United States	Office Of Water	EPA 833-B-96-005
Environmental Protection	(4204)	November 1996
Agency

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ABBREVIATIONS
ACR
Acute-Chronic Ratio
AML
Monthly Average Permit Limit
AWL
Weekly Average Permit Limit
AO
Administrative Order
ANOVA
Analysis of Variance
APO
Administrative Penalty Order
BAT
Best Available Technology
BMP
Best Management Practices
CCC
Criterion Continuous Concentration
CMC
Criterion Maximum Concentration
CFR
Code of Federal Regulations
CV
Coefficient of Variation
CWA
Clean Water Act
DMR
USEPA's Discharge Monitoring Report
DQO
Data Quality Objectives
EC
Effect Concentration
EDW
Effluent Dominated Waters
FAQ
Frequently Asked Questions
IC
Inhibition Concentration
IWC
Instream Waste Concentration
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LC
Lethal Concentration
LOEC
Lowest Observed Effect Concentration
MDL
Maximum Daily Limit
MGD
Million Gallons per Day
MDL
Maximum Daily Limit
MSD
Minimum Significant Difference
MSE
Mean Square Error
NELAC
National Environmental Laboratory Accreditation
NOEC
No Observed Effect Concentration
NOV
Notice of Violation
NPDES
National Pollutant Discharge Elimination System
PAI
Performance Audit Inspection
PE
Performance Evaluation
POTW
Publicly Owned Treatment Works
QA
Quality Assurance
QC
Quality Control
RP
Reasonable Potential
SCTAG
Southern California Toxicity Assessment Group
SETAC
Society of Environmental Toxicology and Chemistry
SOP
Standard Operating Procedure
STR
Salinity/Tolerance Relationship
TAC
Test Acceptability Criteria
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TDS	Total Dissolved Solids
TIE	Toxicity Identification Evaluation
TMDL	Total Maximum Daily Loads
TRE	Toxicity Reduction Evaluation
TSD	USEPA's Technical Support Document for Water Quality-based
Toxics Control
TSERF	Toxicity Standardized Electronic Reporting Format
TU	Toxicity Unit
USEPA	United States Environmental Protection Agency
WET	Whole Effluent Toxicity
WLA	Waste Load Allocations
WQBELs Water Quality Based Effluent Limitations
WQC	Water Quality Criteria
WQS	Water Quality Standards
WWTP	Wastewater Treatment Plant
ZID	Zone of Initial Dilution
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DEFINITIONS
ACUTE TOXICITY is a test to determine the concentration of effluent or ambient waters that
produces an adverse effect on a group of test organisms during a short-term exposure (e.g., 24, 48 or
96 hours). The endpoint is lethality. Acute toxicity is measured using statistical procedures (e.g.,
point estimate techniques or a t-test). Acute toxicity is usually defined as TUa = 100/LC50.
ACUTE-to-CHRONIC RATIO (ACR) is the ratio of the acute toxicity of an effluent or a toxicant to
its chronic toxicity. It is used as a factor for estimating chronic toxicity on the basis of acute toxicity
data, or for estimating acute toxicity on the basis of chronic toxicity data.
ADDnivri Y is the characteristic property of a mixture of toxicants that exhibits a total toxic effect
equal to the arithmetic sum of the effects of the individual toxicants.
AMBIENT TOXICITY is measured by a toxicity test on a sample collected from a receiving
waterbody.
BIOASSAY is a test used to evaluate the relative potency of a chemical or a mixture of chemicals by
comparing its effect on a living organism with the effect of a standard preparation on the same type of
organism. Bioassays frequently are used in the pharmaceutical industry to evaluate the potency of
vitamins and drugs.
CHRONIC TOXICITY is defined as a long-term test in which sublethal effects (e.g., reduced
growth or reproduction) are usually measured in addition to lethality. Chronic toxicity is defined as
TUc = 100/NOEC or TUc = 100/ECp (ICp). The ICp and ECp value should be the approximate
equivalent of the NOEC calculated by hypothesis testing for each test method.
COEFFICIENT OF VARIATION (CV) is a standard statistical measure of the relative variation of a
distribution or set of data, defined as the standard deviation divided by the mean. Coefficient of
variation is a measure of precision within (intralaboratory) and among (interlaboratory) laboratories.
CRITERIA CONTINUOUS CONCENTRATION (CCC) is the EPA national water quality criteria
recommendation for the highest instream concentration of a toxicant or an effluent to which organisms
can be exposed indefinitely without causing unacceptable effect.
CRITERIA MAXIMUM CONCENTRATION (CMC) is the EPA national water quality criteria
recommendation for the highest instream concentration of a toxicant or an effluent to which organisms
can be exposed for a brief period of time without causing an acute effect such as lethality.
CRITICAL LIFE STAGE is the period of time in an organism's lifespan m which it is the most
susceptible to adverse effects caused by exposure to toxicants, usually during early development (egg,
embryo, larvae). Chronic toxicity tests are often run on critical life stages to replace long duration,
life-cycle tests since the most toxic effect usually occurs during the critical life stage.
EFFECT CONCENTRATION (EC) is a point estimate of the toxicant concentration that would
cause an observable adverse effect (e.g., survival or fertilization) in a given percent of the test
organisms, calculated from a continuous model (e.g., USEPA Probit Model).
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HYPOTHESIS TESTING is a technique (e.g., Dunnetts test) that determines what concentration is
statistically different from the control. Endpoints determined from hypothesis testing are NOEC and
LOEC.
Null hypothesis (Ho): The effluent is not toxic.
Alternative hypothesis (HJ: The effluent is toxic.
INHIBITION CONCENTRATION (IC) is a point estimate of the toxicant concentration that would
cause a given percent reduction in a non-quantal biological measurement (e.g., reproduction or
growth) calculated from a continuous model (i.e., USEPA Interpolation Method).
INSTREAM WASTE CONCENTRATION (IWC) is the concentration of a toxicant in the receiving
water after mixing. The IWC is the inverse of the dilution factor.
LC50 is the toxicant concentration that would cause death in 50 percent of the test organisms.
LOWEST OBSERVED EFFECT CONCENTRATION (LOEC) is the lowest concentration of
toxicant to which organisms are exposed in a test, which causes statistically significant adverse
effects on the test organisms (i.e., where the values for the observed endpoints are statistically
significant different from the control).The definitions of NOEC and LOEC in the method manuals
assume a strict dose-response relationship between toxicant concentration and organism response. If
this assumption were always the case, there would be no issue concerning the endpoint definitions
because the NOEC would always be a lower concentration level than the LOEC. However, this strict
dose-response relationship does not exist with all toxicants. When this occurs the test must be
repeated or the lowest NOEC should be reported for compliance purposes.
MINIMUM SIGNIFICANT DIFFERENCE (MSD) is the magnitude of difference from control
where the null hypothesis is rejected in a statistical test comparing a treatment with a control. MSD
is based on the number of replicates, control performance and power of the test.
MIXING ZONE is an area where an effluent discharge undergoes initial dilution and may be
extended to cover the secondary mixing in the ambient waterbody. A mixing zone is an allocated
impact zone where water quality criteria can be exceeded as long as acutely toxic conditions are
prevented.
MONTHLY MEDIAN is the middle value in a monthly distribution above and below which lie an
equal number of values. If the number of values are even, then the monthly median is the average of
the middle two measurements.
NO OBSERVED EFFECT CONCENTRATION (NOEC) is the highest tested concentration of
toxicant to which organisms are exposed in a full life-cycle or partial life-cycle (short-term) test, that
causes no observable adverse effect on the test organisms (i.e., the highest concentration of toxicant at
which the values for the observed responses are not statistically significant different from the
controls). NOECs calculated by hypothesis testing are dependent upon the concentrations selected.
POINT ESTIMATE TECHNIQUES such as Probit, Interpolation Method, Spearman-Karber are
used to determine the effluent concentration at which adverse effects (e.g., fertilization, growth or
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survival) occurred. For example, concentration at which a 25 percent reduction in fertilization
occurred.
REFERENCE TOXICANT TEST indicates the sensitivity of the organisms being used and the
suitability of the test methodology. Reference toxicant data are part of routine QA/QC program to
evaluate the performance of laboratory personnel and test organisms. Reference toxicant tests must
be conducted concurrently with each effluent test (e.g., the reference toxicant required for the red
abalone test method is zinc sulfate).
RWC is the concentration of a toxicant or the parameter toxicity in the receiving water after mixing
at critical low flow conditions. The "instream waste concentration" is an equivalent term.
SIGNIFICANT DIFFERENCE is defined as statistically significant difference (e.g., 95% confidence
level) in the means of two distributions of sampling results.
TEST ACCEPTABILITY CRITERIA (TAC) For toxicity tests results to be acceptable for
compliance, the effluent and the concurrent reference toxicant must meet specific criteria as defined in
the test method (e.g., Ceriodaphnia dubia survival and reproduction test, the criteria are. the test
must achieve at least 80% survival and average 15 young/female in the controls, and achieve a MSD
of 20%).
t-TEST is a statistical analysis comparing only two test concentrations (e.g., a control and 100%
effluent). The purpose of this test is to determine if the 100% effluent concentration is different from
the control (i.e., the test passes or fails).
TOXICITY TESTS are laboratory experiments which employ the use of standardized test organisms
to measure the adverse effect (e.g., growth, survival or reproduction) of effluent or ambient waters.
TOXIC UNIT ACUTE (TUa) is the reciprocal of the effluent concentration that causes 50 percent of
the organisms to die by the end of the acute exposure period (i.e., TUa = 100/LC50).
TOXIC UNIT CHRONIC (TUc) is the reciprocal of the effluent concentration that causes no
observable effect on the test organisms by the end of the chronic exposure period (i e TUc =
100/NOEC).
TOXIC UNITS (TUs) are a measure of toxicity in an effluent as determined by the acute toxicity
units or chronic toxicity units. Higher TUs indicate greater toxicity.
TOXICITY IDENTIFICATION EVALUATION (TIE) is a set of procedures to identify the
specific chemical(s) responsible for effluent toxicity. TTEs are a subset of the TRE.
TOXICITY REDUCTION EVALUATION (TRE) is a site-specific study conducted in a stepwise
process designed to identify the causative agents of effluent toxicity, isolate the sources of toxicity,
evaluate the effectiveness of toxicity control options, and then confirm the reduction in effluent
toxicity.
WHOLE EFFLUENT TOXICITY is the total toxic effect of an effluent or receiving water measured
directly with a toxicity test.
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WASTELOAD ALLOCATION provides a definition of effluent quality that is necessary to meet the
applicable numeric or narrative water quality criteria of the receiving water.
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WET TRAINING
INTRODUCTION
Regulatory Process - Standards to
Permits to Compliance/Enforcement
f/EPA
Purpose of Module and Topics to be Covered:
•	Why we're offering this workshop
•	Foster better understanding of the tool (heavy emphasis) and
its utility in a number of contexts
•	Principal statutory and regulatory underpinnings
•	Provide overview of standards-to-permits process as
background and basis for further discussion of both scientific
(Day 1) and implementation (Day 2) considerations

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Purposes of Workshop:
¦	Provide overview of legal and
regulatory support for use of WET
¦	Foster better understanding of
scientific underpinnings of WET as a
tool
¦	Provide overview of key
implementation considerations
¦	Discuss current WET issues and
concerns
Talking Points:
•	WET has been used as a regulatory tool in the NPDES program
since the early 80's.
•	Efficacy of WET as a predictor of in-stream toxicity has been
sufficiently demonstrated.
•	Much experience has been gained over the last several years
that have led EPA and States to seek to build upon successes
and to undertake any necessary "mid-course corrections" that
may be warranted.
•	The scientific underpinnings of WET were examined in
September 1995 in Pellston, Michigan (sponsored by the
Society of Environmental Toxicology and Chemistry with
support from EPA) and implementation considerations will be
discussed with stakeholders at an upcoming implementation
meeting in the fall of 1996.
•	The workshop won't cover any single topic in exhaustive
detail; rather, the purpose is to provide an overview of current
thought and practice and to indicate where further information
can be obtained.
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Ongoing WET Activities:
¦	SETAC Pellston WET
-	unresolved scientific issues
-	needed research
¦	National meeting to discuss WET
implementation issues
-	standards
-	permitting
Talking Points:
•	The Society of Environmental Toxicology and Chemistry (SETAC)
sponsored Pellston workshop on the technical aspects of the WET
program. The workshop was held in Pellston, Michigan, September 16
- 21, 1995. The workshop focused on discussing unresolved scientific
issues and needed significant research in the area of effluent toxicity
testing and associated receiving system impact predictions.
•	The proceedings of the workshop is published in a book entitled
"Whole Effluent Toxicity Testing: An Evaluation of Methods and
Prediction of Receiving System Responses". (See handout on Pellston
summary conclusions.) The SETAC Special Publications Series was
established by SETAC to provide in-depth reviews and critical
appraisals on scientific subjects relevant to understanding the impacts
of chemicals and technology on the environment. All books in the
series are peer reviewed for SETAC by acknowledged experts. This
publication is available for a cost from the SETAC office by calling at
(904) 469-1500.
•	As a follow-up to the technical review of the WET program, EPA will
held a national meeting for all stakeholders to discuss the
implementation issues of the WET program. This meeting was held on
September 24-26, 1996 in Washington, DC .
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WET Testing for Aquatic Life
Protection
¦	Measures the aggregate toxic effect of
effluent or ambient water
¦	Standardized, surrogate fresh and
marine species: plants, vertebrates
and invertebrates
¦	Acute and chronic tests
Talking Points:
• Provide general overview of what WET testing includes -
standardized acute and chronic tests that measure the
aggregate toxic effect of effluent or ambient water.
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Acute Tests
¦	Duration - 96 hours or less
¦	Endpoint - lethality
Talking Points:
• Acute toxicity tests are used to determine the concentration of
effluent or ambient water that produces an adverse effect on a
group of test organisms during either a 24, 48 or 96 hour
exposure. The endpoint measured is mortality.
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Chronic Tests
¦	Duration - 7 days or less
¦	Endpoints - fertilization, growth, or
reproduction are measured in addition
to possible lethality
Talking Points:
• A chronic toxicity test is defined as a short-term test in which
sublethal endpoints are measured in addition to possible
mortality. The short-term chronic toxicity test methods range
from 40 minutes to a 7 day exposure.
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Why WET?
¦	Whole effluent approach to toxics
control for protection of aquatic life
uses acute and chronic toxicity tests
to measure toxicity of wastewaters
¦	Directly implements narrative criteria
"no toxics in toxic amounts"
Talking Points:
•	WET is a tool to measure the aggregate effects of toxic
impacts.
•	All States have either a narrative or numeric toxicity standard.
WET methods provide an approach to directly implement the
narrative standard that is not afforded by other approaches.
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Integrated Approach to Water
Quality-Based Toxics Control
¦	Chemical-specific approach
¦	Whole effluent toxicity approach
¦	Biological criteria approach
Talking Points:
•	Each approach has advantages and disadvantages which the
practitioner needs to understand ~ none is a panacea (see
TSD pages 20-22).
•	Independent Applicability vs. the Weight-of-Evidence approach
is a hotly contested issue. Water quality standards and
NPDES permits regulations currently support an independent
applicability approach; however, EPA is taking comments on
the issues in its water quality standards proposed rulemaking.
Any movement toward a weight-of-evidence approach would
only be feasible in those cases where high quality data of
various types (WET, bioassessments and chemical specific)
were available.
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Water Quality Standards and
WET
Talking Points:
• The next few slides will deal with the Water Quality Standards
(WQS) program. What are water quality standards and how
they are implemented?
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Water Quality Standards
¦ Section 303 of CWA
Talking Points:
• Section 303 of the CWA requires States to develop and adopt
water quality standards. In cases where States do not adopt
Standards, EPA will promulgate standards. This can include
WET criteria where appropriate.
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Water Quality Standards -
3 Parts
Designated uses
Criteria (numeric and narrative) to
protect designated uses
Antidegradation policy

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Designated Uses
40 CFR 131.10
¦ Examples:
-	warm water fishery
-	cold water fishery
-	fish and wildlife propagation
Talking Points:
• Each State must identify the designated use of the individual
water bodies. This may include decisions based on
biological/ecological issues, socioeconomic issues and
aesthetics.
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Antidegradation Policy
40 CFR 131.12
¦	Existing instream uses
¦	Exceeds water quality
¦	Outstanding national resource water
(ONRW)
¦	Impairment and thermal discharges
Talking Points:
•	Ensures that once a use is achieved it will be maintained.
•	As part of their WQS each State must develop and adopt an
Antidgradation Policy and identify methods for implementing
the policy.
•	At a minimum the policy must address how the State shall
maintain WQ in water bodies where existing uses are being
met, how the State shall maintain WQ in cases where uses are
exceeded (also whether they will allow lower water quality in
cases where it is necessary to accommodate important
economic or social development in the area), how the State
will protect ONRWs, and finally that the policy is consistent
with CWA section 316 for thermal discharges.
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Criteria
40 CFR 131.11
¦	Numeric - individual pollutant or
pollutant parameters
¦	Narrative - free from ..."
-	toxics in toxic amounts
-	objectionable color, odor, taste
Talking Points:
•	Criteria may be expressed as either numeric or narrative. The
criteria must protect the designated uses of the water bodies
for which they are set. In cases where a water body has
multiple designated uses the criteria must protect the most
sensitive use.
•	For numeric criteria, the States are recommended to establish
values based on 304(a) guidance, 304(a) guidance adapted for
site-specific conditions, or use other scientifically defensible
methods.
•	For a narrative toxicity criteria, the States are recommended to
establish criteria based on toxicity test methods where
numeric criteria are not established or to supplement numeric
criteria.

