reasonable potential for the
discharge of Whole Effluent Toxicity. All permit Statement of
Basis should contain a detailed discussion of the reasons for
including, or not including, WET limits in a permit based on a
reasonable potential determination. This will provide an
adequate administrative record should any challenge of the
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determination be made by a discharger or environmental group.
The justification for the inclusion, or exclusion, of limits
should contain a discussion of the following as a minimum:
a.	Existence of a pretreatment program.
b.	Whether or not categorical industries exist in the
system, or in the case of industry, if it is a
categorical industry.
c.	Receiving water characteristics such as classification,
Q7-10, dilution ratios, etc.
d.	Size of the discharge.
e.	Number of commercial and industrial taps.
f.	Compliance history.
g.	History of fish kills in the receiving water.
h.	Actual data showing WET in the discharge.
I. Instream survey data.
Flexibility exists in the type of species selected (the
discharger must first establish that any alternate species has an
equivalent sensitivity), monitoring frequency, and exact dates
for implementation by the permittee. Any deviation from the
diagram must be justified in the Statement of Basis. All major
and minor permits, for which it has been concluded that a
reasonable potential to discharge toxicity exists, must require
two species testing, and an appropriate immediate or delayed
limitation of WET. When permits that have WET limits are
renewed, if the renewed permit requirements are similar to the
old requirements, the limit is effective immediately. The amount
of the delay in the application of limits is discretionary and
dependant on physical characteristics, the amount of existing WET
effluent data, and other restrictions. All major permits, for
which it has been concluded that a reasonable potential to
discharge toxicity does not exist, must require two species
testing and a reopener clause calling for the inclusion of limits
if toxicity occurs. The definition of when toxicity is occurring
at a level to warrant further action is left to the regulatory
authority; a specific definition can be incorporated in the
permit, or it can be left to the judgement of the regulatory
authority, much as it is now for all other permit limitations.
However as a matter of practice, once a second test demonstrates
the continued toxicity problem, initiation of a TRE is warranted.
The example TRE language in Appendix A has been reduced to a
level where it is clear that the responsibility for compliance
with the ultimate WET limits is independent of the quality of any
TRE, or whether or not the permittee has been told to do a TRE.
It is expected that on rare occasions unanticipated events
may occur that will require adjustment in permit conditions to
accommodate these unusual circumstances. Appendix A contains
recommended permit language to provide flexibility in this
eventuality.
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As noted in the introduction, procedures outlined in this
document supplement or supersede previously published guidance
and other non-regulatory national documents. The protocols in
Appendix B shall prevail in any conflict with other such
references. These are from the 40 CFR Part 13 6. Region VIII
specifies that the test type is static renewal, acute test
temperature is 20°C, acute Ceriodaphnia dubia test is conducted
for 48 hours, and acute Pimephales promelas test can be conducted
for 48 or 96 hours. In Pimephales promelas test, if previous WET
tests have shown greater than 20% mortality at 96 hours and not
at 48 hours, then the test should remain at the 96 hour duration.
Once the 48 hour testing has been authorized, if >20% mortality
occurs in subsequent testing, then 9 6 hour duration tests should
be reinstated.
The major purpose of WET controls is to detect and eliminate
toxicity in those cases where its presence is unknown or caused
by interaction between otherwise innocuous substances. It must
be emphasized that if WET is demonstrated, and it is established
that it is due to a known toxicant, as allowed in 40 CFR 122.44
(d)(1)(v), the toxicant must be controlled by WET limits,
specific numerical limits, or by both methods. If the permit
issuing authority feels that the toxicant in question is, or will
be, in compliance with existing water quality standards, WET
testing or sampling procedures may be modified or, in very
unusual cases, the effluent limit may be modified provided it can
be shown that such actions are still sufficient to attain and
maintain applicable numeric and narrative water quality
standards. This will assure that the main purpose of the test
does not continue to be masked by the known toxicant. As an
example, if it is established that whole effluent toxicity is
caused by a metal, and the discharger is on an acceptable
compliance schedule to reduce its metal levels, the whole
effluent protocol could be modified by the permit issuing
authority simply by allowing the use of EDTA in the interim.
Although this modification may mask some other toxicant, the test
will still adhere more closely to its basic intent than it
otherwise would. The most common example of test modification is
the use of the C02 test which is now in the Appendix B protocols.
It should be emphasized that this is the preferred way to control
"creeping pH" during the tests, and that the use of more radical
procedures, such as acid addition or zeolite treatment, has not
been found to be necessary. If pH control is necessary, the
value that is selected to cap the test must reflect the pH value
of the receiving body of water or a value that represents the
combination of effluent and receiving water. During the test the
pH would be allowed to reach this level and then maintained.
There is a program available to calculate this pH value. It
requires the following information for the effluent and the
receiving water: flow, alkalinity, and pH. The information
should represent most of the range of values present in the
receiving waters.
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Storm water permittees do not need to follow the block
diagrams. Test protocols are in Appendix B.
II. Numerical Limits:
Compliance with whole effluent toxicity limits does not
necessarily exclude the imposition of additional numerical limits
on specific pollutants when appropriate. These limits may be
based on numerical water quality standards if they exist; 304 (a)
human health and/or aquatic life criteria (see EPA 440/5-86-001,
"the gold book" or its successor); drinking water maximum
concentration levels (MCLs), or a combination of all three as
circumstances warrant. Concerning toxic pollutants such as
metals; in order to supplant acute whole effluent limits
numerical limitations must be based, as a minimum, on acute
aquatic criteria, and applicable at the end of pipe unless a
diffuser is present.
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REGION VIII WHOLE EFFLUENT TOXIC CONTROL PROGRAM
COLORADO (1)
RECEIVING WATER
LOW FLOW DILUTION
GREATER THAN 9.1,
DISCHARGE IS INTERMITTENT,
OR TO A CLASS 2 STREAM
W/O NUMERIC AQUATIC
STANDARDS
ACUTE AN
D/OR CHRONIC TESTS


DISCHARGE EXCEEDS 20 MGD FOR A
POTW OR 10 MGD FOR AN INDUSTRY
MULTI DILUTIC
)N ACUTE TESTS


DISCHARGE EXCEEDS 20 MGD FOR A
POTW OR 10 MGD FOR AN INDUSTRY



NO
YES
NO
YES
QUARTERLY
TESTING
MONTHLY
TESTING
NO TOXICITY FOR
TWELVE CONSECUTIVE MONTHS
QUARTERLY
TESTING
MONTHLY
TESTING
NO TOXICITY FOR
TWELVE CONSECUTIVE MONTHS
YES
NO
YES
NO
REDUCE TESTING
CONDUCT A TRE;
CONTINUE
TESTING
REDUCE
TESTING
TO ONE
SPECIES
CONDUCT A
TRE;
CONTINUE
TESTING
UP TO
3
YEARS AFTER ISSUANCE
APPLY
AN
EFFLUENT LIMIT OF NO
ACUTE
OR
CHRONIC TOXICITY (2)
UP TO THREE YEARS AFTER PERMIT
ISSUANCE, APPLY AN EFFLUENT LIMIT
OF NO ACUTE TOXICITY
(1) APPLICABLE TO ALL MAJORS AND SIGNIFICANT MINOR PERMITS EXCEPT THOSE WITH NO REASONABLE
POTENTIAL TO DISCHARGE TOXICANTS. MAJOR DISCHARGERS WITH NO REASONABLE POTENTIAL MUST CONTAIN
WET MONITORING AND REOPENER LANGUAGE IN THEIR PERMITS.
m-TF CHRONIC TOXICITY LIMITS ARE USED, CONDUCT CHRONIC TESTING UNLESS THE IN-STREAM DILUTION
EATER THAN 4:1; IF GREATER THAN 4:1 (<20% EFFLUENT), ALSO PROHIBIT ACUTE TOXICITY IN 100%
ENT. IF CHRONIC LIMITS ARE NOT USED, REQUIRE MULTI DILUTION ACUTE TESTS AND TWICE A YEAR
SIC TESTS. SEE EXAMPLE LANGUAGE IN APPENDIX A.
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REGION VIII WHOLE EFFLUENT TOXIC CONTROL PROGRAM
MONTANA (1)
(1) APPLICABLE TO ALL MAJORS AND SIGNIFICANT MINOR PERMITS EXCEPT THOSE WITH NO REASONABLE
POTENTIAL TO DISCHARGE TOXICANTS. MAJOR DISCHARGERS WITH NO REASONABLE POTENTIAL MUST CONTAIN
WET MONITORING AND REOPENER LANGUAGE IN THEIR PERMITS.
(2) IF CHRONIC TOXICITY LIMITS ARE USED, CONDUCT CHRONIC TESTING UNLESS THE IN-STREAM DILUT|
IS GREATER THAN 4:1; IF GREATER THAN 4:1 (<20% EFFLUENT), ALSO PROHIBIT ACUTE TOXICITY IN lu
EFFLUENT. IF CHRONIC LIMITS ARE NOT USED, REQUIRE ACUTE TESTS AND TWICE A YEAR CHRONIC TESTS.
SEE EXAMPLE LANGUAGE IN APPENDIX A.
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REGION VIII WHOLE EFFLUENT TOXIC CONTROL PROGRAM
NORTH DAKOTA (1)
RECEIVING WATER
LOW FLOW DILUTION
GREATER THAN 10:1,
DISCHARGE IS INTERMITTENT
YES
ACUTE A
ND/OR CHRONIC TESTS


DISCHARGE EXCEEDS 20 MGD FOR A
POTW OR 10 MGD FOR AN INDUSTRY



MULTI DILUTION ACUTE TESTS
DISCHARGE EXCEEDS 20 MGD FOR A
POTW OR 10 MGD FOR AN INDUSTRY
NO
YES
NO
YES
QUARTERLY
TESTING
MONTHLY
TESTING
QUARTERLY
TESTING
MONTHLY
TESTING
NO TOXICITY FOR
TWELVE CONSECUTIVE MONTHS
NO TOXICITY FOR
TWELVE CONSECUTIVE MONTHS
YES
NO
YES
NO
REDUCE TESTING
CONDUCT A TRE;
CONTINUE
TESTING
REDUCE
TESTING
TO ONE
SPECIES
CONDUCT A
TRE;
CONTINUE
TESTING
UP TO 3 YEARS AFTER ISSUANCE,
APPLY AN EFFLUENT LIMIT OF NO
ACUTE OR CHRONIC TOXICITY (2)
UP TO THREE AFTER PERMIT
ISSUANCE, APPLY AN EFFLUENT LIMIT
OF NO ACUTE TOXICITY
(1)	APPLICABLE TO ALL MAJORS AND SIGNIFICANT MINOR PERMITS EXCEPT THOSE WITH NO REASONABLE
PC? TAL TO DISCHARGE TOXICANTS. MAJOR DISCHARGERS WITH NO REASONABLE POTENTIAL MUST CONTAIN
V "STORING AND REOPENER LANGUAGE IN THEIR PERMITS.
(2)	IF CHRONIC TOXICITY LIMITS ARE USED, CONDUCT CHRONIC TESTING UNLESS THE IN-STREAM DILUTION
IS GREATER THAN 4:1; IF GREATER THAN 4:1 (<20% EFFLUENT), ALSO PROHIBIT ACUTE TOXICITY IN 100%
EFFLUENT. IF CHRONIC LIMITS ARE NOT USED, REQUIRE ACUTE TESTS AND TWICE A YEAR CHRONIC TESTS.
SEE EXAMPLE LANGUAGE IN APPENDIX A.
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REGION VIII WHOLE EFFLUENT TOXIC CONTROL PROGRAM
SOUTH DAKOTA (1)
RECEIVING WATER
LOW FLOW DILUTION
GREATER THAN 10:1,
DISCHARGE IS INTERMITTENT,
OR TO A CLASS 9 OR 10 STREAM
YES
ACUTE A
lND/OR CHRONIC TESTS |