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Implementation Policy
¦ Explains how states implement the
narrative criterion to establish effluent
limitations to control WET
Talking Points:
• The WQS Regulations allow states to include in their standards
State policies and provisions regarding WQS implementation.
Often these address issues such as mixing zones, variances,
and low flow exemptions. It is recommended that the policy
also include information on the implementation of WET
criteria.
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Water Quality Criteria
Components:
¦	Magnitude: maximum allowable
concentration of WET
¦	Duration: period of time over which
the instream concentration is averaged
¦	Return frequency: designation of how
often the criterion may be exceeded
without impacting the organisms in
the water body
Talking Points:
•	All policies related to criteria development should include
reference to the three criteria components (magnitude,
duration and frequency).
•	Magnitude establishes how much of a pollutant (or pollutant
parameter such as toxicity) expressed as a concentration, is
allowable.
•	Duration establishes the period of time (averaging period) over
which the instream concentration is averaged for comparison
with criteria concentrations. This specification limits the
duration of concentrations above the criteria.
•	Frequency establishes how often criteria may be exceeded.
EPA uses a 3 year return frequency.
•	Magnitude, duration and return frequency provisions of WET
criteria are used in the development of wasteload allocations
and effluent limitations to control the WET of the discharge.
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EPA Has Not Developed a 304(a)
Criterion for WET; EPA
Recommendations in the TSD for
Acute:
magnitude = 0.3 TUa
durations = one hour
frequency = once in three years
Talking Points:
•	Factor of 0.3 is used to adjust the typical LC50 endpoint of an
acute toxicity test to an LC1. (The factor of 0.3 was found to
include 91% of observed LC1 to LC50 ratios in approximately
500 toxicity tests in both industrial and municipal effluents.)
•	The 1 hour averaging period was derived primarily from data
on response time for toxicity to ammonia, a fast-acting
toxicant.
•	EPA selected the 3 year return interval with the intent of
providing a degree of protection roughly equivalent to a 7Q10
design flow condition, with some consideration of rates of
ecological recovery from a variety of stresses.
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EPA Has Not Developed a
304(a) Criterion for WET; EPA
Recommendations in the TSD
for Chronic:
magnitude = 1.0 TUc
durations = 4 days
frequency = once in three years
Talking Points:
•	A chronic criterion of 1.0 TUc is recommended by EPA for
application outside of the mixing zone to prevent any chronic
toxicity in the receiving water outside the mixing zone.
•	EPA selected the 4-day averaging period based on the shortest
duration in which chronic effects are sometimes observed for
certain species and toxicants; and thus should be fully
protective even for the fastest-acting toxicants.
•	EPA's frequency recommendations apply to both acute and
chronic criteria.
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TUa
= 100/LC50
TUc
= 100/NOEC or ECp
Talking Points:
•	Since toxicity involves an inverse relationship with the effect
concentration (the lower the effect concentration, the higher
the toxicity of the effluent), it is often more convenient to
translate concentration-based toxicity measurements into toxic
units (TUs).
•	It's important to note that two different types of toxic units
are used and they are not interchangeable.
•	Acute and chronic toxic units can be used to quantify the
toxicity of an effluent as well as to specify water quality
criteria based on toxicity.
•	An important consideration with specification of criteria for the
parameter toxicity is that permit limits designed to ensure that
the criteria are met, may actually be below detection levels for
the whole effluent toxicity test (this is particularly important in
connection with acute toxicity). This subject will be covered
in more detail in the permit limit development module.

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Critical Low Flows and Mixing
Zones
¦	State WQS protect designated uses
during critical low flow periods
¦	Mixing zones are allowed in some
WQS
Talking Points:
•	Mixing zones are small areas in the receiving water near
certain discharge outfalls where ambient concentrations above
the otherwise applicable state water quality criteria are
allowed.
•	EPA recommends that standards clearly address mixing zones;
whether allowed or not allowed, and procedures for defining
mixing zones
•	CWA does not require attaining WQ criteria at the end-of-pipe.

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Statutory Requirements for
Development of WQBELs for WET
¦	CWA 101(a): Goals of the CWA; to
restore and protect the nation's waters
¦	CWA 101(a)(3): Prohibits discharge of
toxic pollutants in toxic amounts
¦	CWA 301(a): Discharges to waters of the
US must be in accordance with an NPDES
permit (per Section 402)
¦	CWA 301(b)(1)(C): Permits must assure
that water quality standards are met
Talking Points:
•	CWA 101 (a) states that the goal of CWA is to restore and
maintain the chemical, physical, and biological integrity of the
nation's waters. A final goal of zero discharge is established,
along with an interim fishable/swimmable goal.
•	CWA 101(a)(3) provides a specific statutory basis for water
quality standards and WQBELs designed to prohibit discharges
of toxic pollutants in toxic amounts.
•	CWA 301 (a) requires that a permit be obtained before anyone
may discharge pollutants into waters of the US; Section 402
sets forth the basic structure for the NPDES program.
•CWA 301(b)(1)(C) requires that dischargers with permits meet
all technology-based requirements of the CWA and any more
stringent requirements necessary to achieve state water
quality standards. This statutory provision was the primary
basis for development of EPA's WQBEL regulations at
122.44(d).
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Regulatory Requirements for
Development of WQBELs for
WET
¦ 40 CFR 122.44(d)(1) describes
requirements for NPDES permitting
authorities to follow in developing
WQBELs, including those necessary to
control WET

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40 CFR 122.44(d)(1): Basis for
Reasonable Potential (RP)
Determinations and Development of
WQBELs:
¦	List of factors to be considered for RP
determination
¦	Limits must be developed where there's a positive
RP determination for either a numeric criterion for
an individual chemical or WET, or a narrative WET
criterion
¦	Where a RP finding is made with respect to a
narrative criterion, the permitting authority may
control WET or the causative toxicant(s).
Talking Points:
•	These regulatory provisions (the principal WET portions of
which are summarized above) provide a very clear and strong
regulatory basis for "reasonable potential" (RP) determinations
and permit limit development.
•	It's important to note that, while providing a firm regulatory
basis, the regulations also allow an appropriate level of
flexibility for regulatory authorities.
•	The regulatory authority need only "consider" a number of
factors when making a RP determination. The relative weight
given each factor in this decision as well as the type and
quantity of information considered are at the discretion of the
regulatory authority. Similarly, once an RP determination has
been made, the specific permit limit derivation procedures are
a matter for discretion of the permitting authority.
•	Each of the steps in the RP and permit limit derivation process
will be considered in greater detail in the Permit Development
module.
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WET Control Policy
July 1994
(1)	To promote uniform, nationwide
compliance with statutory and
regulatory requirements for the
control of WET; and
(2)	To assist permit writers in
implementing these requirements
Talking Points:
• See handout copy of the WET Control Policy.
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Summary of the Eight

Statements of Policy
(1)
Basis for WET controls
(2)
Evaluation of dischargers for reasonable

potential
(3)
Evaluating reasonable potential
(4)
Consequences of establishing

reasonable potential
Talking Points:
•	The permitting authority should evaluate WET water quality
criteria attainment for acute WET at the edge of the acute
mixing zone and for chronic WET at the edge of the chronic
mixing zone except where the state has different requirements
for evaluating WET criteria. The permitting authority will
develop WET effluent limitations based upon the more
stringent of the acute or chronic criterion applied at the edge
of the respective mixing zone or alternatively, on both.
•	At a minimum, the permitting authority should review all major
dischargers for reasonable potential to cause or contribute to
exceedances of WET water quality criteria.
•	The permitting authority will consider available WET testing
data and other information in evaluating whether a discharger
has the reasonable potential to cause or contribute to
exceedance of WET water quality criteria.
•	Upon finding reasonable potential to cause or contribute to
exceedance of WET water quality criteria, the permitting
authority will impose effluent limitations to control WET.
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Summary of the Eight
Statements of Policy (cont.)
(5)	WET monitoring
(6)	Compliance schedules in NPDES
permits
(7)	WET controls and the pollutants
ammonia and chlorine
(8)	WET controls and POTWs
Talking Points:
•	Where appropriate, the permitting authority should impose
WET monitoring conditions upon dischargers that do not have
effluent limitations to control WET.
•	Where allowed under state and federal law, NPDES permits
may contain schedules for compliance with WET effluent
limitations.
•	The requirements of the water quality permitting regulations
apply without regard to the pollutant(s) that may be causing
toxicity, including ammonia and chlorine.
•	The requirements of the water quality permitting regulations
apply to all dischargers, including POTWs.
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Conclusions
¦	WET is a "tried and true" tool with
great usefulness in water quality
control programs
¦	The statutory and regulatory basis for
WET is well established and defensible
in the context of NPDES permitting
Talking Points:
• Having established this basis for the WET program, "the stage
is now set" for a deeper discussion of the tests methods
themselves and important QA/QC issues.
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Appendix
SUMMARY OF PRINCIPAL PROVISIONS OF 122.44(d)
WQBELs AND REASONABLE POTENTIAL DETERMINATIONS
•	Limitations must control all pollutants or pollutant parameters (either conventional,
nonconventional, or toxic pollutants) which the director determines are or may be
discharged at a level which will cause, or contribute to an excursion above any state
water quality standard, including state narrative criteria for water quality.
•	When determining whether a discharge, has the reasonable potential to cause, or
contributes to an instream excursion above a narrative or numeric criteria within a
state water quality standard, the permitting authority shall use procedures which
account for existing controls on point and nonpoint sources of pollution, the
variability of the pollutant or pollutant parameter in the effluent, the sensitivity of the
species to toxicity testing (when evaluating WET) and where appropriate, the
dilution of the effluent in the receiving water.
•	When the permitting authority determines, using the procedures in paragraph
(d)(1)(ii) of this section, that a discharge causes, has the reasonable to cause, or
contributes to an in-stream excursion above the allowable ambient concentration of
a state numeric criteria within a state water quality standard for an individual
pollutant, the permit must contain effluent limits for that pollutant.
•	When the permitting authority determines, using the procedures in paragraph
(d)(1)(ii) of this section, that a discharge causes, has the reasonable potential to
cause, or contributes to an in-stream excursion above the numeric criterion for
whole effluent toxicity, the permit must contain effluent limits for whole effluent
toxicity.
•	Except as provided in this subparagraph, when the permitting authority determines,
using the procedures in paragraph (d)(1)(ii) of this section, toxicity testing data, or
other information, that a discharge 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 permit must contain effluent limits for WET. Limits
on whole effluent toxicity are not necessary where the permitting authority
demonstrates in the fact sheet or statement of basis of the NPDES permit, using the
procedures in paragraph (d)(1)(ii) of this section, that chemical-specific limits for the
effluent are sufficient to attain and maintain applicable numeric and narrative state
water quality standards.
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UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, O.C. 20460
JUL t m
OFFICE OF
WATER
MEMORANDUM
SUBJECT: New Whole Effluent Toxicity (WET) Policy
for National Pollutant Discharge Elimination System
(NPDES) Permits
Attached is EPA's new policy governing the development of
effluent limitations in NPDES permits to control WET for the
protection of aquatic life. It consists of eight policy
statements and additional background materials. The policy in
part reaffirms EPA's strong continuing commitment to the existing
Clean Water Act provisions and water quality permitting
regulations in 40 C.F.R. 122.44(d)(1). The policy also addresses
some specific areas where questions have arisen regarding the
implementation of these statutory and regulatory requirements. I
believe adherence to the policy will help ensure greater national
consistency in the control of WET.
In addition, a notice of availability of the policy is
scheduled for publication in the Federal Register soon.
Additional copies of the policy can be obtained by contacting the
U.S. EPA, National Center for Environmental Publications and
Information, P.O. Box 42419, Cincinnati, Ohio 45242-2419
(Document Number EPA 833-B-94-002).
If you have any questions, please call me or Cynthia C.
Dougherty, Director, Permits Division, at (202) 260-9545.
Attachment
cc: Robert Perciasepe
Tudor Davies
Robert Wayland
Robert Van Heuvelen
Elaine Stanley
Susan Lepow
Regional Water Permits Branch Chiefs
FROM: Michael B. Cook, Directo
Office of Wastewater Man
(4201)
TO:
Water Management Division Directors, Regions I-X
State NPDES Directors
n»qnfd/n*yc Hbl»
Ti \S PiMad«OiSefC*noiaMionMK«rirMi
commaImhSO*rvcycuo

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United Sates
Environmental Protection Office of Water EPA 833-B-94-002
	(42Q3)	July 1994
oEFA WHOLE EFFLUENT TOXICITY
(WET) CONTROL POLICY
POLICY FOR THE DEVELOPMENT OF EFFLUENT LIMITATIONS IN
NATIONAL POLLUTANT DISCHARGE ELIMINATION SYSTEM PERMITS
TO CONTROL WHOLE EFFLUENT TOXICITY FOR THE PROTECTION
OF AQUATIC LIFE

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CLARiriClTTOMty
l. XSSSZE: The definition of whole effluent toxicity in 40
C.F.R. S 122.2*
CLARIFTCATTOW*
EPA defined whole effluent toxicity in 40 C.F.R. S 122.2 as
the "aggregate toxic effect of an effluent measured directly by a
toxicity test." The petitioners were concerned that this
definition, in conjunction with the requirement in 40 C.F.R. c
122.44(d)(l)(iv) and (v) that states implement narrative criteria
by imposing limits on whole effluent toxicity, could be read
expansively to require states to impose whole effluent toxicity
limits prohibiting discharges which evoke any response in test
organisms, no matter how slight, as measured by toxicity tests.
The petitioners stated that such an interpretation could deprive
a state of the authority to define what it considers to be
acceptable levels of toxicity in a discharger's effluent
consistent with applicable water quality standards. EPA does not
interpret the definition of whole effluent toxicity in section
122.2, or the requirements of section 122.44(d)(1)(iv) and (v)
as imposing any substantive water quality standard for what
constitutes an acceptable level of whole effluent toxicity.
Rather, these sections indicate when the permitting authority
must establish permit limits on whole effluent toxicity for
purposes of achieving water quality standards (either numeric of
narrative water quality criteria).
2. ISSVE? The enforceability of limitations based upon
single toxicity test results, as discussed at 54 Fed. Reg.
CLARIFICATTOW*
In the preamble to the final rule, at 54 Fed. Reg. 23,871,
EPA stated that:
A limit on whole effluent toxicity refers to a numeric
effluent limitation expressed in terms such as toxic units,
no observed effect level (NOEL), LC 50, or percent
mortality. Effluent limitations may be expressed as chronic
toxicity or acute toxicity (or both). Regardless of how the
numeric limitations for whole effluent toxicity are
expressed, any single violation of an effluent limit is a
violation of the NPDES permit and is subject to the full
range of state and Federal enforcement actions.
EPA interprets this paragraph and existing regulations to
provide that violation of an effluent limit for whole effluent
toxicity is enforceable, whether that limit is expressed in terms
of a numeric effluent limit or, where setting a numeric effluent
l

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limit is infeasible, best management practices.1 (For example
some storm water discharges have volumes and pollutant	'
concentrations that fluctuate wildly with storm events, making it
difficult to document resulting water quality impacts.).• The
preamble statement does not address the issue of how permit
limits may be derived. For example, when used appropriately,
permit limits may include averages (e.g.~ monthly averages) which
may be exceeded by an individual measurement so long as the
average of the individual measurements is not above the limit and
any applicable daily maximum is complied with. Permit limits,
however expressed, must be designed to protect water quality
standards.
3. issue: The requirement for limitations on all pollutants
and the use of indicators, as set forth at 40 C.F.R. c
122.44(d) (l)(i).
CLARIFICATION:
40 c.F.R. S 122.44(d)(l)(i) requires that permits contain
effluent limitations to control pollutants that "are or may be"
discharged at levels having the Reasonable potential to cause,
or contribute to an excursion above any State water quality
standard, including State narrative criteria for water quality."
EPA did nc.t intend to require water quality-based permit
limitations on all pollutants contained in a discharge through
the promulgation of the June 2, 1989 regulation; nor do we
believe that the regulation has that effect. The proper
interpretation of the regulations is that developing water
quality-based limitations is a step-by-step process. First, the
permitting authority must evaluate all available information to
determine at what level pollutants are expected to exist in the
current discharge. This determination is governed by 40 C.F.R. §
122.44(d)(l)(ii). The goal of this step is to estimate the
levels of pollutants in the effluent as discharged at the time of
permit application, or with any projected increases in the
discharge.
Under 40 c.F.R. S 122.44(d)(l)(ii), the permitting authority
must take into account the likely variability of the pollutant in
the effluent, other current discharges (from both point and non-
point sources as well as natural background), and (where
appropriate) dilution. At the end of this step the permitting
authority will have estimated an in-strean level of the pollutant
(or pollutant parameter) of concern that has the reasonable
potential to occur as a result of the discharge. (Most of this
1 The technological or economic feasibility of a
discharger meeting numeric limitations is not relevant to this
determination.
2

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step may have already been completed as a part of the total
maximum daily load and vasteload allocation calculation.) If the
estimated in-stream levels (vhich may occur, but will not
necessarily cccur) would exceed any applicable water quality
criterion, including the narrative criteria, then the permitting
authority must go to the next step and establish a water quality-
based limit in accordance with paragraphs 122.44(d)(1)(iii)-(vi).
EPA does not interpret section 122.44(d)(1)(i) as requiring
that permits contain water quality-based limitations on every
pollutant that may be present in a given effluent. Rather, water
quality-based limits are established where the permitting
authority reasonably anticipates the discharge of pollutants by
the permittee at levels that have the reasonable potential to
cause or contribute to an excursion above any state water quality
criterion, including state narrative criteria for water quality.
40 C.F.R. S 122.44(d)(1)(i). The permitting authority should
evaluate the reasonable potential for an excursion above a water
quality criterion in light of the character of the effluent as
discharged.
4. ISSUE; The use of a state policy or regulation
interpreting state narrative water quality criteria, as set
forth at 40 C.F.R. S 122.44(d)(1)(vi)(A).
CLARIFICATION:
The final rule provides that a permitting authority must
establish permit limits using one or more of several options
whenever a specific chemical for which the state has not
established a water quality criterion is present in an effluent
at a concentration that causes, has the reasonable potential to
cause, or contributes to an excursion above a state narrative
criterion. 40 C.F.R.*S 122.44(d)(1)(vi). The rule then
prescribes several options for establishing permit limitations,
including "explicit State policy or regulation interpreting [the
State's] narrative water quality criterion . . . ." 54 Fed. Reg.
at 23,896, codified at 40 C.F.R. S 122.44(d)(1)(vi)(A).
EPA interprets section 122.44(d)(1)(vi) as requiring permit
writers to use a formally adopted state regulation or policy
(including any state waste load allocation approved by EPA or
established by EPA using formally-adopted state regulations or
polices, where available) for deriving a chemical-specific
numeric water quality-based effluent limitation from an
applicable narrative standard in lieu of the other options for
interpreting a narrative standard set forth in that section, if
such a formally-adopted state regulation or policy exists. Such
a regulation or policy would typically be part of either a
state's water quality standards or total maximum daily load for
the water body in question, and would be subject to EPA approval
or disapproval in accordance with 40 C.F.R. Parts 130 or 131. If
3

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the state had not formally adopted a state regulation or policy
pursuant to 40 C.F.R. Parts 130 or 131, or if it has not been
approved as part of the state NPDES program, the permit writer
must develop limits, using any one of the options set forth in
section 122.44(d)(lj(vi). Some of the industry petitioners in
American Paper Institute v. U.S. EPA (D.C. Cir. No. 89-1499) and
consolidated cases do not agree that a formally adopted state
regulation or policy must be subject to CPA approval or
disapproval before permit writers would be required to use the
policy in developing limits. CPA expects this issue to be
litigated in the permit context.
When a permit writer interprets a narrative standard, the
method of interpretation used will be available for public
comment as a part of the permit and typically may be appealed
through administrative and judicial procedures available for
review of NPDCS permit conditions.
5. ISSUE: The standards for listing waters on the list of
Clean Water Act ("CWA") section 304(1)(1)(B), 33 U.S.C. S
1314(1)(1)(B), as set out at 40 C.F.R. S 130.10(d)(5.
CLARIFICATION!
Section 304(1) (1)(B) of the CWA, 33 U.S.C. S 1314(1)(1)(B),
provides that the state should list waters where an applicable
water quality standard is exceeded "due entirely or
substantially" to point sources. CPA's final rule requires
listing of a water under section 304(1) (l)(B) where (l) water
quality-based limits on one or more point sources would result in
the water quality standard for a toxic pollutant being achieved,
or (2) discharges from one or more point sources would be
sufficient to cause or are expected to cause an exceedence of the
water quality standard for a toxic pollutant, regardless of any
contribution of the same pollutant from nonpoint sources. 54
Fed. Reg. at 23,897, codified at 40 C.F.R. 5 130.10(d)(5).
The conditions in 40 C.F.R. S 130.10(d)(5) govern only the
determination of whether or not a given water should be listed
under section 304(1)(1)(B). Section 130.10 (d)(5) does not
dictate the limitations to be included in an individual control
strategy ("ICS"). ZCSs may be developed'in light of permit
limits and nonpoint souree requirements established through the
total maximum daily load ("TMDL") process. The TMDL is a
quantification of the capacity of a waterbody to assimilate
pollutants based on the applicable water quality standard. The
TMDL consists of the sum of wasteload allocations for point
sources, load allocations for nonpoint sources, and natural
background, with a margin of safety to account for uncertainty.
Subject to CPA approval, if a state determines that reductions in
the discharge of pollutants from a point source would be
inequitable or prohibitively expensive, the state may adopt a
4

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TMDL for achieving the water quality standards which relies in
whole or in part upon control requirements on nonpoint sources.
See 40 C.F.R. Section 130.7
5

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July 1994
POLICY FOR THE DEVELOPMENT OF EFFLUENT LIMITATIONS IN NATTnM&T-
POLLUTANT DISCHARGE ELIMINATION SYSTEM (NPDES) PERMITS TO CONTROT.
WHOLE EFFLUENT TOXICITY (WET) FOR THE PROTECTION OF AOUATTC LTFF
INTRODUCTION
EPA is today publishing a national policy on the control of
WET in NPDES permits. This policy is intended (i) to promote
uniform, nationwide compliance with statutory and regulatory
requirements for the control of WET, and (ii) to assist permit
writers in implementing these requirements.
This policy reflects EPA's experience in implementing the
regulations at 40 C.F.R. § 122.44(d)(1), which were originally
published at 54 Fed. Reg. 23868 (June 2, 1989) (also referred to
as "the water quality permitting regulations"). In part, this
policy restates those regulations and reaffirms EPA's strong,
continuing commitment to their prompt and complete
implementation. The water quality permitting regulations, as
well as the statutory provisions restated in this policy
document, are fully binding on EPA Regions as well as States
authorized to administer the NPDES program.
This policy also provides guidance to permit writers on
implementation of the statutory and regulatory requirements for
the control of WET.1 EPA permit writers are expected to follow
the portions of this policy that provide such guidance, although
decisions on individual permit provisions should be made on a
case-by-case basis, applying the law and regulations to specific
facts and justifying decisions in the record for the permit.
Similarly, EPA Regions will consider this policy in determining
whether State-issued NPDES permits adequately implement the
statutory and regulatory requirements for the control of WET.
This policy addresses some specific areas where questions
have arisen regarding the implementation of statutory
regulatory requirements. It does not address all areas where the
regulatory agency will exercise judgment in the implementation of
statutory and regulatory requirements. For the questions
addressed, it provides EPA's view as to the best course of action
in most instances. This policy does not establish or affect
legal rights or obligations and is not finally determinative of
the issues addressed. Most importantly, nothing in this policy
should be interpreted as providing any relief from the statutory
and regulatory requirement that permits include conditions as
necessary to assure attainment of water quality standards.
XA brief summary of existing Agency policy and guidance
addressing WET issues is contained in Appendix One to today's
policy.

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SUMMARY OF STATEMENTS OF POLICY
Eight statements of policy appear below. Explanations of
each statement follow.
1.	Basis for WET Controls
The permitting authority should evaluate WET water quality
criteria attainment for acute WET at the edge of the acute mixing
zone and for chronic WET at the edge of the chronic mixing zone
except where the State has different requirements for evaluating
WET criteria.2 The permitting authority will develop WET
effluent limitations based upon the more stringent of the acute
or chronic criterion applied at the edge of the respective mixing
zone, or, alternatively, on both.
2.	Evaluation of Dischargers for Reasonable Potential
At a minimum, the permitting authority should review all
major dischargers for reasonable potential to cause or contribute
to exceedance of WET water quality criteria.
3.	Evaluating Reasonable Potential
The permitting authority will consider available WET testing
data and other information in evaluating whether a discharger has
reasonable potential to cause or contribute to exceedance of WET
water quality criteria.
4-	Consequences of Establishing Reasonable Potential
Upon finding reasonable potential to cause or contribute to
exceedance of WET water quality criteria, the permitting
authority will impose effluent limitations to control WET.
5-	Whole Effluent Toxicity Monitoring
Where appropriate, the permitting authority should impose
WET monitoring conditions upon dischargers that do not have
effluent limitations to control WET.
6. Compliance Schedules in NPDES Permits
Where allowed under State and federal law, NPDES permits may
contain schedules for compliance with WET effluent limitations.
^Throughout this policy, the term "WET water quality
criteria" refers to State numeric water quality criteria for WET
and State narrative water quality criteria for toxicity such as
"no toxics in toxics amounts" in State water quality standards.
2

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7.	Whole Effluent Toxicity Controls and the Pollutants Ammoi-pa
and Chlorine
The requirements of the water quality permitting regulations
apply without regard to the pollutant(s) that may be causing
toxicity, including ammonia and chlorine.
8.	Whole Effluent Toxicity Controls and Publicly Owned
Treatment Works (POTWs)
The requirements of the water quality permitting regulations
apply to all dischargers, including POTWs.
3

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EXPLANATION OF STATEMENTS OF POLICY3
1. Basis for WET Controls
The permitting authority should evaluate WET water
quality criteria attainment for acute WET at the edge
of the acute nixing zone and for chronic WET at the
edge of the chronic nixing zone except where the state
has different requirements for evaluating WET criteria.
The pemitting authority will develop WET effluent
limitations based upon the nore stringent of the acute
or chronic criterion applied at the edge of the
respective nixing zone, or, alternatively, on both.
This policy statement describes the procedure permitting
authorities should use to evaluate WET water quality criteria
attainment and to develop effluent limitations to control WET.
In the absence of more specific State requirements, EPA believes
this approach most appropriately assures compliance with State
water quality standards.',5 The permitting authority must
evaluate WET water quality criteria attainment according to the
applicable State water quality standard(s). Permitting
authorities should assess WET concentrations as diluted in the
receiving water at the edge of the acute and chronic mixing zones
*To aid the reader in using this policy, Appendix Two
contains some background materials on WET testing, the State
water quality standards process and WET, and federal statutory
and regulatory requirements for development of water quality-
based permit limitations for WET.
4state water quality standards or implementation procedures
may (l) specify whether and how it is appropriate to account for
dilution in establishing WET controls; (2) require the applicable
criteria to apply at the outfall point of discharge or may
contain a specific policy approved by EPA allowing or prohibiting
mixing zones; as well as (3) specify exposure factors for WET
which are relevant to the application of this policy statement,
such as critical flow requirements for the receiving water,
appropriate modeling techniques for determining the fate of the
pollutant or pollutant parameter in stream, or required
techniques for evaluating the mixing of the pollutant or
pollutant parameter in the stream.
sNPDES permitting authorities traditionally measure
compliance with effluent limitations at the outfall point of
discharge. By issuing this policy statement, EPA does not intend
to disturb this well-established permitting practice. Permitting
authorities are familiar with procedures for determining the
concentration of toxicity in-stream and establishing end-of-pipe
effluent limitations on the basis of the information.
4

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and apply the more stringent of the acute criterion at the edge
of the acute mixing zone or the chronic criterion at the edge of
the chronic mixing zone in developing" WET effluent limitations.
If there is uncertainty as to which of the two criterion so
applied is more stringent for the discharge, however, the
permitting authority will apply both.
The statement reflects the specific requirement of 40 C.F.R.
§ 122.44(d)(1)(ii) that "where appropriate, [the permitting
authority will consider] the dilution of the effluent in the
receiving water" in determining whether a discharge causes, has
the reasonable potential to cause, or contributes to exceedance
of WET water quality criteria. This statement should assist
permitting authorities in establishing WET controls which meet
the requirements of sections 301(b)(1)(C) and 402 of the Clean
Water Act (CWA) and 40 C.F.R. S 122.44(d)(1).
40 C.F.R. S 122.44(d)(l)(iv) and (v) require the permitting
authority to impose effluent limitations to control WET where it
determines that a discharge causes, has the reasonable potential
to cause, or contributes to exceedance of WET water quality
criteria. 40 C.F.R. S 122.44(d)(1)(vii) also requires permitting
authorities to establish effluent limitations on point sources
which are consistent with the requirements of applicable State
water quality standards. This is a basic premise of this policy
statement. Where the applicable State water quality standard or
implementation procedure requires a different basis for
establishing WET controls, the permitting authority must follow
applicable State requirements.6
The second component of the policy statement also reflects
the principle of section 301(b)(1)(C) of the CWA that effluent
limitations must assure compliance with all State water quality
standards. Here, the permitting authority will establish WET
controls for the particular discharge based upon the more
stringent of the acute or chronic criterion (or both) applied at
the edge of their respective mixing zones in order to achieve
both criteria.
Consistent with this policy statement, the permitting
authority will establish two independent zones for controlling
6For example, some State water quality standards or
implementation procedures do not allow mixing zones at all or
restrict mixing zone use for certain dischargers. Where this is
the case, the permitting authority will not use the procedure
provided in policy statement one concerning the application of
mixing zones. The permitting authority must still ensure that
the permit includes WET limitations as necessary to achieve the
applicable State requirements.
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acute and chronic WET.7 The first zone, the acute mixing zone,
immediately surrounds the discharge outfall. The acute mixing
zone is normally sized to prevent lethality (sometimes also
described as "acute effects") to passing organisms. The permit
must include effluent limitations as necessary to meet numeric or
narrative water quality criteria for acute toxicity at the edge
of the acute mixing zone. The second zone, the chronic mixing
zone, is typically a larger zone which surrounds the acute mixing
zone. The chronic mixing zone is normally sized to protect the
ecology of the water body as a whole from all point-source
related stresses including WET. The permit must include effluent
limitations as necessary to meet numeric or narrative water
quality criteria for chronic toxicity at the edge of the chronic
mixing zone.1
Once it is determined what the appropriate mixing zones are,
the permitting authority will take several additional steps
consistent with this policy statement. The permitting authority
will (l) evaluate the receiving water concentration of acute WET
at the edge of the acute mixing zone and of chronic WET at the
edge of the chronic mixing zone for the particular discharge, (2)
determine which of the acute criterion or the chronic criterion
applied at the edge of the appropriate mixing zone is the more
stringent of the two for the particular discharge, and (3)
establish effluent limitations to assure attainment of the more
stringent criterion (or both where it is unclear which is more
stringent). The Technical Support Document for Water Quality-
based Toxics Control, as revised in March 1991 (EPA/505/2-90-001)
(the TSD) at 3.3 and 5.4, illustrates how to apply this procedure
7This policy does not address what is acute or chronic WET.
40 C.F.R. § 122.2 defines "whole effluent toxicity." Appendix
Two, which provides an overview of the water quality standards
process and WET, describes traditional acute and chronic toxicity
tests and EPA's recommended magnitudes for acute and chronic WET.
States may interpret narrative water quality criteria for
toxicity in State implementation procedures. In the absence of
such implementation procedures, EPA's recommended magnitudes for
WET are .3 acute toxic unit and 1.0 chronic toxic unit at the
edge of the appropriate mixing zone. Technical Support Document
for Water Oualitv-based Toxics Control, as revised in March 1991
(EPA/505/2-90-001), at 2.3.3 and 2.3.4 (the TSD1.
*The implementation of this policy requires permitting
authorities to establish mixing zones unless State standards or
implementation procedures direct otherwise; however, the specific
size of a particular mixing zone depends on a variety of factors
which can also be specified in the State water quality standard
or implementation procedure. See generally the Water Quality
Standards Handbook at 2-7 (1983); the TSD at 2.2.2, for
discussions of this issue.
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to evaluate a particular discharge for reasonable potential and
to develop effluent limitations.
2. Evaluation of Dischargers for Reasonable Potential
At a minibub, the permitting authority should review all
major dischargers for reasonable potential to cause or
contribute to exceedance of VET water quality criteria.
40 C.F.R. S5 122.44(d)(1)(iv) and (v) require permitting
authorities to impose effluent limitations to control WET
whenever a discharge causes, has the reasonable potential to
cause, or contributes to an instream excursion of applicable
water quality criteria.9 This policy statement identifies which
dischargers the permitting authority should, as a first priority,
assess for reasonable potential.10
The group of dischargers which the permitting authority
should evaluate first for reasonable potential are "major"
facilities. EPA defines a major POTW as a POTW having a design
flow of one million gallons per day or greater, a service
population of 10,000 or greater, or a significant impact on water
quality. EPA identifies a major industrial discharger on the
basis of a combination of factors, including size, toxic
pollutant potential, and stream flow volume." EPA believes that
these facilities (either POTWs or industrial facilities) have the
greatest opportunity for impacting surface water quality and
therefore should be evaluated for "reasonable potential" to
exceed an applicable State water quality standard.
Permitting authorities should continue to evaluate other
dischargers of concern for reasonable potential to exceed WET
water quality criteria. Factors which permitting authorities may
consider in deciding whether a particular discharge is "of
'Throughout this policy, any reference to "reasonable
potential" includes both reasonable potential to cause and
reasonable potential to contribute to an excursion of numeric
water quality criteria for WET or narrative water quality
criteria.
,0This policy statement continues to reflect EPA's position
on this matter articulated in the January 25, 1989, memorandum of
Rebecca W. Hanmer, Acting Assistant Administrator for Water, to
Regional Administrators entitled "Whole Effluent Toxicity Basic
Permitting Principles and Enforcement Strategy."
"See the June 27, 1990, memorandum "New NPDES Non-Municipal
Permit Rating System" from James R. Elder, Director of the Office
of Water Enforcement and Permits, to Regional Water Management
Division Directors, which is Appendix Three to today's policy.
7