DISCHARGE EXCEEDS 2 0 MGD FOR A
POTW OR 10 MGD FOR AN INDUSTRY
MULTI DILUTION ACUTE TESTS
DISCHARGE EXCEEDS 20 MGD FOR A
POTW OR 10 MGD FOR AN INDUSTRY
NO
YES
NO
YES
QUARTERLY
TESTING
MONTHLY
TESTING
QUARTERLY
TESTING
MONTHLY
TESTING
NO TOXICITY FOR
TWELVE CONSECUTIVE MONTHS
NO TOXICITY FOR
TWELVE CONSECUTIVE MONTHS
YES
NO
YES
NO
REDUCE
TESTING
CONDUCT A TRE;
CONTINUE TESTING
REDUCE
TESTING
TO ONE
SPECIES
CONDUCT A
TRE;
CONTINUE
TESTING
UP TO 3 YEARS AFTER ISSUANCE,
APPLY AN EFFLUENT LIMIT OF NO
ACUTE OR CHRONIC TOXICITY (2)
UP TO THREE YEARS AFTER PERMIT
ISSUANCE, APPLY AN EFFLUENT LIMIT
OF NO ACUTE TOXICITY
(1)	APPLICABLE TO ALL MAJORS AND SIGNIFICANT MINOR PERMITS EXCEPT THOSE WITH NO REASONABLE
POTENTIAL TO DISCHARGE TOXICANTS. MAJOR DISCHARGERS WITH NO REASONABLE POTENTIAL MUST CONTAIN
WET MONITORING AND REOPENER LANGUAGE IN THEIR PERMITS.
(2)	IF CHRONIC TOXICITY LIMITS ARE USED, CONDUCT CHRONIC TESTING UNLESS THE IN-STREAM DILUH
IS GREATER THAN 4:1; IF GREATER THAN 4:1 (<20% EFFLUENT), ALSO PROHIBIT.ACUTE TOXICITY IN 1^
EFFLUENT. IF CHRONIC LIMITS ARE NOT USED, REQUIRE ACUTE TESTS AND TWICE A YEAR CHRONIC TESTS.
SEE EXAMPLE LANGUAGE IN APPENDIX A.
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REGION VIII WHOLE EFFLUENT TOXIC CONTROL PROGRAM
UTAH (1)
(1)	APPLICABLE TO ALL MAJORS AND SIGNIFICANT MINOR PERMITS EXCEPT THOSE	WITH NO REASONABLE
Pfi ~ TIAL TO DISCHARGE TOXICANTS. MAJOR DISCHARGERS WITH NO REASONABLE	POTENTIAL MUST CONTAIN
Vi JNITORING AND REOPENER LANGUAGE IN THEIR PERMITS.
(2)	IF CHRONIC TOXICITY LIMITS ARE USED, CONDUCT CHRONIC TESTING UNLESS	THE IN-STREAM DILUTION
IS GREATER THAN 4:1; IF GREATER THAN 4:1 (<20% EFFLUENT), ALSO PROHIBIT ACUTE TOXICITY IN 100%
EFFLUENT. IF CHRONIC LIMITS ARE NOT USED, REQUIRE ACUTE TESTS AND TWICE A YEAR CHRONIC TESTS.
SEE EXAMPLE LANGUAGE IN APPENDIX A.
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REGION VIII WHOLE EFFLUENT TOXIC CONTROL PROGRAM
WYOMING (1)
RECEIVING WATER
LOW FLOW DILUTION
GREATER THAN 10:1,
DISCHARGE IS INTERMITTENT,
OR TO A CLASS IV STREAM
ACUTE A
>ND/OR CHRONIC TESTS |