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concern" obviously would include those factors which are
described in Chapter 3 of the TSD as factors for assessing
reasonable potential (including WET data, chemical-specific data,
instream survey data, industry or publicly owned treatment work
type, compliance history, receiving water type,
designated/existing uses, and dilution calculations). Under
§ 122.44(d)(1)(iv) and (v), permitting authorities must impose
effluent limitations to control WET where reasonable potential is
established. In addition, the permitting authority should
consider WET controls, where appropriate, in issuing general
permits.
3. Evaluating Reasonable Potential
The permitting authority will consider available WET
testing data and other information in evaluating
whether a discharger has reasonable potential to cause
or contribute to exceedance of VET water quality
criteria.
This policy statement describes vhat information is
considered in evaluating whether a specific discharger has the
reasonable potential to cause or contribute to excursion of WET
water quality criteria. The permitting authority first
determines whether valid WET testing data is available that is
relevant to the particular discharge.11 Whole effluent toxicity
data may be available from previous monitoring. Additionally,
under 40 C.F.R. § 122.21(j), certain POTWs are required to submit
WET testing as part of the permit application. The permitting
authority may also decide to require the permittee to generate
WET data prior to permit issuance or as a condition of the
permit. See policy statement five below. If valid WET testing
data is available that is relevant to the particular discharge,
the permitting authority uses this data to determine if the
discharge exhibits reasonable potential under §§ 122.44(d)(l)(iv)
or (v).13 Where such WET data exist and demonstrate reasonable
potential, the permitting authority does not need to gather or
>2The permitting authority determines whether available WET
testing is valid and addresses concerns relative to toxicity for
the particular discharge. For example, where a facility
discharges to a low flow stream, submission of acute WET testing
data showing no toxicity is insufficient (absent conversion of
the acute results to chronic results using an acute-to-chronic
ratio, as explained in the TSD) to assess reasonable potential
for chronic toxicity.
"if additional factors also demonstrate reasonable potential
(see main text discussion below), the permitting authority should
also discuss these factors in the fact sheet or statement of
basis accompanying the permit.
8

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generate other information to verify or support the WET results.
EPA believes it is appropriate to assess reasonable potential on
the basis of WET testing. Whole effluent toxicity testing is
comparable in precision to chemical analytical measurements in
wide use. See discussions of these questions in 55 Fed. Reg.
30082, 30112-30115 (July 24, 1990); 54 Fed. Reg. 23858, 23874
(June 2, 1989); the TSD at 1.3 and 3.3.
The permitting authority should also consider whether other
factors establish reasonable potential for the discharge. The
TSD at 3.2 offers a discussion of factors other than facility-
specific WET monitoring data which a permitting authority may
consider in making a reasonable potential determination for a
particular discharge. These factors include 1) industry type
(primary, secondary, raw materials used, products produced, best
management practices, control equipment, treatment efficiencies,
etc.), 2) publicly owned treatment work type (pretreatment,
industrial loadings, number of taps, unit processes, treatment
efficiencies, chlorination/ammonia problems, etc.), 3) compliance
history, 4) existing chemical data from discharge monitoring
reports and applications, 5) available instream survey data, 6)
receiving water type and designated/existing uses, 7) available
dilution, etc. For each individual permit, the permitting
authority must include a clear explanation in the statement of
basis or fact sheet accompanying the permit of the specific
factors considered in evaluating reasonable potential for the
particular discharge.
EPA believes this approach to assessing reasonable potential
implements the requirements of sections 301(b)(1)(C) and 402 of
the CWA and 40 C.F.R. S 122.44(d)(1). 40 C.F.R.
§ 122.44(d)(1)(ii), (iv), and (v) require the permitting
authority to use valid procedures which account for at least the
following four factors in establishing whether a discharge
causes, has the reasonable potential to cause, or contributes to
an exceedance of WET water quality criteria: (1) existing
controls on point and nonpoint sources of pollution, (2) the
variability of the pollutant or pollutant parameter in the
effluent, (3) the sensitivity of the test species when evaluating
WET, and (4) the dilution of the effluent in the receiving water
where appropriate. 40 C.F.R. S 122.44(d)(1)(v) also explicitly
provides that the permitting authority must establish an effluent
limitation to control WET where it determines, using "toxicity
testing data, or other information," that the discharge causes,
has the reasonable potential to cause, or contributes to an
exceedance of a narrative water quality criterion.
4. Consequences of Establishing Reasonable Potential
Upon finding reasonable potential to cause or
contribute to exceedance of WET water quality criteria,
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the permitting authority will impose efflueat
limitations to control WET.
This policy statement reiterates the requirements of
sections 301(b)(l)(C) and 402 of the CWA as well as 40 c F r
§§ 122.44(d)(1)(iv) and (V). 40 C.F.R. §§ 122.44(d)(l)(iv) and
(V) require the permitting authority to establish effluent
limitations in a permit to control WET where it determines that a
discharge has the reasonable potential to cause or contribute to
an mstream excursion above a numeric criterion for WET or a
narrative criterion.14
The permitting authority can either modify the permit or
reissue the permit upon expiration, as appropriate, to
incorporate effluent limitations to control WET. In no instance
will the permitting authority reissue the permit without
including appropriate effluent limitations to control WET In
appropriate cases, the permitting authority may also reouire the
discharger to conduct a toxicity identification
eyaluation/toxicity reduction evaluation to identify and
eliminate the cause of the toxicity as part of a compliance
schedule to comply with effluent limitations to control WET.
5- Whole Effluent Tonicity Monitoring
Where appropriate, the permitting authority should impose
WET monitoring conditions upon dischargers that do not have
effluent limitations to control WET.
WFT ™?!:e appropriate' the permitting authority should impose
ET monitoring conditions upon those dischargers for which it
did not determine reasonable potential and did not impose
effluent limitations to control WET. Where the permitting
authority concludes that a continued monitoring requirement is
warranted based upon the particular circumstances^^
discharger, the permitting authority should require WET
monitoring for a reasonable period of time and evaluate the
monitoring results at the conclusion of this period."
"Paragraph (v) provides that where the permitting authority
determines that a discharge causes, has the reasonable potential
to cause, or contributes to an instream excursion above a
watfr quality criterion, the permit must contain (l) a
liUent	or (2) a pollutant-specific limitation,
where the permitting authority demonstrates that a pollutant-
specific limitation is sufficient to attain and maintain
applicable numeric and narrative water quality standards.
"40 C.F.R. s 122.21(j) requires many new and existing POTWs
to collect WET data for submission to the permitting authority at
time of application or reapplication for an NPDES-permit. Where
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EPA and authorized NPDES States have broad authority under
the CWA to require continued monitoring to assure attainment of
water quality criteria. Under sections 308 and 402 of the CWA,
EPA or a State with an authorized NPDES program can require NPDES
permittees to provide WET testing data to assure State water
quality standards will be attained and maintained. The
permitting authority can impose a requirement on the discharger
to collect monitoring data through conditions in the NPDES permit
or through CWA section 308 letters. Under sections 301(b)(l)(C)
and 402 of the CWA as well as 40 C.F.R. §§ 122.44(d)(1)(iv) and
(v), EPA or a State with an authorized NPDES program must impose
effluent limitations to control WET where continued monitoring
results in a determination of reasonable potential to exceed WET
water quality criteria.
6. Compliance Schedules in NPDES Permits
Where allowed under state and federal lav, NPDES permits may
contain schedules for compliance with WET effluent
limitations.
This policy statement reflects the principles for allowing
compliance schedules in NPDES permits which were articulated in
In re star-Kist Caribe. Inc.. NPDES Appeal No. 88-5 (May 26,
1992) (order denying modification request).16 Section
301(b)(l)(C) of the CWA establishes a deadline of no later than
July 1, 1977, for compliance with effluent limitations developed
to meet State water quality standards. In light of this CWA
provision, EPA has determined that all permits must reflect this
deadline, unless the following requirements are met.17 NPDES
permits may contain schedules of compliance beyond July 1, 1977,
to meet water quality-based effluent limitations if two
requirements are met. The two requirements are: 1) the permit
appropriate, the permitting authority may, in its discretion,
require more frequent WET monitoring of POTWs or industrial
dischargers. For example, it may be appropriate to impose a
continued WET monitoring obligation upon a major, industrial
discharger for which WET testing data is not available.
Similarly, it may be appropriate to impose a monitoring
obligation upon a discharger for which available WET data is
limited or for which later information raises the possibility of
reasonable potential.
"40 C.F.R. S 122.2 defines a "schedule of compliance" as a
"schedule of remedial measures included in a 'permit', including
an enforceable sequence of interim requirements . . . leading to
compliance with the CWA and regulations."
"This entire discussion does not apply to permit limitations
which are governed by section 304(1) of the CWA.
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effluent limitation must be based either on a post-July l, 1977
State water quality standard or a new or revised interpretation
of a pre-July l, 1977 State water quality standard; and (2) the
applicable State water quality standard or implementing
regulations must explicitly authorize schedules of compliance.
40 C.F.R. § 122.47 also governs compliance schedules in
NPDES permits. The regulation authorizes, where appropriate,
schedules requiring compliance with effluent limitations as soon
as possible and no later than the applicable CWA statutory
deadline. The regulation imposes certain restrictions on
allowing schedules of compliance for new sources, new
dischargers, and recommencing dischargers. The regulation
establishes requirements for interim dates for certain schedules
of compliance and for permittee reporting. Any compliance
schedules developed for WET limitations must also satisfy
§ 122.47, if applicable.
Thus, to decide whether to allow a compliance schedule in an
NPDES permit for effluent limitations to control WET, the
permitting authority must answer these questions:
1.	Was the applicable State water quality criterion
promulgated or interpreted after July 1, 1977?
At this time, most permitting authorities
establish effluent limitations to control WET on the
basis of state narrative water quality criteria. Most
State narrative water quality criteria for toxicity
were adopted before July 1, 1977. Where this is the
case, the permitting authority can only allow a
schedule of compliance in the NPDES permit where the
State has made a new or revised interpretation of the
applicable narrative water quality criterion after
July 1, 1977. Where the permitting authority establishes an
effluent limitation to control WET on the basis of a State
numeric water quality criterion for WET, it is more likely
that the criterion is a post-July l, 1977 criterion.
2.	Do the State water quality standards or implementing
regulations explicitly authorize schedules of compliance?
The State must include an explicit statement
authorizing compliance schedules in the State water quality
standard or implementing regulations. If the State water
quality standard or implementing regulations are silent on
whether schedules of compliance are authorized for NPDES
permits, the permitting authority cannot place a schedule of
compliance in the NPDES permit. Permit writers may find the
express authorization in the State statute or water quality
standards, water quality planning, or NPDES regulations.
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3. Do other relevant provisions of State or federal lav or
policy allov the schedule of compliance?
Here, for example, the permitting authority should
consider whether allowing a schedule of compliance for the
specific discharge meets the requirements of 40 C.F.R.
§ 122.47, if applicable, or any other requirements of State
law.
Where the permitting authority answers yes to each of these
questions, it may allow a schedule of compliance in the NPDES
permit.
The permitting authority, however, is not compelled to
establish a schedule of compliance in the NPDES permit where so
authorized. The permitting authority should impose a schedule of
compliance only where appropriate under the specific conditions
of discharge. It has long been EPA's policy that EPA and
authorized NPDES States should require compliance with State
water quality standards as soon as possible in order to further
the goals of the CWA.
7- Whole Effluent Toxicity Controls and the Pollutants Ammonia
and Chlorine
The requirements of the vater quality permitting regulations
apply without regard to the pollutant(s) that may be causing
toxicity, including ammonia and chlorine.
This policy statement is designed to address several
questions which permitting authorities have encountered in
establishing WET controls where ammonia or chlorine is the
primary cause of toxicity. The questions typically arise on the
following set of facts: A permittee discharges to a stream for
which the State has not established numeric water quality
criteria for ammonia or chlorine; the State has adopted a
narrative water quality criterion for toxicity which is
applicable to the stream; the permittee conducts WET monitoring;
and the permittee exceeds the narrative criterion due to ammonia
or chlorine. The permitting authority must answer several
questions in permitting this discharge: What effluent
limitations must it establish in the permit? Will the permitting
authority require the permittee to control ammonia or chlorine,
so that the permittee does not exceed the narrative criterion due
to ammonia or chlorine? Is there a basis to treat ammonia or
chlorine differently from other pollutants in applying
§ 122.44(d)(1) to these facts?
The requirements of § 122.44(d)(1) apply to all pollutants
and pollutant parameters, including ammonia and chlorine. The
Agency sees no basis upon which to treat WET due to ammonia or
chlorine differently from WET due to other pollutants in applying
13

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the requirements of 40 C.F.R. § 122.44(d)(1) or other provisions
of federal law." Several provisions of 40 C.F.R. § 122.44(d)(1)
provide the answers to the questions posed in this policy
statement.
40 C.F.R. § 122.44(d)(l)(v) requires the permitting
authority to establish effluent limitations to control WET which
causes, has the reasonable potential to cause, or contributes to
an excursion above a State narrative water quality criterion.
Under § 122.44(d)(1)(v), the permitting authority must establish
either a WET effluent limitation designed to meet the narrative
criterion or a pollutant-specific effluent limitation where the
permitting authority is satisfied it will assure compliance with
applicable narrative and numeric criteria to control the WET due
to ammonia or chlorine. 40 C.F.R. S 122.44(d)(1)(vi) allows the
permitting authority to use one of three options for developing
effluent limitations for a pollutant determined to cause an
exceedance of a State narrative criterion where the State has
developed no numeric water quality criterion for the specific
pollutant. These options are (l) establish an effluent
limitation using a calculated numeric water quality criterion
which will .attain and maintain the applicable narrative criteria
and fully protect the designated use; (2) establish an effluent
limitation on a case-by-case basis using EPA criteria
supplemented by other relevant information where necessary; and
(3) establish an effluent limitation for an indicator parameter
provided certain factors are established.
8- Whole Effluent Toxicity Controls and POTWs
The requirements of the water quality permitting
regulations apply to all dischargers, including POTWs.
This policy statement reaffirms EPA's longstanding policy of
treating	on an equal basis in imposing and
enforcing effluent limitations to control all pollutants and
pollutant parameters. Under sections 301(b)(1)(C) and 402 of the
*This policy statement does not mean that permitting
authorities may disregard technical factors which are unique to
ammonia and chlorine in implementing regulatory requirements.
For example, holding pH constant in the laboratory during a WET
test may be necessary to assure a representative WET sample,
where WET is due to ammonia. The permitting authority may also
use judgment in interpreting testing results and setting limits
where temperature has a significant impact upon WET, which may be
the case for ammonia discharges during winter. In addition, the
permitting authority may require WET testing prior to
chlorination if a facility is under a schedule to dechlorinate.
Once dechlorination is implemented, then WET testing should be
conducted on the final effluent.
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CWA as well as 40 C.F.R. § 122.44(d)(1), all dischargers must
meet effluent limitations designed to attain and maintain
applicable State water quality standards. Under the current
NPDES program, EPA exercises enforcement discretion where
appropriate for particular violations of effluent limitations
designed to meet State water quality standards, and provides
technical guidance and support to dischargers in seeking
solutions to water quality-permitting and compliance problems.
See Attachment 2 to the January 25, 1989, memorandum from Rebecca
w. Hanmer, Acting Assistant Administrator for Water, to EPA
Regional Administrators entitled "Whole Effluent Toxicity Basic
Permitting Principles and Enforcement Strategy." EPA's
Enforcement Management System for the National Pollutant
Discharge Elimination System (1989) also discusses how EPA
exercises such enforcement discretion.
Authorized NPDES States and EPA will work with
municipalities (as well as other permittees) to solve water
quality-related problems, including those resulting from WET. In
1991, EPA published the revised 2SE, which provides comprehensive
technical guidance for assessing and regulating the discharge of
toxic substances to the waters of the United States. In 1989,
the Agency published a specific toxicity reduction manual for the
municipal discharger, the Toxicity Reduction Evaluation Protocol
for Municipal Wastewater Treatment Plants (EPA 600/2-88/062 April
1989). Since 1985, EPA's National Effluent Toxicity Assessment
Center in Duluth, Minnesota has provided assistance to regulatory
authorities in connection with dischargers that have complex
toxicity problems. EPA has published the Toxicity Identification
Evaluation; Characterization of Chronically Toxic Effluents.
Phase I (EPA-600/6-91/005F) (May 1992), a guidance document
designed to assist dischargers and their consultant laboratories
in conducting chronic aquatic toxicity identification
evaluations.
CONCLUSION
The fundamental premises of today's policy are not new.
Because of the importance of WET controls, however, EPA is taking
this opportunity to reiterate key principles associated with
implementation of existing statutory and regulatory requirements
for WET. The publication of this policy is designed to foster
consistent nationwide compliance with existing statutory and
regulatory provisions for the control of WET, to facilitate the
development of appropriate controls for WET in NPDES permits, and
to help assure attainment of water quality standards throughout
the nation.
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APPENDIX ONE
HISTORY OF FEDERAL REGULATION OF WHOLE EFFLUENT TOXICITY fWET)
Since 1984, the Environmental Protection Agency, the States
and the regulated community have employed an integrated strategy'
consisting of both biological and chemical means to control toxic
effects upon water quality beyond Clean Water Act (CWA)
technology-based requirements in order to achieve and maintain
State water quality standards. One method for measuring the
biological effects of toxic effluents upon aquatic life is WET
testing. EPA and the States have used the data derived from WET
testing to assess compliance with State water quality standards
and to establish National Pollutant Discharge Elimination System
(NPDES) permit effluent limitations necessary to attain and
maintain those standards.
In the past ten years, the Agency has published regulations,
policy statements, and guidance documents which address a variety
of issues associated with WET controls in NPDES permits. In
1984, EPA issued the "Policy for the Development of Water
Quality-Based Permit Limitations for Toxic Pollutants" (published
at 49 Fed. Reg. 9016 (March 9, 1984)). This policy addresses the
use of biological and chemical methods to assure that toxic whole
effluent discharges are regulated consistent with federal and
State requirements. The document discusses such specific issues
as integration of chemical and biological approaches; chemical,
physical, and biological testing requirements (WET requirements);
use of data; setting of effluent limitations; and monitoring.
On January 25, 1989, Rebecca W. Hanmer, Acting Assistant
Administrator for Water, sent to EPA Regional Administrators the
memorandum entitled "Whole Effluent Toxicity Basic Permitting
Principles and Enforcement Strategy." A group of Regional and
State representatives developed the strategy, which discussed the
minimum acceptable national requirements for WET permitting.
In 1989, the Agency revised existing 40 C.F.R.
§ 122.44(d)(1), which previously required NPDES permits to
contain any more stringent requirements necessary to achieve
State water quality standards. Seg 54 Fed. Reg. 23868
(June 2, 1989). The revised regulation described in greater
detail requirements for NPDES permitting authorities to follow in
developing NPDES effluent limitations to assure compliance with
State water quality standards. On August 14, 1992, Michael B.
Cook, Director, Office of Wastewater Enforcement and Compliance,
and Robert H. Wayland, III, Director, Office of Wetlands, Oceans
and Watersheds, transmitted a memorandum to the Water Management
Division Directors, Regions I-X, entitled "Clarifications
Regarding Certain Aspects of EPA's Surface Water Toxics Control
Regulations." (See Appendix Four.) In 1990, EPA also published
new 40 C.F.R. § 122.21(j), which established a requirement for
1

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r£& of'valid	«>•
See 55 Fed. Reg. 30082 (July 24 i9Qrn ™°nS r NPDES Permits,
regulatory revisions and the 1984 noli™ preambles to these
explanation of leoal and	- policy provide a detailed
effluent lim^atfS. * * SUPPOrt for WET testi"9 and
documents which' adcSessTthe subiec^o?6^**10118 9111(14006
control, including WET control See'°fi^ef. ?uaJity-based toxics
Technic*i support^f •'"Particular, the
Contra^	nrigiiuii/ ^1^1.!!" ^ qed Toyl°g
revised in March 1991 (EPA/505/2^90-001*" ®ePteaber 1985 and was
as^„vsasai3H,r^?^« ~
quality-based pemit li^itaSons teS^K'SE ^SSSf.
Biological11Assessments Sd SKiftaTS1?*" «» <*
Program.w m discussing inSSSSn ^^ ^ater Quality
methods, the 1991 policy refill»««*.various assessment
application." in essence "indona^ ! °n "independent
appropriate regulatory action shSSd®*!	means that
biosurvey, pollutant-specific or	whf" »"/ one of
that an applicable water gualitJ s^Sd^f
2

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APPENDIX TWO
BACKGROUND MATERIALS ON WHOLE EFFLUENT TOXICITY fWETl TESTING.
THE STATE WATER QUALITY STANDARDS PROCESS AND WET. FEDERAL
STATUTORY AND REGULATORY REQUIREMENTS FOR WET. AND WET REDUCTION
GUIDANCE
Overview of WET Testing for
Aquatic Life Protection
The WET approach to toxics control for the protection of
aquatic life involves the use of acute and chronic WET testing to
measure the toxicity of wastewaters. Whole effluent toxicity
tests typically use standardized, surrogate freshwater or marine
plants, vertebrates, or invertebrates to measure the aggregate
toxic effect of an effluent. An acute WET test is typically a
test of 96-hours or less in duration in which lethality is the
measured endpoint. A chronic WET test is typically a longer-term
test in which sublethal effects, such as fertilization, growth,
and reproduction can be measured in addition to lethality. On
December 4, 1989, EPA published proposed Part 136 methods for
conducting short-term acute and chronic WET testing for marine
and freshwater species. See 54 Fed. Reg. 50216. Once these
methods are final, they will constitute approved Part 136 test
methods for the NPDES program.
Overview of the State Water Quality Standards Process and WET
Section 303 of the Clean Water Act (CWA) establishes the
statutory basis for the current State water quality standards
program. Under this provision of the CWA, States bear primary
responsibility for adopting water quality standards. State water
quality standards represent the means by which EPA and authorized
NPDES States control point source discharges when technology-
based controls for point source discharges are inadequate.
A water quality standard defines the water quality of a
water body by designating the uses to be made of the water, by
setting criteria necessary to protect the uses, and by
establishing antidegradation policies and implementation
procedures that serve to maintain and protect water quality. See
section 303(c) of the CWA and 40 C.F.R. Part 131. States adopt
water quality standards to protect public health or welfare,
enhance the quality of the water, and serve the purposes of the
CWA. Among other requirements, State water quality criteria must
protect aquatic life.
Under section 303 of the CWA and 40 C.F.R. Part 131, EPA
must approve State water quality standards or disapprove State
water quality standards and overpromulgate with federal water
quality standards. State water quality standards are effective
until EPA overpromulgates with federal standards. Once
1

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quality standardfor^he^tate"3 are the applIcable uater
numeric concentrations^f^iiutants^r^IuStant'1 aS constituent
as narrative statements represwtiM a	J Parameters or
supports a particular use ThiTiS^r^X °f "?ter that
parameter WET. Several stafoe	t^ue of the pollutant
WET. Most evaluation and control "SeTI? ^!r^fflcriJeria for
is based upon maintenance of the State's	% i ' however'
are statements of a desired water gualitv^oai t°xicity which
State waters must, at allti£es1md1io£ free'?,™
substances that are toxic to humans or aouaSe liJ® ?
considers narrative criteria to aoDlv	\ ****
all flows unless specified otherwise in th! i ?? uses at
standards. EPA regards narrative	*	? water quality
both short-term and long-term WET eff»S / r4.tOXlcity to cover
effects, respectively)?	effects (acute and chronic
dete™Jninr^r=pici£ichpoU«^ta«i^S *•**«¦«*• for
water quality stands?	attajj>
upon the failure of the staS mi iSi. I .tte State 
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frequency for WET- The duration and frequency of the discharae
may also be defined by the design stream flow appropriate to the
criterion. Magnitude is the maximum allowable concentration of
WET, which is typically expressed as a concentration of toxicity
mstream; duration is the period of time over which the instream
concentration is averaged for comparison with criteria
concentrations, in order to limit the durations of concentrations
above the criterion; and return frequency is a designation of how
often the criterion may be exceeded without impacting the
organisms in the water body. This information is needed because
ambient water quality typically varies in response to changes in
effluent quality, stream flow, and other factors. Accordingly
organisms in the receiving water typically experience fluctuating
exposure to pollutants, including some periods of exposure to
high pollutant concentrations, which may have adverse effects
For this reason, criteria indicate a time period over which
exposure is to be averaged, as well as a maximum concentration,
thereby linuting the duration of exposure to.elevated
concentrations. In addition, to predict or ascertain the
attainment of criteria it is necessary to specify the allowable
frequency for exceeding the WET criteria. The permitting
authority uses the magnitude, duration, and return frequency
provisions of WET criteria to develop wasteload allocations and
effluent limitations to control the WET of the discharge.
Whole effluent toxicity criteria as adopted by the State or
as implemented by the permitting authority on a case-by-case
basis typically consist of two expressions of magnitude of
toxicity to surrogate test species, one to protect against acute
toxicity effects and one to protect against chronic toxicity
effects, along with a duration and return frequency for each. As
of the publication date of this policy, EPA has not developed a
recommended section 304(a) criterion for WET. The Technical
Support Document for Water Qua!itv-basad Toxics Control . Uhlh
was originally published in September 1985 and was revised in
March 1991 (EPA/505/2-90—001) (the JSJ>), contains recommended
f?f-?n eS' ^ura^^-ons» and return frequencies for assessing acute
WET (.3 acute toxic unit to the most sensitive of at least three
species, with an averaging period of one hour and a once in
three-year return frequency) and chronic WET (1.0 chronic toxic
unit to the most sensitive of at least three species, with an
avera^in^ period of 4 days and a once in three—year return
frequency). See the ISfi at 2.3.3 to 2.3.5.
At their discretion, States may adopt certain policies for
implementation of water quality standards, such as critical low
flow and mixing zone policies. EPA has the authority to review
and approve or disapprove such policies. See 40 C.F.R. § 131.13.
Additionally, EPA and States may establish a Technical Agreement
describing procedures that will be used in developing TMDLs and
wasteload allocations. TMDLs, wasteload allocations, and permit
limitations developed consistent with Technical Agreements are
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subject to a lesser degree of EPA review. State water quality
standards protect water quality for designated uses in critical
low flow situations. Under § .131.13/ States may designate
critical low flows below which numeric water quality criteria do
not apply. Mixing zones are small areas in the receiving water
near certain discharge outfalls where ambient concentrations
above the otherwise applicable State water quality criteria are
allowed. See generally the Water Quality Standards HandhrvMr at
2-7 (1983)? the JSg at 2.2.2. Some States prohibit mixing zones
entirely for all pollutants or pollutant parameters. Others may
allow mixing zones in general, but provide spatial dimensions to
limit the areal extent of the mixing zones. Permitting
authorities may allow mixing zones on a case-by-case basis for
individual discharges. EPA strongly recommends that States have
a definitive statement in their water quality standards on
whether or not mixing zones are allowed and clearly explain the
procedures for defining mixing zones where allowed.
Overview of Federal Statutory and Regulatory RecmirPments fnr
Development of Water Quality-based Permit Limitations for WET
Section 101(a) of the CWA establishes a national policy of
restoring and maintaining the chemical, physical, and biological
integrity of the Nation's waters, in addition, section 101(a)(3)
of the CWA states the national policy that the discharge of toxic
pollutants in toxic amounts is prohibited. Under sections 301
and 402 of the CWA, point source dischargers must obtain an NPDES
permit before discharging into waters of the United States.
Under sections 301(b) (1) (C) and 402 of the CWA, dischargers with
NPDES permits must meet all of the technology-based requirements
of the CWA as well as any more stringent requirements necessary
to achieve State water quality standards established under
section 303 of the CWA.
Xn 40 C.F.R. § 122.2, EPA defines "whole effluent toxicity"
as "the aggregate toxic effect of an effluent measured directly
by a toxicity test." EPA has published 40 C.F.R. § 122.44(d)(1),
which describes requirements for NPDES permitting authorities to
follow in developing water quality-based effluent limitations,
including those necessary to control WET. The regulation
consists of seven subparagraphs:
1. Section 122.44fdlm fi)
"Limitations must control all pollutants or pollutant
parameters (either conventional, nonconventional, or
toxic pollutants) which the Director determines are or
may be discharged at a level which will cause, have the
reasonable potential to cause, or contribute to an
excursion above any State water quality standard,
including State narrative criteria for water quality."
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2. Section 122.44 fdMlWii)
"When determining whether a discharge causes, has the
reasonable potential to cause, or contributes to an in-
stream excursion above a narrative or numeric criteria
within a state water quality standard, the. permitting
authority shall use procedures which account for
existing controls on point and nonpoint sources of
pollution, the variability of the pollutant or
pollutant parameter in the effluent, the sensitivity of
the species to toxicity testing (when evaluating whole
effluent toxicity), and where appropriate, the dilution
of the effluent in the receiving water."
3.	Section 122.44(d)(1)fiiil
"When the permitting authority determines, using the
procedures in paragraph (d)(1)(ii) of this section,
that a discharges causes, has the reasonable potential
to cause, or contributes to an in-stream excursion
above the allowable ambient concentration of a State
numeric criteria within a State water quality.standard
for an individual pollutant, the permit must contain
effluent limits for that pollutant."
4.	section 122 .44 (d) (lWiv)
"When the permitting authority determines, using the
procedures in paragraph (d)(1)(ii) of this section,
that a discharges causes, has the reasonable potential
to cause, or contributes to an in-stream excursion
above the numeric criterion for whole effluent
toxicity, the permit must contain effluent limits for
whole effluent toxicity."
5.	Section 122.44(d) m fvl
"Except as provided in this subparagraph, when the
permitting authority determines, using .the procedures
in paragraph (d)(l)(ii) of this section, toxicity
testing data, or other information, that a discharge
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 permit must contain effluent limits for
whole effluent toxicity. Limits on whole effluent
toxicity are not necessary where the permitting
authority demonstrates in the fact sheet or statement
of basis of the NPDES permit, using the procedures in
paragraph (d)(1)(ii) of this section, that chemical-
specific limits for the effluent are sufficient to
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attain and maintain applicable numeric and narrative
State water quality standards."
6. Section 122.44 fdWIWvi 1
"Where a State has not established a water quality
criterion for a specific chemical pollutant that is
present in an effluent at a concentration that causes,
has the reasonable potential to cause, or contributes
to an excursion above a narrative criterion within an
applicable State water quality standard, the permitting
authority must establish effluent limits using one or
more of the following options:
(A)	Establish effluent limits using a calculated
numeric water quality criterion for the pollutant which
the permitting authority demonstrates will attain and
maintain applicable narrative water quality criteria
and will fully protect the designated use. Such a
criterion may be derived using a proposed State
criterion, or an explicit State policy or regulation
interpreting its narrative water quality criterion,
supplemented with other relevant information which may
include: EPA's Water Quality Standards Handbook,
October 1983, risk assessment data, exposure data,
information about the pollutant from the Food and Drug
Administration, and current EPA criteria documents; or
(B)	Establish effluent limits on a case-by-case
basis, using EPA's water quality criteria, published
under section 304(a) of the CWA, supplemented where
necessary by other relevant information; or
(C)	Establish effluent limitations on an indicator
parameter for the pollutant of concern, provided:
(1)	The permit identifies which pollutants are
intended to be controlled by the use of the effluent
limitation;
(2)	The fact sheet required by § 124.56 sets
forth the basis for the limit, including a finding that
compliance with the effluent limit on the indicator
parameter will result in controls on the pollutant of
concern which are sufficient to attain and maintain
applicable water quality standards;
(3)	The permit requires all effluent and ambient
monitoring necessary to show that during the term of
the permit the limit on the indicator parameter
continues to attain and maintain applicable water
quality standards; and
6

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(4) The permit contains a reopener clause
allowing the permitting authority to modify or revoke
and reissue the permit if the limits on the indicator
parameter no longer attain and maintain applicable
water quality standards."
7. Section 122.44 fdWlWvii)
"When developing water quality-based effluent limits
under this paragraph the permitting authority shall
ensure that:
(A)	The level of water quality to be achieved by
limits on point sources established under this
paragraph is derived from, and complies with all
applicable water quality standards; and
(B)	Effluent limits developed to protect a
narrative water quality criterion, a numeric water
quality criterion, or both, are consistent with the
assumptions and requirements of any available wasteload
allocation for the discharge prepared by the State and
approved by EPA pursuant to 40 CFR 130.7."
Overview of Toxicity Reduction Guidance
\
The purpose of a toxicity reduction evaluation (TRE),
including a toxicity identification evaluation (TIE), is to
investigate the causes and determine corrective actions for WET
problems. The permitting authority may require the permittee to
conduct these evaluations in specific cases. Section 5.8 of the
TSD contains a detailed discussion of EPA's recommended approach
for conducting TIEs and TREs, including a list of guidance
documents EPA has developed describing methods and procedures for
conducting TIEs and TREs.
7