DISCHARGE EXCEEDS 20 .MGD FOR A
POTW OR 10 MGD FOR AN INDUSTRY


MULT I DILUTIC
)N ACUTE TESTS


DISCHARGE EXCEEDS 2 0 MGD FOR A
POTW OR 10 MGD FOR AN INDUSTRY



NO
YES
NO
YES
QUARTERLY
TESTING
MONTHLY
TESTING
QUARTERLY
TESTING
MONTHLY
TESTING
NO TOXICITY FOR
TWELVE CONSECUTIVE MONTHS
NO TOXICITY FOR
TWELVE CONSECUTIVE MONTHS
YES
NO
YES
NO
REDUCE
TESTING
CONDUCT A TRE;
CONTINUE TESTING
REDUCE
TESTING
TO ONE
SPECIES
CONDUCT A
TRE;
CONTINUE
TESTING
UP TO THREE YEARS AFTER ISSUANCE
APPLY AN EFFLUENT LIMIT OF NO
ACUTE OR CHRONIC TOXICITY (2)
UP TO THREE YEARS AFTER PERMIT
ISSUANCE, APPLY AN EFFLUENT LIMIT
OF NO ACUTE TOXICITY
(1) APPLICABLE TO ALL MAJORS AND SIGNIFICANT MINOR PERMITS EXCEPT THOSE WITH NO REASONABLE
POTENTIAL TO DISCHARGE TOXICANTS. MAJOR DISCHARGERS WITH NO REASONABLE POTENTIAL MUST CONTAIN
WET MONITORING AND REOPENER LANGUAGE IN THEIR PERMITS.
(2)IF CHRONIC TOXICITY LIMITS ARE USED, CONDUCT CHRONIC TESTING UNLESS THE IN-STREAM DILUTION-
IS GREATER THAN 4:1; IF GREATER THAN 4:1 (<20% EFFLUENT), ALSO PROHIBIT ACUTE TOXICITY IN l|
EFFLUENT. IF CHRONIC LIMITS ARE NOT USED, REQUIRE ACUTE TESTS AND TWICE A YEAR CHRONIC TES"
SEE EXAMPLE LANGUAGE IN APPENDIX A.
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TOXICITY REDUCTION EVALUATIONS
Actual procedures to be followed in a TRE are unique in
every individual situation. In addition, the discharger will
always be more familiar with his operation than the regulatory
agency and an excessive amount of procedural detail may inhibit
an innovative approach. In any event, a TRE in most cases should
pursue the following elements, most of which are self-evident.
If a problem is established, and if prior knowledge or
intuition is not helpful, initial efforts should be expended on
characterization and identification of the toxicant(s).
Procedures exist for rapidly narrowing the possibilities to
certain groups of pollutants such as metals, non polar organics,
oxidants, etc. In many cases, it is anticipated that the TRE may
essentially terminate at this point if it is conclusively shown
that the problem is due to one distinct pollutant whose source,
and method of correction, is known. This pollutant may be
already controlled through a compliance schedule linked to a
numerical limit. Alternatively, a numerical limit and/or
compliance schedule may be subsequently imposed on the permittee.
Once the problem has been identified and located, the
ultimate objective is elimination by process controls,
pretreatment, combined waste stream treatment, local enforcement,
or whatever other measure may be necessary.
It is anticipated that toxicity problems will be controlled
in most cases by following the above procedures. However, as
noted earlier, there may be situations when a well done and
acceptable TRE will reveal a problem requiring additional time
consuming activity before final resolution. Under these
circumstances, relief may be granted through the language noted
in Appendix A. It is emphasized that, under these circumstances,
the discharger must convince the regulatory agency that a
thorough TRE has been done and more time is needed to address the
problem. Only then should permit relief be granted.
EPA has developed guidance on TIE/TRE procedures
(6) (7) (8) (9). This information has been widely distributed, and
is available from the Region VIII EPA Office or from National
Center for Environmental Publications and Information, PO Box
42419, Cincinnati, OH 45242.
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BIBLIOGRAPHY
1.	"Technical Support Document for Water Quality Based Toxics
Control", EPA 505/2-90-001, U.S. EPA, March, 1991.
2.	"Methods for Measuring Acute Toxicity of Effluent and
Receiving Waters to Freshwater and Marine Organisms," Fourth
Edition EPA 600/4-90/027F, U.S. EPA, August 1993.
3.	"Short Term Methods for Estimating the Chronic Toxicity of
Effluents and Receiving Waters to Freshwater Organisms,
Third Edition", EPA 600/4-91/002, U.S. EPA, July 1994.
4.	"Permit Writers Guide to Water Quality Based Permitting for
Toxic Pollutants", Office of Water, U.S. EPA, 1987.
5.	"Development of Water Quality-based Permit Limitations for
Toxic Pollutants; National Policy", U.S. EPA Federal
Registerf Vol. 49, No. 48, March 9, 1984.
6.	"Methods for Aquatic Toxicity Identification Evaluations,
Phase I Toxicity Characterization Procedures",Second
Edition, EPA/600/6-91/003, February 1991.
7.	"Methods for Aquatic Toxicity Identification Evaluations,
Phase II Toxicity Identification Procedures for Samples
Exhibiting Acute and Chronic Toxicity",
EPA/600/R-92/080, September, 1993.
8.	"Methods for Aquatic Toxicity Identification Evaluations,
Phase III Toxicity Confirmation Procedures for Samples
Exhibiting Acute and Chronic Toxicity",
EPA/600/R-92-/081, September, 1993.
9.	"Toxicity Identification Evaluation: Characterization of
Chronically Toxic Effluents, Phase I",
EPA 600/6-91/005F, May, 1992.
10.	Whole Effluent Toxicity: Guidelines Establishing Test
Procedures for the Analysis of Pollutants. Federal Register.
Vol. 60. No. 199, October 16, 1995.
11.	Whole Effluent Toxicity (WET) Control Policy,
EPA 833-B-94-002, July 1994.
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APPENDIX A
EXAMPLE PERMIT LANGUAGE
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A. Definitions (Continued)
13. "Acute Toxicity" occurs when 50 percent or more
mortality is observed for either species (See Part
I.C.) at any effluent concentration. Mortality in
the control must simultaneously be 10 percent or
less for the effluent results to be considered
valid.
14.	"Chronic toxicity" occurs when during a chronic
toxicity test, the 25% inhibition concentration
(IC25) calculated on the basis of test organism
survival and growth, or survival and reproduction,
is less than or equal to the effluent dilution
designated in this permit ( see Part I.C.)
15.	"IC2511 (Inhibition concentration) is a point
estimate of the toxicant concentration that would
cause a 25% reduction in a nonlethal biological
measurement of the test organism, such as
reproduction or growth.
16. "NOEC" (no observer effect concentration) is the
highest tested concentration of an effluent or a
toxicant at which no adverse effects are observed
on the aquatic test organism at a specific time of
observation. Determined using hypothesis testing.