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SETAC PELLSTON WET WORKSHOP SUMMARY AND CONCLUSIONS:
OBJECTIVES AND ORGANIZATION OF THE WORKSHOP
The objectives of the workshop were:
•	Evaluate the appropriateness of endpoints in the routinely used WET methods for
assessing effluent toxicity.
•	Evaluate the degree and causes of variability associated with routinely used methods
for assessing effluent toxicity.
•	Establish the scientific framework for assessing the nature of aquatic responses to
effluent, measuring field or community responses, and determining how biotic
characteristics influence measured field response(s) in refence and affected areas.
•	Critically examine the relationships between effluent toxicity, ambient toxicity, and
field bioassessments.
OVERALL WORKSHOP CONCLUSIONS
1 WET exposure methods are technically sound and require no immediate modifications.
2.	WET testing is an effective tool for predicting impact in lotic receiving systems. Additional
laboratory to field validation is not essential for the continued use of WET testing.
3.	The guidance provided in the U.S. EPA's Technical Support Document for Water Quality
Based Toxics Control must be followed closely to meet the objectives of the WET testing
program.
4.	A number of problems with WET tests are caused by misapplication of the tests,
misinterpretation of data, quality of the WET test laboratory, and the lack of training and
experience of laboratory personnel, regulators and permitees.
5.	Current WET permit limits have sufficient margins of safety so that episodic exceedences
should not cause receiving water impacts. The significance of an exceedence of WET limits
depends on receiving water conditions, especially dilution at the time of the exceedence, and
the duration of the toxic event.
6.	Variability in the use of both WET test methods and bioassessment techniques and influences
test interpretation and acceptability and the extrapolation of WET test results to field impacts.
7 The largest sources of variability in WET testing are the level of analyst expertise and
judgment and test organism conditio^ health. Deviation from established methods can be
controlled by an effective QA/QC program.
8. Currendy used statistical methods are widely used and accepted. However, improvements are
1

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available that should be considered.
9.	Biological assessment approaches, when properly designed, can accurately assess
environmental impact to aquatic biota.
10.	Bioassessments are needed to compensate for the limitations of WET tests to predict
phytotoxicity, sediment toxicity, bioaccumulation, genotoxicity, indirect biotic effects, and
effects of persistent chemicals.
11.	In addition to WET testing, results from in situ testing, ambient toxicity testing, and
bioassessments are useful to evaluate WET limits and margins of safety.
12.	The relationship between WET tests and receiving water impacts is based largely on animal
effects in streams. Minimal data exist describing the effect of effluent toxicity exposure in
wetlands, estuaries, and large rivers.
13.	Careful thought must be given to selecting appropriate reference conditions for field
assessments. Regional reference conditions strengthen assessments of receiving water impacts
and facilitate characterization of natural variation
14.	Effluent toxicity is one of several factors that can adversely impact biological communities
and is not always the major cause of observed community impacts.
2

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STATUTORY AND REGULATORY CONSIDERATIONS
A primary objective of the NPDES and water quality standards programs is to control the discharge
of toxics. The CWA and EPA regulations authorize and require the use of the "integrated strategy"
to achieve and maintain water quality standards. Relevant provisions that provide statutory authority
for using toxicity testing and WET limitations include the following:
° Section 101(a) of the CWA sets forth the "goal of restoring and maintaining the
chemical, physical, and biological integrity of the Nation's waters" and, at section
101(a)(3), prohibits "the discharge of toxic pollutants in toxic amounts."
° Section 502(15) of the CWA defines biomonitoring as the "determination of the
effects on aquatic life, including accumulation of pollutants in tissue, in receiving
waters due to the discharge of pollutants (A) by techniques and procedures, including
sampling of organisms representative of appropriate levels of the food chain
appropriate to the volume and physical, chemical, and biological characteristics of the
effluent, and (B) at appropriate frequencies and locations."
° Section 304(a)(8) requires EPA to "...develop and publish information on methods
for establishing and measuring water quality criteria for toxic pollutants on other bases
than pollutant-by-pollutant criteria, including biological monitoring and assessment
methods."
0 Section 303(c)(2)(B) requires States to adopt criteria for toxic pollutants and criteria
based on biological monitoring or assessment methods EPA develops. However,
"nothing in this section shall be construed to limit or delay the use of effluent
limitations or other permit conditions based on or involving biological monitoring or
assessment methods ."
0 Section 302(a) provides authority to EPA and the States to establish water quality-
based effluent limitations on discharges that interfere with the attainment or
maintenance of that water quality which shall assure protection of public health, public
water supplies, and the protection and propagation of a balanced population of
shellfish, fish and wildlife
° Sections 301(b)(1)(C) and 402 require that all NPDES permits must comply with
any more stringent limitations necessary to meet applicable water quality standards,
whether numeric or narrative. Section 301(b)(1)(C) states that "In order to carry out
the objective of this Act there shall be achieved...any more stringent limitations
including those necessary to meet water quality standards..., or required to implement
any applicable water quality standard...".
° Sections 308(a) and 402 provide authority to EPA or the State to require that
NPDES permittees/applicants use biological monitoring methods and provide chemical
toxicity and instream biological data when necessary for the establishment of effluent
limits, the detection of violations, or the assurance of compliance with water quality
standards Section 308(a) states "whenever required to carry out the objective of this
Act, including but not limited to (1) developmg or assisting in the development of any
effluent limitation (2) determining whether any person is in violation of any such

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effluent limitation ..(A) the
Administrator shall require the
owner or operator of any point
source to...(m) install, use, and
maintain such monitoring
equipment or methods (including
where appropriate, biological
monitoring methods) . "
0 Section 510 provides
authority for States to adopt or
enforce any standards or effluent
limitations for the discharge of
pollutants only on the condition
that such limitations or standards
are no less stringent than those
in effect under the CWA
REGULATOR YCONSIDER ATTONS
40 CFR Part 122.44(d)(l)(i) -
reflects EPA's water quality-
based approach
40 CFR Part 122.44(d)(1)(H) -
presents procedures for water
quality-based limits
considerations
40 CFR Part 122.44(d)(l)(iv) -
requires WET limits where WET
standards are exceeded
40 CFR Part 122.44(d)(l)(v) -
requires WET limits if the
narrative standard is exceeded
40 CFR Part 122.44(d)(l)(vii) -
requires permit conditions to
assure compliance with water
quality standards and WLAs
40 CFR Part 122.21(j) - requires
POTWs to submit biomonitonng
data with permit application
40 CFR Part 130.7 - requires
TMDLsusing specific pollutants
or biomonitonng approach
STATUTQRYCQNSIDERATTONS
0 Section 101(a) - states national goals
° Section 502(15) - defines biomonitonng
0 Section 304(a)(8) - develops biomonitonng
methods
° Section 303(c)(2)(B) -outlines biological
methods for standards
° Section 302(a) - requires effluent limits to
protect aquatic life
° Section 301(b)(1)(C) - requires limits
necessary to meet water quality standards
including narrative
° Section 308(a) -provides authority to
require permittees to use biological methods
0 Section 402 - sets out requirements of
NPDES permits program
o Section 510 - requires states to adopt
standards at least as stringent as those in
effect under the Act
A.AStatutory basis for WET controls
On May 26, 1989, the EPA Deputy Administrator
signed regulations that implemented section 304(1) of
the CWA (54 FR 23868, June 2, 1989). Commonly
referred to as the 304(1) regulations, these regulations
did more than implement section 304(1). While 40
CFR Parts 130.10 and 123.46 were modified
specifically for 304(1) requirements, 40 CFR Part
122.44(d) was modified to clarify and reinforce
EPA's existing regulations governing water quality-
based permitting. The following parts of 40 CFR
Part 122.44(d) pertain to the requirements for WET
limits in NPDES permits.
- 40 CFR Part 122.44(d)(l)(i) was expanded
to reflect EPA's approach to water quality-
based permitting, an approach that includes
all parameters (conventional,
nonconventional, and toxics) and all
applicable standards, both narrative and
numeric.
A.ARegulatory basis for WET controls
2

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-	40 CFR Part 122.44(d)(l)(n) discusses procedures to be used to determine if a discharge
causes, has a reasonable potential to cause, or contributes to an excursion of a water quality
standard. The procedures include consideration of four general factors: "...existing controls
on point and nonpoint sources .variability of the pollutant...in the effluent, the sensitivity of
the species to toxicity testing...and...the dilution of the effluent in the receiving stream."
-	40 CFR Part 122 44(d)(l)(iv) requires effluent limits for whole effluent toxicity when it has
been shown that a discharge causes, has a reasonable potential to cause, or contributes to an
excursion of a numeric WET criterion.
-	40 CFR Part 122.44(d)(l)(v) requires limits for WET when it has been shown that a
discharge causes, has a reasonable potential to cause, or contributes to an excursion of a
narrative WET criterion However, WET limits are not necessary if it can be demonstrated
satisfactorily that chemical specific limits are sufficient to maintain all applicable standards.
-	40 CFR Part 122.44(d)(l)(vn) requires that all permit limits and conditions assure
compliance with water quality standards and wasteload allocations.
The regulations described above were subsequently challenged and upheld. In the Natural Resources
Defense Council. Inc v EPA, court case, at 859 F.2d 156 (D.C. Cir., 1989), several issues with
regard to WET implementation were reviewed. The Court held that EPA has the authority to express
permit limitations in terms of toxicity as long as the limits reflect the appropriate requirements of the
CWA, as provided in 40 CFR 125 3(c)(4) [More detail on this case can be found in Appendix B-6
Of the TSD.]
In addition to the May 1989 changes to 40 CFR Part 122.44(d)(1), on July 3, 1990, the EPA
Administrator signed final regulations that modified the permit application regulations at (55 FR
30082, July 24, 1990) 40 CFR Part 122 21(j). This section now requires large publicly-owned
treatment works (POTWs) to provide the results of valid whole effluent biological toxicity testing with
their application for a permit. This requirement applies to the following POTWs:
-All POTWs with a design flow of greater than or equal to 1 MGD (major facilities)
-All POTWs with approved pretreatment programs or POTWs required to develop a
pretreatment program, and
-Any other POTW as determined by the State Director
Further regulations at 40 CFR Part 130 7 require total maximum daily loads (TMDLs) and wasteload
allocations (WLAs) be developed for water quality-limited stream segments. A pollutant-by-pollutant
or biomonitoring approach may be used to establish TMDLs.
3

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ACUTE AND CHRONIC
TOXICITY TEST
METHODS

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Module Objectives
History of method testing program
Define acute and chronic tests
Elements of a test method
Test precision of methods

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History of Testing Program
¦	Acute testing began in the 1950s
¦	Short-term testing began in the 1980s
Talking Points:
•The development of acute WET testing began in the 1950s.
• Development of the short-term chronic tests for freshwater
species began in 1980 and development of the saltwater
species began in 1983. Site studies to examine the
predictability of freshwater tests were initiated in 1981 and
1984 for saltwater tests. The site studies were designed to
determine if a reasonable correlation exists between WET test
results and receiving water impacts.

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Acute/Chronic Toxicity
¦	Acute toxicity
-	endpoint - mortality
-test duration - either 24, 48 or 96 hours
¦	Chronic toxicity
-	endpoint - growth, reproduction, and
mortality
-	test duration - 7 days or less
Talking Points:
•	Acute toxicity tests are used to determine the concentration of effluent or ambient water
that produces an adverse effect on a group of test organisms during either a 24, 48 or 96
hour exposure. The endpoint measured is mortality. In an acute toxicity test, an effluent
sample is collected, diluted and placed in test chamber with the chosen test species.
After 24, 48 or 96 hours, the number of live organisms remaining in each test
concentration and a control is recorded.
•	A chronic toxicity test is defined as a short-term test in which sublethal effects, such as
growth, length, reproduction, fertilization, or larval development are measured in addition
to sometimes mortality. The short-term chronic toxicity test methods developed range
from a 40 minute to 7 day exposure.
•	Another aspect to consider when testing for acute toxicity testing is whether the
permittee is currently conducting a chronic toxicity test which also includes a survival
endpoint, such as the Pimephales promelas 7 day growth and survival test. In this
situation, compliance with acute and chronic requirements can be jointly evaluated; the
chronic toxicity at the end of the 7 day test and acute toxicity at either 48 or 96 hours
into the 7 day test. This is also known as a "dual endpoint" test, this is an effective use
of both time and financial resources.
•	The chronic test methods that could be evaluated for both acute and chronic requirements
are the topsmelt, the silverside, the Pacific mysid and the Atlantic mysid. The chronic
water flea test method, Ceriodaphnia dubia, cannot be analyzed for both acute and
chronic requirements because the test design is not amenable to calculation of a lethal
concentration (LC50) value. However, if an NOEC endpoint is used, then this test can be
used for both acute/chronic toxicity.
•	The first decision for a permit writer to make in selecting the appropriate toxicity tests is
whether to measure acute or chronic effects. The next question to answer is whether to
test with freshwater or marine species. Once that decision has been made, the following
parameters should be considered when selecting the appropriate test species: taxonomic
diversity; type of facility and toxicants; and seasonal and temporal effects.

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Approved Test Methods
¦ EPA promulgated methods in Part 136
-	existing permits as cited in the permit
-	new permits must use the approved
methods
Talking Points:
• EPA has recently added new biological testing methods to the
list of approved and standardized analytical methods for testing
wastewater pollutants. This information was published in the
Federal Register as a final rulemaking (amendment) to the 40
CFR Part 136 analytical methods. This rule became effective
on November 15, 1995.
•The test methods standardized in this rule will replace
unapproved test methods for NPDES permits issued after the
effective date of this rule. Existing NPDES permits will not be
re-opened to include test methods from this rule, unless the
permitting authority wants to re-open existing permits.
However, the NPDES permittee may request to the permitting
authority to replace existing methods with the newly
promulgated methods or the west coast chronic marine
methods.
5

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Species Selection Decision Tree
Receiving Waterbody (% salinity)
Freshwater
T
TDS
T
Taxa Diversity
I
Toxicant of Concern
Marine
Salinity
TaxaDrver|it^j
Toxicant of Concern
Native Species
I
Species Availability
6

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Method Manuals Covers
¦
Health and safety
¦
Quality assurance
¦
Facilities, equipment, supplies
¦
Test organisms
¦
Dilution water
¦
Effluent sampling and handling
¦
Test endpoints
¦
Report preparation
¦
Test methods
Talking Points:
• Each test method covers a chapter on each of these topics.
The chronic and acute test method manuals are consistent in
requirements for the sections on health and safety; quality
assurance; facilities, equipment, supplies; dilution water;
effluent sampling and handling; and report preparation. The
sections on test organisms and test endpoints differ with the
different test species and endpoints used for the method. The
chapter on test endpoints is consistent among the test
methods for consistently of statistical analysis.

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Summary of Test Conditions
¦ Each method has recommended test
conditions:
-	test design; replicates, test
concentrations, chambers, volume
-	test duration and type
-temperature, light, DO, salinity
-	age of organisms, feeding,
-	dilution water
-	sample requirements
-	test acceptability criteria
Talking Points:
{insert an example summary tables here}

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Table 3. Summary of Test Conditions and Test Acceptability Cntena for Daphnid,
Cenodaphnia Dubia, Survival and Reproduction Toxicity Tests with Effluents
and Receiving Waters
1
Test type
Static renewal
2
Temperature ( C)
25 ± 1 C
3
Light quality
Ambient laboratory illumination
4
Light intensity
10-20 E/m2/s. or 50-100 ft-c (ambient laboratory levels)
5
Photopenod
16 h light 8 h dark
6
Test chamber size
30 mL (minimum)
7
Test solution volume
15 mL (minimum)
8
Renewal of test


solutions
Daily
9
Age of test organisms
Less than 24 h and all released within a 8-h period
10
Number neonates per


test chamber
1
11
Number replicate test


chambers per


concentration
10
12
Number Neonates per


test concentration
10
13
Feeding regime
Feed 0 1 mL each of YCT and algal suspension per test chamber daily
14
Cleaning
Use freshly cleaned glass beakers or new plastic cups daily
9

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Table 3 Summary of Test Conditions and Test Acceptability Criteria for Daphnid,

Cenodaphnia Dubia, Survival and Reproduction Toxicity Tests with Effluents

and Receiving Waters (continued)
15
Aeration
None
16
Dilution water
Uncontammated source of receiving or other natural water, synthetic
water prepared using MILLIPORE MILLhQ® or equivalent deiomzed
water and reagent grade chemicals or DMW (see Section 7,

Oil ut ion
Water)
17
Test concentrations
Effluents Minimum of 5 and a control Receiving Water 100%
receiving water or minimum of 5 and a control
18
Dilution factor
Effluents 0 5
Receiving Waters None or 0 5
19
Test duration
Until 60% of surviving control organisms have three broods (maximum
test duration 8 days)
20
End points
Survival and reproduction
21
Test acceptability criteria
80% or greater survival and an average of 15 or more young per
surviving female in the control solutions 60% of surviving control
organisms must produce three broods
22
Sampling requirements
For on-site tests, samples collected daily, and used within 24 h of the
time they are removed from the sampling device For off-site tests, a
minimum of three samples collected on days one, three, and five with

a
maximum holding time of 36 h before first use (see Section 8, Effluent
and Receiving Water Sampling. Sample Handling, and Sample
Preparaton for Toxicity Tests. Subsection 8 5 4)
23
Samp volume
1 LVdoy

10

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Clarification Regarding
Flexibility in Test Methods
¦ Tudor Davies Memo dated April 10,
1996 addresses:
-	pH and ammonia control
-	temperature
-	hardness
-test dilution concentrations
-	acceptance criteria for Champia parvula
Talking Points:
•	Purpose of this memo is to provide clarifications regarding the
flexibility in the regulations regarding promulgated at 60
Federal Register 53529 (October 16, 1995), which establish
analytical test methods for the determination of WET test
methods (see attached memo).
•The toxicity test methods use discretionary terms such as
"may" or "should" with the understanding that the lab analyst
must have flexibility to optimize successful test completion. In
other instances, the manuals use stronger, compulsory terms
such as "shall" or "must" to prescribe procedures necessary
for nationwide standardization.
•	In the majority of these instances, we anticipate that silence in
the manual will indicate flexibility for the individual analyst
such as pH and ammonia control, temperature, hardness, test
dilution concentrations and acceptance criteria for Champia
parvula. See the attached Tudor Davies memo.
11

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Acute Test Methods [EPA/608/4-90-027F]

Receiving
Water Type
Species
Toxicant
{including, but
not limited to)
Salinity Range of Effluent
Fish
Freshwater
Fathead minnow,
Pimephales promelas
ammonia
1-6%

Freshwater
Rainbow trout,
Oncorhynchus mykiss
ammonia
1-2%

Marine
Silverside, Menidia
beryflina
ammonia
1-36%
Note: Can be used at end of pipe
testing, if the effluent is J> 5%

Marine
Topsmelt, Atherinops
affinis
ammonia
5-36%
Note: Can be used at end of pipe
testing, if the effluent is >_ 5%
Invertebrate
Freshwater
Water flea,
Ceriodaphnia dubia
pesticides
1-3%

Freshwater
Water flea, Daphnia
pufex and Daphnia
magna
pesticides
1 -6%

Marine
Atlantic mysid,
Mysidopsis bahia
metals
15-36%

Marine
Pacific mysid,
Holmesimysis costata
metals,
insecticides
32-36%
Note: Any of these test mehtods can be used as either static non-renewal or static renewal tests with test durations of
24, 48, or 96 hours. Lethality is the only endpoint. In Appendix B, the manual contains the supplemental list of
acute toxicity test species. This list specifies the test temperature, salinity for marine species and life stage to be
tested. For example, the topsmelt is included in this list, therefore, this species should be conducted the same as
o for the the silverside except for the parameters of test temperature, salinity, and life stage.

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Chronic East Coast Marine Test Methods [EPA/608/4-91-003]

Species
Test Type
Toxicants*
Salinity
Range of
Effluent
Test Endpoint
Fish
Inland silverside,
Menidia beryllina
7-day renewal
Surfactants,
ammonia
5-36%
growth and
survival
Invertebrate
Atlantic mysid,
Mysidopsis bahia
7-day renewal
metals
5-36%
growth,
fecunidity and
survival
* Including, but not limited to.
Note. These test species and methods are only to be used when the west coast species list in Table 2-A
are not available and with approval from the permitting authority.

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Chronic Freshwater Test Methods [EPA/608/4-91-002]

Species
Test Type
Toxicants*
Test Endpoint
Fish
Fathead minnow,
Pimepha/es promelas
7-day renewal test
surfactants,
ammonia
growth and survival
Invertebrate
Water flea, Ceriodaphnia
dubia
7-day renewal test
pesticides,
surfactants
reproduction and
survival
Plant
Green alga, SeJenastrum
capricornutum
96-hour
non-renewal
metals,
herbicides
growth
* Including, but not limited to.

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Test Design
¦	Test solutions:
-	negative control - dilution water
-	positive control - reference toxicant
-	effluent concentrations
¦	Replicates
-	test specific
Talking Points:
•	Test design is covered in the section on test procedures in each test method.
Typically, the test consists of at least five effluent concentrations plus a dilution
water control. Tests that use sea salts or brine to adjust salinity must also
contain a brine control.
•	For freshwater tests, the dilution water may be uncontaminated receiving water, a
standard synthetic (reconstituted) water, or some other uncontaminated natural water.
For marine tests, the dilution water should be uncontaminated 1 -um filtered natural
seawater or hypersaline brine prepared from uncontaminated natural seawater plus
reagent water. This water is used in all dilution steps and as the control water.
•	The selection of effluent test concentrations should be based on the objectives of the
study. EPA recommends that one of the five effluent treatments must be a
concentration of effluent mixed with dilution water which corresponds to the
permittee's instream waste concentration (IWC). At least two of the effluent
treatments must be of lesser effluent concentration than the IWC, with one being at
least one-half the concentration of the IWC.
•	Reference toxicant tests should be conducted as described in the Quality Assurance
section of the test method manual. Reference toxicant tests are used to indicate the
sensitivity of the test organisms being used and demonstrate a lab ability to obtain
consistent test results with the method. Frequency of testing with reference toxicant
tests is depends on whether the organisms are cultured in-house or by an outside
supplier. If the test organisms to be used are cultured in-house, then the reference
toxicant can be conducted once during that month. If the test organisms used are from
an outside source (e.g., commercial supplier), then the reference toxicant test must be
conducted concurrently with the effluent test.
•	The test method specifies the number of test organisms for each test solution and the
number of replicates required for each test concentration. The test method also
specifies the recommended test chamber size and test solution volume.
15

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Control
0%
50%
Replicates
6.25%
Mk M
12.5%
/jsSsssL
25%

100%
-W.
16

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Selection of Test Duration
¦	Acute duration - either 24, 48 or 96
hours in length
¦	Chronic duration - range from 40
minutes to 7 days
Talking Points:
•	The acute test methods can be conducted as either 24, 48 or
96 hours in duration, if the toxicant is fast acting, then select
either a 24 or 48 hour duration. These tests are usually
conducted as static non-renewal tests. Non-renewal testing is
important when it may be difficult to collect effluent renewals
such as stormwater or overseas samples. If the mode of
toxicant is unknown as is the case with most effluents, then
select a 96-hour test with a renewal at 48 hours.
•	The test duration for the chronic tests range from 40 minutes
to 7 days. The chronic test methods specify the duration of the
test, such as 7 days for the Ceriodaphnia dubia survival and
reproduction test.
17

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Selection of Test Type
¦	Chronic is specified in test method
¦	Acute may be conducted as either:
-	static non-renewal
-	static renewal
-	flow-through
Talking Points:
•	Static non-renewal tests: The test organisms are exposed to the same
test solution for the duration of the test.
•	Static renewal tests: The test organisms are exposed to a fresh test
solution of the same concentration of sample every 24-hour or other
prescribed interval, either by transferring the test organism from one test
chamber to another, or by replacing all or a portion of solution in the test
chambers.
•	Flow-through tests: (1) sample is pumped continuously from the
sampling point directly to the dilutor system; or (2) grab or composite
samples are collected periodically, and then placed in a tank to the
dilutor system.
•	The chronic test methods specifies whether the test is to be conducted
as static non-renewal or as static renewal.
•	The acute test methods can be conducted as either static non-renewal,
static renewal or flow through tests. See Diamond et al., 1995 for a
description of a flow-through system design using larval fish. The acute
test manual highlights some advantages and disadvantages of the test
types to consider when determining whether to use static non-renewal,
static renewal or flow through for acute toxicity testing [USEPA 1993,
p.45],
18

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Test Conditions
¦	Temperature
¦	Light
¦	Dissolved oxygen
¦	Conductivity and salinity
Talking Points:
•	The test temperature is specified for each test method. For example, the
chronic fathead minnow test temperature is 25 ±1 C.
•	Light quality, intensity and photoperiod are specified for each test method.
•	Dissolved oxygen requirements are specified for each test method. Dissolved
oxygen concentration in the effluent samples should be near saturation prior
to use. Aeration may be necessary to bring the dissolved oxygen into
equilibrium with air, minimize oxygen demand, and stabilize the pH. Aeration
can reduce the apparent toxicity of the test solutions by stripping them of
highly volatile toxic substances, or increase the toxicity by altering the pH.
However, the DO in the test solutions must not be allowed to fall below 4.0
mg/L. If aeration is necessary the rate is specified in the test method.
•	For freshwater tests, it is recommended that at a minimum, pH, conductivity
and total residual chlorine are measured in the undiluted effluent or receiving
water and pH and conductivity are measured in the dilution water. It is also
recommended that total alkalinity and total hardness be measured in the
undiluted effluent test water and the dilution water. For marine tests, at a
minimum, pH or salinity and total residual chlorine are measured in the
undiluted effluent or receiving water and pH and salinity are measured in the
dilution water.
19

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Test Organisms
¦	Culture and handling
¦	Age of organisms
¦	Source of food and feeding regime
Talking Points:
•	Each test method has detailed information on how to handle
and culture test organisms or obtain broodstock (e.g., for the
sea urchin fertilization test).
•	Each test method specifies an age requirement. For example,
the chronic fathead minnow test method requires newly
hatched larvae less than 24 hours old. If shipped, then not
more than 48 hours old and a 24 hour range in age.
•	Each test method has detailed information on source of food
and feeding regime if necessary for the test method. For
example, the chronic fathead minnow test method, the fish are
fed newly hatched brine shrimp, Artemia nauplii (less than 24
hours old).
20

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Selection of Dilution Water
¦	Dilution water may be either standard
lab water or receiving water
¦	Depends on the objectives of the test
Talking Points:
•	As stated in the testing manuals the selection of dilution water is based on the
objectives of the test.
•	The use of dilution water is an important part of toxicity testing. Dilution water may be
either standard laboratory water and/or receiving water. The type of dilution water
used in effluent toxicity tests will depend largely on the objectives of the test. These
objectives are:
(1)	If the objective of the test is to estimate the absolute acute or chronic toxicity
of the effluent, which is the primary objective of NPDES permit-related toxicity
testing, a standard laboratory dilution water as defined in each test method is
used.
(2)	If the objective of the test is to estimate the toxicity of the effluent in
uncontaminated receiving water, the test may be conducted using dilution
water consisting of a single grab sample of receiving water (if non-toxic),
collected either upstream and outside the influence of the outfall, or with other
uncontaminated natural water (ground or surface) or standard dilution water
having approximately the same characteristics (hardness and/or salinity) as the
receiving water.
(3)	If the objective of the test is to determine the additive or mitigating effects of
the discharge on already contaminated receiving water, the test is performed
using dilution water consisting of receiving water collected immediately
upstream or outside the influence of the outfall.
Note: If the test organisms have been cultured in water which is different from the test
dilution water, a second set of controls, using culture water should be included in the test.
21

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Sample Requirements
¦	Type of effluent samples
¦	Test initiation time
¦	Sampling location
Talking Points:
•	The effluent or receiving water samples are collected as either a grab or
composite sample (see Chapter on Effluent and Receiving Water Sampling,
Sample Handling and Sample Preparation for Toxicity Tests in a test methods
manual). Composite samples (for example, flow-proportional or timed
composites) should be collected using an iced or refrigerated collection device.
Effluent samples must be maintained at 4° from collection until utilized in the
toxicity testing procedure. The single allowable exception is when a grab sample
is collected and delivered to the performing laboratory for test initiation no later
than 4 hours following the time of collection. All other samples must be
received by the laboratory at a temperature at 4°C or the sample should be
considered invalid.
•	The maximum elapsed time between the collection of a sample and its first use is 36
hours for offsite testing . The composite sample begins at time zero when the last
composite in a 24-hour composite is collected. EPA believes that 36 hours is adequate
time to deliver the sample to the laboratory performing the test in most cases. In the
isolated cases, where the permittee can document that this delivery time cannot be
met, the permitting authority can allow an option for an extension of shipped sample
holding time such as for overseas shipping. The request for a variance in sample
holding time must include supportive data which show that the show that the toxicity
of the effluent sample is not reduced (e.g., because of volatilization and/or sorption of
toxics on the sample container surfaces) by extending the holding time beyond 36
hours.
•	The sampling site should be located below the last waste treatment process, including
disinfection. There may be no removal of chlorine or any other effluent constituent by
either chemical or physical methods prior to testing without approval from the
permitting authority. The collection container should be filled with no headspace and
closed immediately to minimize loss of volatiles.
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Test Acceptability Criteria
¦ Controls
-	minimum survival
-	minimum growth, length, reproduction
Talking Points:
•	Each test method has a specific test acceptability criteria
(TAC). For example, the chronic fathead minnow test method
requires 80% or greater survival in the control(s) and an
average dry weight per surviving organism of equal or greater
than 0.25 mg/L.
•	All the acute test methods require 90% or greater survival in
the control(s).
•	All the TAC must be met in order for a test method to be
acceptable for compliance purposes. If a test does not meet
the TAC, then the permittee must repeat the test as soon as
possible.
23

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Test Precision
¦	Intra lab precision
¦	Inter lab precision
¦	Statistical measurements of precision
%CV = standard deviation/mean *100
Talking Points:
• Precision is a measure of test consistency or repeatability both
with a lab (intra lab) and among several labs (inter lab) using
the same test method and reference toxicant. Precision is
quantified by a variety of measures including the coefficient of
variation (CV = standard deviation/mean *100) of point
estimates (e.g., LC50 for acute endpoints and EC/IC25 for
chronic endpoints) from multiple tests conducted with the
same test method and reference toxicant.
24

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Take Home Points
Must use approved test methods
Labs must follow the summary table
Test methods are precise

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WET TEST COSTS
An informal survey of Region 9 WET testing laboratories yielded
the following information. Costs for definitive freshwater acute
non-renewal tests range from $250-$500, while marine acute non-
renewal test costs range from $250-$750 (the higher cost was for
Mysidopsis bahia). Costs for definitive freshwater chronic
renewal tests range from $950-$1250. Costs for definitive marine
chronic renewal tests range from $800-$2250 (the higher cost was
for Mysidopsis bahia since this test has three endpoints). Costs
depend on: (1) the organism supplies, costs and availability,
(2) ease of working with test organisms, and (3) amount of time
in calculating test endpoint (e.g., microscope time), etc.
Acute Toxicity Test Costs:
TEST SPECIES
RANGE OF COSTS
Ceriodaphnia dubia, Daphnia pulex,
Daphnia magna, Pimephales promelas,
Oncorhynchus mykiss, Menidia beryllina
$225 - 500
Mysidopsis bahia
$600 - 750
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Chronic Toxicity Test Costs
TEST SPECIES
RANGE OF COSTS
Selenastrum capricornutum growth test
$600 - 950
Ceriodaphnia dubia survival and
reproduction, Pimephales promelas
survival and growth, Menidia beryllina
survival and growth, Atherinops affinis,
survival and growth
$1000 - 1250
Mytilus spp. and Strongylocentrotus
purpuratus larval development
$800 - 1100
Strongylocentrotus purpuratus and
Dendraster excentricus fertilization
$500 - 1100
Haliotis rufescens larval development
test
$1000 - 1250
Macrocystis pyrifera germination and
germ-tube length
$1000 - 1250
Mysidopsis bahia survival, fecundity and
growth
$1100 - 2250*
The fecundity endpoint can be optional, since there are two
sublethal endpomts (growth and fecundity) . This must be
approved by the permitting authority.
2

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AMBIENT TOXICITY TESTING PROGRAMS
The following contains a brief summary of the uses and findings of selected laboratory toxicity testing
projects with ambient water samples collected in California during the last eight years with the
objectives of screening for and identifying water quality problems. Ambient water toxicity testing has
been used by Regional and State Water Boards, not as a compliance measure, but rather as a
screening tool which can be followed up with Toxicity Identification Evaluations (TIEs) and analytical
chemistry procedures to identify the specific chemical causes of water quality problems. There is no
officially designated ambient water toxicity testing program in California.
COLUSA BASIN DRAIN-PESTICIDES USED IN RICE CULTIVATION
In the spring Colusa Basin Drain (CBD) receives large quantities of tailwater discharged from rice
field floodings. CBD, in turn, discharges into the Sacramento River and, during this tune, can
constitute up to one third of the river flow.
Acute toxicity tests were conducted with water samples collected from CBD before, during, and after
the release of tailwater from rice fields. Tests organisms were Ceriodaphnia, Neomysis, and striped
bass larvae and eggs. These toxicity tests clearly identified toxicity associated with the discharge of
tailwater from rice fields. TIEs and associated chemical analyses specifically identified some of the
pesticides used in rice cultivation as the causes of toxicity to Ceriodaphnia and Neomysis.
As a result of these findings, the Central Valley Regional Water Quality Control Board and the
Department of Pesticide Regulation (DPR) initiated actions which resulted in alterations of irrigation
practices on rice fields (e.g., increased holding times of irrigation water following the application of
pesticides). The increased on-field holding times resulted in decreased frequency and magnitude of
toxicity, as well as lower concentrations of pesticides, in CBD and Sacramento River water samples
during the release of rice irrigation tailwater. Water quality of the CBD discharge was clearly
improved as a consequence of the information gained from toxicity testing and TIE data
IMPERIAL COUNTY - ALAMO RIVER
There is extensive irrigation of Imperial County agriculture with Colorado River water via the All-
American Canal. The Alamo River, which discharges into the Salton Sea, consists primarily of
agricultural irrigation tailwater. For over two years, water samples have been collected at up to 11
stations along the 50 mile course of the Alamo River. These samples have been screened for water
quality using 96-hour acute toxicity tests with Ceriodaphnia and Neomysis.
Although the head water of the Alamo River in the United States has never tested toxic, frequent and
high magnitude acute lethality has been seen in water samples taken along the entire length of the
river which receives irrigation tailwater. TIEs and associated analytical chemistry have clearly
demonstrated that five pesticides, chlorpyrifos, diazinon, carbofuran, malathion, and carbaryl, are
major contributors to the toxicity in many of the Alamo River toxic samples.
Staff from the Colorado River Basin Regional Water Quality Control Board and the State Water
Resources Control Board (SWRCB) and DPR have agreed to work cooperatively to reduce pesticide
concentrations in the Alamo River. These three agencies, along with the Imperial Irrigation District,
will attempt to convene an Interagency Coordinating Committee (ICC) of interested parties to assist in
1