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USE THIS PAGE WHEN ONLY ACUTE TOXICITY IS LIMITED
C. Specific Limitations and Self-Monitoring Requirements
3.	Whole Effluent Toxicity Testing - Acute Toxicity
Starting in the 	 quarter of calendar year	, the permittee
shall, at least once each calendar quarter, conduct acute
static replacement toxicity tests on a sample of the
discharge. Quarterly samples shall be collected on a two day
progression; i.e., if the first quarterly sample is on a
Monday, during the next quarter, sampling shall be on a
Wednesday, etc.
The replacement static toxicity tests shall be conducted in
accordance with the procedures set out in the latest revision
of "Methods for Measuring the Acute Toxicity of Effluents to
Freshwater and Marine Organisms", EPA-600/4-90/027F
(Rev. August 1993)and the "Region VIII EPA NPDES Acute Test
Conditions -Static Renewal Whole Effluent Toxicity Test". In
the case of conflicts, the Region VIII Document will
prevail. The permittee shall conduct an acute 48-hour static
toxicity test using Ceriodaphnia dubia and an acute 96
hour static toxicity test using Pimephales promelas.
Acute toxicity occurs when 50 percent or more mortality is
observed for either species at any effluent concentration. If
more than 10 percent control mortality occurs, the test shall
be repeated until satisfactory control survival is achieved.
If acute toxicity occurs, an additional test shall be
conducted within two weeks of the date of when the permittee
learned of the test failure. If only one species fails,
retesting may be limited to this species. Should acute
toxicity occur in the second test, testing shall occur once a
month until further notified by the permit issuing authority.
Quarterly test results shall be reported along with the
Discharge Monitoring Report (DMR) submitted for the end of
the reporting calendar quarter (e.g., whole effluent results
for the calendar quarter ending March 31 shall be reported
with the DMR due April 28, with the remaining reports
submitted with DMRs due each July 28, October 28, and January
28). Monthly test results shall be reported along with the
DMR submitted for that month. The format for the report shall
be consistent with the latest revision of the "Region VIII
Guidance for Acute Whole Effluent Reporting", and shall
include all chemical and physical data as specified.
If the results for four consecutive quarters of testing
indicate no acute toxicity, the permittee may request the
permit issuing authority to allow a reduction to quarterly
acute toxicity testing on only one species on an alternating
basis. The permit issuing authority may approve or deny the

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request based on the results and other available information
without an additional public notice. If the request is
approved, the test procedures are to be the same as specified
above for the test species.

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USE THIS PAGE (AND THE PRECEDING PAGE) WHEN ONLY ACUTE TOXICITY IS
LIMITED BUT TWICE A YEAR CHRONIC TESTING IS ALSO REQUIRED
C. Specific Limitations and Self-Monitoring Requirements
4. Whole Effluent Toxicity Testing - Chronic Toxicity
Starting 	, the permittee shall, at least once
during the six month period from January through June, and at
least once during the six month period from July through
December, conduct chronic short term toxicity tests on a
sample of the final effluent. If only two samples are taken,
the interval between samples shall be at least three months.
The chronic toxicity tests shall be conducted in accordance
with the procedures set out in the latest revision of "Short
Term Methods for Estimating the Chronic Toxicity of Effluents
and Receiving Waters to Freshwater Organisms", EPA/600/4-
91/002, and the "Region VIII EPA NPDES Chronic Test Conditions
- Static Renewal Whole Effluent Toxicity Test". In case of
conflicts, the Region VIII procedure will prevail. Test
species shall consist of Ceriodaphnia dubia and Pimephales
promelas. If acceptable control performance criteria are not
met, the test shall be considered invalid. Chronic toxicity
occurs when during a chronic toxicity test, the 25%
inhibition concentration (IC25) calculated on the basis of test
organism survival and growth or survival and reproduction, is
less than or equal to 	 effluent dilution.
Test results shall be reported with the Discharge Monitoring
Report (DMR) submitted for the end of the calendar period
during which the whole effluent test was run (e.g. results
shall be reported with the next routine DMR covering the month
in which the sample was taken). The format for the report
shall be consistent with the latest revision of the "Region
VIII Guidance for Chronic Whole Effluent Reporting", and shall
include all the physical and chemical testing as specified.
If the results for four consecutive samples of chronic whole
effluent testing indicate no chronic toxicity, the permittee
may request the permit issuing authority to allow the
permittee to reduce testing frequency, and/or reduce testing
to one species on an alternating basis, and/or modify testing
to the acute test program. The permit issuing authority may
approve, partially approve, or deny the request based on
results and other available information. If approval is
given, the modification will take place without a public
notice.

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USE THIS PAGE WHEN CHRONIC TOXICITY IS LIMITED
C. Specific Limitations and Self-Monitoring Requirements
3. Whole Effluent Toxicity Testing - Chronic Toxicity
Starting in the 	1__ quarter of calendar year 	, the
permittee shall, at least once each quarter, conduct chronic
short term toxicity tests on the final effluent. There shall
not be chronic toxicity in 	 percent concentration of the
final effluent.
The monitoring frequency shall be quarterly. Quarterly
samples shall be collected on a two day progression; i.e., if
the first quarterly sample is on a Monday, during the next
quarter, the sampling shall begin on a Wednesday. If chronic
toxicity is detected, an additional test shall be conducted
within two weeks of the date of when the permittee learned of
the test failure. The need for any additional samples shall be
determined by the permit issuing authority.
The chronic toxicity tests shall be conducted in accordance
with the procedures set out in the latest revision of "Short
Term Methods for Estimating the Chronic Toxicity of Effluents
and Receiving Waters to Freshwater Organisms", EPA/600/4-
91/002, and the "Region VIII EPA NPDES Chronic Test Conditions
- Static Renewal Whole Effluent Toxicity Test". In case of
conflicts, the Region VIII procedure will prevail. Test
species shall consist of Ceriodaphnia dubia and Pimephales
promelas. A multi dilution test consisting of five
concentrations and a control is required. If test
acceptability criteria is not met for control survival,
growth, or reproduction, the test shall be considered invalid.
Chronic toxicity occurs when, during a chronic toxicity test,
the 25% inhibition concentration (IC25) calculated on the basis
of test organism survival and growth or survival and
reproduction, is less than or equal to 	 effluent
concentration.
Test results shall be reported along with the Discharge
Monitoring Report (DMR) submitted for the end of the calendar
period during which the whole effluent test was run (e.g.
results for the calendar quarter ending March 31 shall be
reported with the DMR due April 28, with the remaining reports
submitted with DMRs due each July 28, October 28, and January
28). Monthly test results shall be reported along with the
DMR submitted for that month. The format for the report shall
be consistent with the latest revision of the "Region VIII
Guidance for Chronic Whole Effluent Reporting", and shall
include all the physical and chemical testing as specified.
If the results for one year (four consecutive quarters) of
whole effluent testing indicate no chronic toxicity, the
permittee may request the permit issuing authority to allow

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the permittee to reduce testing frequency, and/or reduce
testing to one species on an alternating basis, and/or modify
testing to the acute test program. The permit issuing
authority may approve, partially approve, or deny the request
based on results and other available information. If approval
is given, the modification will take place without a public
notice.

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USE THIS PAGE (AND THE APPROPRIATE PRECEDING PAGE) IF BOTH ACUTE
TOXICITY AND CHRONIC TOXICITY ARE LIMITED
C. Specific Limitations and Self-Monitoring Requirements
4. Whole Effluent Toxicity Testing - Acute Toxicity
Starting in 	, the permittee shall conduct quarterly
acute static replacement toxicity tests on a effluent sample
of the discharge. The effluent shall be obtained from the
sample required for the chronic toxicity tests as noted in
Part 	of this permit.
The replacement static acute toxicity tests shall be conducted
in accordance with the procedures set out in the latest
revision of "Methods of Measuring the Acute Toxicity of
Effluents to Freshwater and Marine Organisms", EPA-600/4-
90/027F (Rev. August 1993) and the "Region VIII EPA NPDES
Acute Test Conditions - Static Renewal Whole Effluent Toxicity
Test." In the case of conflicts, the Region VIII procedures
will prevail. The permittee shall conduct the acute 48-hour
static toxicity test using Ceriodaphnia dubia, and the acute
96 hour static toxicity test using Pimephales promelas.
Acute toxicity occurs when 50 percent or more mortality is
observed for either species at any effluent concentration. If
more than 10% control mortality occurs, the test shall be
repeated until satisfactory control survival is achieved.
If acute toxicity occurs, an additional test shall be
conducted within two weeks of the date of when the permittee
learned of the test failure. If only one species fails,
retesting may be limited to this species. Should toxicity
occur in the second test, testing shall occur once a month
until further notified by the permit issuing authority.
Quarterly test results,shall be reported along with the
Discharge Monitoring Report (DMR) submitted for the end of the
reporting calendar quarter (e.g., whole effluent results for
the calendar quarter ending March 31 shall be reported with
the DMR due April 28, with the remaining reports submitted
with DMRs due each July 28, October 28, and January 28).
Monthly test results shall be reported along with the DMR
submitted for that month. The format for the report shall be
consistent with the latest revision of the "Region VIII
Guidance for Acute Whole Effluent Reporting", and shall
include all chemical and physical data as specified.
If the results for four consecutive quarters of testing
indicate no acute toxicity, the permittee may request the
permit issuing authority to allow a reduction to quarterly
acute toxicity testing on only one species on a alternating
basis. The permit issuing authority may approve or deny the
request based on the results and other available information

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without an additional public notice. If the request is
approved, the test procedures are to be the same as specified
above for the test species.

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C. Specific Limitations and Self-Monitoring Requirements
5. Toxicity Reduction Evaluation (TRE)
Toxicity Identification Evaluation (TIE)
Should acute toxicity and/or chronic toxicity be detected in
the permittee's discharge, a TIE-TRE shall be undertaken by
the permittee to establish the cause of the toxicity, locate
the source(s) of the toxicity, and develop control of, or
treatment for the toxicity. Failure to initiate, or conduct
an adequate TIE-TRE, or delays in the conduct of such tests,
shall not be considered a justification for noncompliance
with the whole effluent toxicity limits contained in Part
I.C.l. of this permit. A TRE plan needs to be submitted to
the permitting authority within 45 days after confirmation
of the continuance of effluent toxicity.
6. Chronic Toxicity Limitation-Reopener Provision
This permit may be reopened and modified (following proper
administrative procedures) to include chronic whole effluent
toxicity limitations if any other information or data are
developed indicating that chronic whole effluent toxicity
limits are needed as required under 40 CFR 122.44 (d). Also
see Part IV.P. of this permit for additional whole effluent
toxicity reopener provisions.
If acceptable to the permit issuing authority, and if in
conformance with current regulations, this permit may be
reopened and modified to incorporate TRE conclusions
relating to additional numerical limitations, a modified
compliance schedule, and or modified whole effluent
protocol.

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USE THIS LANGUAGE WHEN A TOXICITY LIMIT IS IN THE PERMIT
Specific Limitations and Self-Monitoring Requirements
Toxicity Limitation-Reopener Provision.
This permit may be reopened and modified (following proper
administrative procedures) to include a new compliance date,
additional or modified numerical limitations, a new or
different compliance schedule, a change in the whole effluent
protocol, or any other conditions related to the control of
toxicants if one or more of the following events occur:
a.	Toxicity was detected late in the life of the permit near or
past the deadline for. compliance.
b.	The TRE results indicate that compliance with the toxic
limits will require an implementation schedule past' the date
for compliance and the permit issuing authority agrees with
the conclusion.
c.	The TRE results indicate that the toxicant(s) represent
pollutant(s) that may be controlled with specific numerical
limits, and the permit issuing authority agrees that
numerical controls are the most appropriate course of
action.
d.	Following the implementation of numerical controls on
toxicants, the permit issuing authority agrees that a
modified whole effluent protocol is necessary to compensate
for those toxicants that are controlled numerically.
e.	The TRE reveals other unique conditions or characteristics
which, in the opinion of the permit issuing authority,
justify the incorporation of unanticipated special
conditions in the permit.
USE THIS LANGUAGE WHEN WHOLE EFFLUENT TOXICITY MONITORING IS IN THE
PERMIT BUT A WHOLE EFFLUENT TOXICITY LIMIT IS NOT IN THE PERMIT
Specific Limitations and Self-Monitoring Requirements
Toxicity Limitation-Reopener Provision
This permit may be reopened and modified (following proper
administrative procedures) to include whole effluent toxicity
limitations if whole effluent toxicity is detected in the
discharge.

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APPENDIX B
REGION VIII ACUTE AND CHRONIC
TESTING PROTOCOLS

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REGION VIII EPA NPDES ACUTE TEST CONDITIONS
STATIC RENEWAL WHOLE EFFLUENT TOXICITY TEST
1. Temperature
2 . Light duration and
type
3.	Container size
4.	Volume of test
solution
5.	Age of test organisms
6.	No. of organisms
per container
7.	No. of replicates
8.	Renewal frequency
9.	No. of dilutions
Ceriodaphnia
dubia
20° c ± 1°
16 hours-ambient
30 ml minimum
15 ml minimum
less than 24 hr
5
4
daily
5	(+control)
Pimephales promelas
(Fathead minnows)
20° c ± 1°
16 hours-ambient
250 ml minimum
200 ml minimum
1	to 14 days (a)
10
2	(minimum)
daily
5 (+control)
10. Feeding
11. Test duration
12 . Acceptable control
mortality
none
4 8 hours
10 % or less
0.1 ml brine shrimp prior to
selection, and at 48 hrs (if
necessary)
9 6 hours (b)
10% or less
13.	Dilution Series: 100%, 75%, 50%, 25%, 12.5%, 6.25%, 0% (control).
14.	Dilution water: Dilution water shall consist of receiving water if it is not toxic.
If receiving water is unsuitable, use moderately hard reconstituted water.
15.	Holding time: A maximum of 36 hours from the conclusion of sampling until the
initiation of testing. Samples must be maintained at 4°C or less. Do not freeze
samples.
16.	C02 atmospheres: Permittees may receive individual case by case permission to
introduce C02 enriched atmospheres if necessary to inhibit "rising pH creep". Target
pH must reflect receiving water and effluent values. pH shall not be allowed to fall
more than 0.2 units below the target value.
(a)	All fish used in a test series must be born within 24 hours of each other.
(b)	If previous testing has shown greater than 20% mortality at 96 hours and not at 48
hours, test duration should be 96 hours. Once the 48 hour testing has been authorized,
if >20% mortality occurs in subsequent testing, then 96 hour duration tests should be
reinstated.

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REGION VIII EPA NPDES CHRONIC TEST CONDITIONS
STATIC RENEWAL WHOLE EFFLUENT TOXICITY TEST
Ceriodaphnia
dubia
Pimephales promelas
(Fathead minnows)
1.	Temperature
2.	Light duration and
Type
3.	Test container Size
4.	Test solution volume
5.	Age of test organisms
6.	No. of organisms per
container
7.	No. of replicates
8.	Renewal frequency
9.	No. Of Dilutions
10	Feeding
11.	Test duration
12. Acceptable control
Performance:
25° C ± 1°
16 hours-ambient
30 ml (minimum)
15 ml (minimum)
less than 24 hr (a)
1
10
daily (c)
5 (+ control)
(d)
until 60% have 3
broods in the
control
20% mortality or
less; three brood
average total of 15
or more.
25° C ± 1°
16 hours-ambient
500 ml (minimum)
250 ml (minimum)
less than 24 hr(b)
10 (minimum)
4	(minimum)
daily (c)
5	(+ control)
(e)
7 days
20% mortality or less;
avg. dry weight gain per
fish = 0.25 mg or more.
13.	Dilution Series: 100%, 50%, 25%, 12.5%, 6.25%, 0% (control).
14.	Dilution Water: Dilution water shall consist of receiving water if not toxic. If
receiving water is unsuitable, use moderately hard reconstituted water.
15.	Holding time: A maximum of 36 hours from the conclusion of sampling until the
initiation of testing should be maintained. Samples must be maintained at 4°C or
less Do not freeze.
(a)	Ceriodaphnia used in a test series must be born within 8 hrs of each other.
(b)	If fish are shipped in, they must be less than 48 hours old.
(c)	Permittee shall use a minimum of three fresh effluent samples taken at intervals of
two or three days depending on weekend shipping arrangements.
(d)	Recommendation: 0.1 ml/day of "YCT" and up to 0.1 ml/day of Selenastrum suspension
containing 3-4 x 107 cells.
(e)	0.1 ml of brine shrimp three times a day at four hour intervals or longer, or
0.15 ml twice a day at a six hour interval or longer.

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1.
2 .
3 .
4 .
5 .
6.
7 .
8 .
9 .
10
12
13
14
15
RgGION VIII NPDES CHRONIC TEST CONDITIONS
STATIC WHOLE EFFLUENT TOXICITY TESTS
Selenastrum capricornuturn
Temperature
Light quality
25 ± 1°
"Cool white" florescent lighting
Light intensity
Test container Size
Test solution volume
Age of test organisms
Initial cell density
in test containers
No. of replicates
Renewal frequency
Shaking rate
Test duration
Acceptable control
performance
400 ± 40 foot candles or 4306 lux
125 ml or 250 ml
50 or 100 ml
4 to 7 days
10,000 cells/ml
4
none
100 cpm continuous, or twice daily by hand
96 hours
2 x 105 cells/ml without EDTA or 1 x 106 cells/ml with
EDTA, Variability of controls should not exceed 20%
Dilution Series: 100%, 50%, 25%, 12.5%, 6.25%, 0% (control).
Dilution water: Algal stock culture medium with and/or without EDTA.
Holding time: A maximum of 36 hours from the end of sampling until the initiation of
testing should be maintained. Samples must be maintained at 4°C or less. Do not
freeze.

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APPENDIX C
ACUTE AND CHRONIC REPORTING GUIDANCE

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REGION VIII GUIDANCE FOR ACUTE WHOLE EFFLUENT REPORTING
BPMIT NAME				 NPDES No 	 OUTFALL NO.
MORTALITY TEST: 	 PASS 	 FAIL LC50 	% Tua
Test Species 	 Sample Time & Date 	
Sample temperature upon arrival at laboratory: 	
Analysis Time & Date: Begin 	 End 	
Dilution water used: 	
Initial Total Residual Cl2 in 100% Effluent: 	
Initial NH3 (as N) in 100% Effluent: 	
pH in 100% Effluent: Initial 	 After 24 Hours:
pH in 0% Control: Initial 	 After 24 Hours:
Dilutions (% Effluent) *
NUMBER
ALIVE
0%
6.25%
12 .5%
25%
50%
75%
100%
Start of
test







After 24
hrs







After 48
hrs







After 72
hrs







After 96
hrs







* normally, a minimum of five plus control (0%)
COMMENTS: 	
ANALYST'S NAME 	
LABORATORY 	 SIGNATURE/DATE

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REGION VIII GUIDANCE FOR CHRONIC WHOLE EFFLUENT REPORTING
CERIODAPHNIA DUBIA
PERMIT NAME	 NPDES No 	 OUTFALL NO.
IC25	 NOEC 		PASS	 FAIL		Tuc 	
Sample Time, & Dates	Analyses: Time & Date
No 1 		 Beginning 	
No 2	 Ending 	
No 3	
Sample temperature upon arrival at laboratory 	 	 	
Dilution Water Used: 	
Initial NH3(as N) in 100% Effluent	 Initial TRC in 100% Effluent	
Initial pH in 100% Effluent 	 pH after 24 hours 	
Initial pH in Control 	 pH after 24 hours 	
CERIODAPHNIA	
Total Number of Young per Adult
% Dilution

6 .25
12 .5
2R
50
ino
Replicate
A






B






C






D






E






F






6






H






I






J






MEAN






No. Live
Adults






COMMENTS:
ANALYST'S NAME
LABORATORY 	
SIGNATURE/DATE

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REGION VIII GUIDANCE FOR CHRONIC WHOLE EFFLUENT REPORTING
PIMEPHALES PROMELAS (FATHEAD MINNOW)
PERMIT NAME	
IC25	 NOEC
PASS
NPDES NO
FAIL 	
OUTFALL NO.
Tu.
Sample Time, & Dates
No 1 	
No 2	
No 3	
Dilution Water Used:
Analyses: Time & Date
Beginning 	
Ending 	
Sample temperature upon arrival at laboratory-
Initial NH3(as N)in 100% Effluent.
Initial pH in 100% Effluent 	
Initial pH in Control 	
Initial TRC in 100% Effluent.
pH after 24 hours	
pH after 24 hours.
p-rmgpwaT.ES PROMELAS	
Mean Weight | Number of surviving organisms
% Dilution
Control
6 .25
12 .5
25
50
100
Replicates
A
1
1
1
1
1
1
B
1
1
1
1
1
1
C
1
1
1
1
1
1
D
1
1
1
1
1
1
Mean
1
1
1
1
1
1
COMMENTS
A] ST1 S NAME
Lf ATORY 	
SIGNATURE/DATE

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REGION VIII GUIDANCE FOR CHRONIC WHOLE EFFLUENT REPORTING
SELENASTRUM CAPRICORNUTUM
PERMIT NAME	 NPDES No 	 OUTFALL NO.
IC25	 NOEC 	 PASS 	 FAIL 	 TUC	
Sample Time & Date	Analyses: Time & Date
No 1 		Beginning 	
Ending 	
Sample temperature upon arrival at laboratory 	
Dilution Water Used: 	
Initial NH3(as N) in 100% Effluent	 Initial TRC in 100% Effluent	
Initial pH in 100% Effluent 	 Initial pH in Control 	
EDTA Used 	
SELENASTRUM CAPRICORNUTUM	
Cell Density or Chlorophyll Content
% Dilution
Control
6 .25
12 .5
25
50
100
Replicate
A






B






C






D






Mean






COMMENTS:
ANALYST'S NAME
LABORATORY 	
SIGNATURE/DATE

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