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the development of practices aimed at reducing pesticides in the Alamo River to nontoxic levels. This
ICC could include the above entities, as well as the Imperial County Agricultural Commissioner, the
Farm Bureau, grower organizations, pesticide advisors, applicators organizations, and the Soil
Conservation District.
SAN JOAQUIN RIVER WATERSHED
The San Joaquin River has the second largest watershed in California and, due to extensive
hydrological manipulations, this river now receives large volumes of agricultural tile drain water, as
well as irrigation tailwater. The San Joaquin and its tributaries were extensively sampled from
February 1988 through June 1990. The samples were screened using the EPA chronic freshwater
three species methods, Ceriodaphnia, Pimephales, and Selenastrum.
A pattern of frequent and high magnitude acute mortality to Ceriodaphnia was demonstrated in a 43
mile stretch of the San Joaquin River between its confluence with the Merced and Stanislaus Rivers.
Based on chemical analyses of the toxic samples, the primary causes of the toxicity water quality
problem were attributed to pesticides, including diazinon, chlorpyrifos, carbofuran, carbaryl and
parathion. The US Geological Survey and DPR performed subsequent studies on the San Joaquin
River which confirmed extensive pesticide contamination
Although no regulatory actions have been initiated to address these water quality problems, the San
Joaquin County Agricultural Commissioner has been conferring with the Central Valley Regional
Water Quality Control Board staff regarding these problems.
SACRAMENTO-SAN JOAQUIN DELTA ESTUARY
The Sacramento-San Joaquin delta estuary is of monumental ecological, aesthetic, and economic
significance in California. Over the past 21 months there has been extensive sampling (approximately
24 sites, sampled monthly) in the delta estuary. These samples have been screened with the EPA
chronic freshwater three species methods, Ceriodaphnia, Pimephales, and Selenastrum. The data
collected to date demonstrate periodic and widespread water quality problems in this critical area.
Although the causes of the toxicity have not been completely identified, TIEs and chemical analyses
reveal that chlorpyrifos, diazinon, and carbofuran contribute to the toxicity seen at some times during
the year. These data are currently being incorporated into a draft report which will be circulated for
technical review.
ORCHARD RUNOFF IN THE CENTRAL VAT ] FY
Considerable acreage in the Sacramento, Feather, and San Joaquin River watersheds is devoted to
fruit and nut growing. Acute toxicity screening tests of water samples collected at multiple sites
throughout these watersheds indicated water quality problems during January and February.
Specifically, many of the samples collected during this time yielded Ceriodaphnia mortality.
Follow up analytical chemistry and immunosorbant analyses pointed to diazinon, a pesticide applied to
dormant orchards during December and January for the control of a bud boring insect, as a water
quality problem in these watersheds. The concentrations of diazinon measured in samples collected
during this period frequently exceeded the acute mortality LC50 of several aquatic species. These
2

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studies also suggested that certain orchard areas surrounding the Feather, Sacramento, and San
Joaquin Rivers were the geographic source of diazinon.
To date no regulatory actions have been taken to control the offsite movement of this pesticide.
However, DPR, UC Davis Extension (the BIOS project) and Ciba-Geigy Corporation (a manufacturer
of diazinon) have conducted some exploratory studies on practices which could reduce the offsite
movement of diazinon. These studies included the voluntary cooperation of growers.
REVOLON SLOUGH/MUGU LAGOON
Mugu Lagoon is considered a significant ecological area which may be at high risk. Revolon Slough,
in Ventura County, receives large volumes of agricultural irrigation tail water. Water was collected at
sites on this slough over the course of a year and screened with the EPA chronic freshwater three
species methods, Ceriodaphnia, Pimephales, and Selenasrrum.
Data from this study revealed periodic toxicity to each of the three species. Based on these initial
data, another year of testing has been initiated which will include TIEs and chemical analyses to
identify the causes of water quality problems.
ANAHEIM/NEWPORT BAYS
Four freshwater streams and channels discharging into the sensitive Anaheim and Newport Bays were
sampled. Four sites were sampled twice between November through February. Water quality in
these samples was screened using the EPA chronic freshwater three species methods, Ceriodaphnia,
Pimephales, and Selenasirum.
Periodic acute and chronic toxicity were detected in these samples. Discharges into Newport Bay
were primarily toxic to Ceriodaphnia. TIEs suggested that the toxicants were organic chemicals and,
although pesticides were detected in these toxic samples, there was no confirmation as to the causes of
toxicity. Freshwater discharges into Anaheim Bay proved to be toxic to all three test species, but
there was no identification of the causative chemicals. Funds were not available to specifically
identify the causes of toxicity or to follow up these initial findings.
FINAL COMMENTS
Despite low, and ever-declining, funding, toxicity testing of surface waters has proved to be powerful
water quality screening tool. Given the relative short time this tool has been used, it has an
exceptional record for indicating water quality problems. Specifically, toxicity testing with
subsequent Tits and chemical analyses have an excellent record in locating the geographic source,
land use practices, and chemical causes of water quality problems.
Surface water quality toxicity testing studies plus TIE results also have evoked several Department of
Fish and Game hazard assessments for specific pesticides. These assessments include the
development of water quality criteria for the pesticides. In the last ten years, ambient water toxicity
testing in association with lit and analytical chemistry results have yielded the potential for several
changes in land/water use practices.
3

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REFERENCES
TEST METHODS, TRE AND TIE DOCUMENTS
Acute toxicity test methods
USEPA Methods for Measuring the Acute Toxicity of Effluents to Freshwater and Marine Organisms
(EPA/600/4-90-027F). Note, see Appendix B of the acute toxicity test manual for the supplemental list
of acute test species.
Freshwater tests
Vertebrates:
•	Fathead minnow, Pimephales promelas
•	Rainbow trout, Oncorhynchus mykiss
•	Brook trout, Salvelinus fontinalis
Invertebrates:
•	Water flea, Ceriodapbma dubia
•	Water flea, Daphnia pulex and D. magna
Marine tests
Vertebrates:
•	Inland silverside, Menidia berylhna
•	Topsmelt, Atherinops affinis
Invertebrates.
•	Atlantic mysid, Mysidopsis bahia
•	Pacific mysid, Hobnesimysis costaia
Chronic toxicity test methods
Freshwater tests
USEPA Short-Term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to
Freshwater Organisms (EPA/600/4-91-002).
Vertebrate:
•	Fathead minnow, Pimephales promelas
Invertebrate:
•	Water flea, Ceriodaphnia dubia
Plant:
•	Green alga, Selenastrum capricornutum
Marine tests
USEPA Short-Term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to
1

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Marine and Estuarine Organisms (EPA/600/4-91-003).
USEPA Short-Term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to
West Coast Marine and Estuarine Organisms (EPA/600/R-95/136, August 1995).
Vertebrates:
•	Inland silverside, Menidia beryllina (EPA/600/4-91-003)
•	Topsmelt, Atherinops affinis (EPA/600/R-95/136)
Invertebrates:
•	Atlantic mysid, Mysidopsis bahia (EPA/600/4-91-003)
•	Red abalone, Haliotis rufescens (EPA/600/R-95/136)
•	Bivalves, Crassostrea gigas and Mytilus spp. (EPA/600/R-95/136)
•	Purple urchin, Strongylocentrotus purpuratus and Sand dollar, Dendrasrer excentricus
(EPA/600/R-95/136)
Plants:
•	Giant kelp, Macrocystis pyrifera (EPA/600/R-95/136)
Toxicity reduction/identification evaluation methods
TRE
USEPA Toxicity Reduction Evaluation Protocol for Industrial Treatment Plants (EPA/600/2-88/070).
USEPA Toxicity Reduction Evaluation Protocol for Municipal Wastewater Treatment Plants (EPA/600/2-
88/062).
TIE
USEPA Toxicity Identification Evaluation: Characterization of Chronically Toxic Effluents, Phase I
(EPA/600/6-91-05F).
USEPA Methods for Aquatic Toxicity Identification Evaluations: Phase n Toxicity Identification
Procedures for Samples Exhibiting Acute and Chronic Toxicity (EPA/600/R-92-080).
USEPA Methods for Aquatic Toxicity Identification Evaluations: Phase in Toxicity Confirmation
Procedures (EPA/600/R-92-81).
Other documents
USEPA Technical Support Document for Water Quality-based Toxics Control (EPA/505/2-90-001).
Office of Water. Washington, DC.
USEPA Manual for the Evaluation of Laboratories Performing Aquatic Toxicity Tests (EPA/600/4-90-
031).
USEPA Methods for Chemical Analysis of Water and Wastes (EPA/600/4-79/020). Revised March,
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1983.
STATISTICAL PROGRAMS
Dunnett Program (Version 1.5)
Inhibition Concentration (ICp) Approach (Version 2.0)
Probit Analysis (Version 1.5)
Trimmed Spearman-Karber (Version 1.5)
Note: If you are interested in obtaining any of these statistical programs, please send a formatted
3.5" disk to James Lazorchak, EPA EMSI^Ci, 3411 Church Street, Cincinnati, OH 45244.
SPREADSHEETS
Contact: Madonna Narvaez, USEPA, Region 10, OW-130, 1200 Sixth Avenue, Seattle, WA 98101.
Telephone: (206) 553-1774; FAX: (206)553-1280.
VIDEOS
USEPA Freshwater Culturing Methods for Ceriodaphnia dubia and Pimepholes promelas.
USEPA Test Methods for Freshwater Effluent Toxicity Tests.
USEPA Culturing and Toxicity Test Methods for Marine and Estuarine Effluents for Mysidopsis bahia.
Note: If you are interested in obtaining these three videos at a cost, please call The National
Audiovisual Center at (800) 788-6282.
DATABASES
AQUIRE - (AQUatic Information REtrieval database)
ASTER - (Assessment Tools for the Evaluation of Risk)
The AQUIRE database now contains more than 127,000 individual test records for 5,525 chemicals and
2,791 freshwater and marine organisms. Over 9,000 publications have been reviewed for AQUIRE.
These data are also available from the ASTER Database System. Both AQUIRE and ASTER now have
the electronic capability of sending help text and reports to an internet address.
For information about logging onto these databases, contact the Environmental Research
Laboratory-Duluth at (218) 720-5602; fax (218) 720-5539; and internet at
outreach@du4500.dul.epa.gov.
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TABLE 1. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA
FOR FATHEAD MINNOW, PIMEPHALES PROMELAS, LARVAL SURVIVAL AND
GROWTH TOXICITY TESTS WITH EFFLUENTS AND RECEIVING WATERS
1.	Test type:
2.	Temperature (°C):
3.	Light quality:
4.	Light intensity:
5.	Photoperiod:
6.	Test chamber size:
7.	Test solution volume:
8.	Renewal of test
solutions:
9.	Age of test organisms:
Static renewal
25 ± 1°C
Ambient laboratory illumination
10-20 ^E/m2/s (50-100 ft-c)(ambient
laboratory levels)
16 h light, 8 h darkness
500 mL (minimum)
250 mL (minimum)
Daily
Newly hatched larvae less than 24 h old.
If shipped, not more than 48 h old, 24 h
range in age
10. No. larvae per test chamber: 15 (minimum of 10)
11.	No. replicate chambers
per concentration:
12.	No. larvae per
concentration:
13.	Source of food:
14. Feeding regime:
4 (minimum of 3)
60 (minimum of 30)
Newly hatched Artemia nauplii (less than
24 h old)
Feed 0.1 g newly hatched (less than 24-h
old) brine shrimp nauplii three times
daily at 4-h intervals or, as a minimum,
0.15 g twice daily, 6 h between feedings
(at the beginning of the work day prior to
renewal, and at the end of the work day
following renewal). Sufficient nauplii
are added to provide an excess. Larvae
fish are not fed during the final 12 h of
the test
l

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TABLE 1. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA
FOR FATHEAD MINNOW, PIMEPHALES PROMELAS, LARVAL SURVIVAL AND
GROWTH TOXICITY TESTS WITH EFFLUENTS AND RECEIVING WATERS
15.	Cleaning:
16.	Aeration:
17.	Dilution water:
18.	Test concentrations:
19.	Dilution factor
20.	Test duration:
21.	Endpoints:
22.	Test acceptability
criteria:
23. Sampling
requirements:
Siphon daily, immediately before test
solution renewal
None, unless DO concentration falls below
4.0 mg/L. Rate should not exceed
100 bubbles/min
Uncontaminated source of receiving or
other natural water, synthetic water
prepared using MILLIPORE MILLI-Q® or
equivalent deionized water and reagent
grade chemicals, or DMW (see Section 7,
Dilution Water)
Effluents: Minimum of 5 and a control
Receiving Water: 100% receiving water
minimum of 5 and a control
or
Effluents: > 0.5
Receiving waters:
7 days
None or > 0.5
Survival and growth (weight)
80% or greater survival in controls;
average dry weight per surviving organism
in control chambers equals or exceeds 0.25 mg
For on-site tests, samples collected
daily, and used within 24 h of the time
they are removed from the sampling device;
For off-site tests, a minimum of
three samples collected on days one,
three and five with a maximum holding time
of 36 h before first use (see Section 8,
24. Sample volume required: 2.5 L/day
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TABLE 3. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR
DAPHNID, CERIODAPHN1A DUBIA, SURVIVAL AND REPRODUCTION TOXICITY
TESTS WITH EFFLUENTS AND RECEIVING WATERS
1.	Test type:
2.	Temperature (°C):
3.	Light quality:
4.	Light intensity:
5.	Photoperiod:
6.	Test chamber size:
7.	Test solution volume:
8.	Renewal of test solutions:
9.	Age of test organisms:
10.	No. neonates per
test chamber:
11.	No. replicate test
chambers per concentration:
12.	No. neonates per
test concentration:
13.	Feeding regime:
14.	Cleaning:
15.	Aeration:
16.	Dilution water:
Static renewal
25 ± re
Ambient laboratory illumination
10-20 n£/mz/s, or 50-100 ft-c
(ambient laboratory levels)
16 h light, 8 h dark
30 mL (minimum)
15 mL (minimum)
Daily
Less than 24 h; and all released
within a 8-h period
1
10
10
Feed 0.1 mL each of YCT and algal
suspension per test chamber daily
Use freshly cleaned glass beakers or
new plastic cups daily
None
Uncontaminated source of receiving or
other natural water, synthetic water
prepared using MILLIPORE MILLI-Q® or
equivalent deionized water and reagent
grade chemicals or DMW (see Section
7, Dilution Water)
3

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TABLE 3. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA
FOR DAPHNID, CERIODAPHNIA DUBIA, SURVIVAL AND REPRODUCTION
TOXICITY TESTS WITH EFFLUENTS AND RECEIVING WATERS (CONTINUED)
17.	Test concentrations:
18.	Dilation factor:
19.	Test duration:
20.	Endpoints:
21.	Test acceptability criteria:
22.	Sampling requirements:
Effluents: Minimum of 5 and a control
Receiving Water: 100% receiving water
or minimum of 5 and a control
Effluents: >0.5
Receiving Waters: None or > 0.5
Until 60% of surviving control
organisms have three broods (maximum
test duration 8 days)
Survival and reproduction
80% or greater survival and an average
of 15 or more young per surviving
female in the control solutions. 60%
of surviving control organisms must
produce three broods.
For on-site tests, samples collected
daily, and used within 24 h of the
time they are removed from the
sampling device. For off-site tests,
a minimum of three samples collected
on days one, three, and five with a
maximum holding time of 36 h before
first use (see Section 8, Effluent and
Receiving Water Sampling, Sample
Handling, and Sample Preparation for
Toxicity Tests, Subsection 8.5.4)
22. Sample volume required:
1 L/day

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TABLE 13. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR
FATHEAD MINNOW, P1MEPHALES PROHELAS, ACUTE TOXICITY TESTS WITH
EFFLUENTS AND RECEIVING WATERS1
1.	Test type:
2.	Test duration:
3.	Temperature:2
4.	Light quality:
5.	Light intensity:
6.	Photoperiod:
7.	Test chamber size:
8.	Test solution volume:
9.	Renewal of test
solutions:
10.	Age of test organisms:
11.	No. organisms per
test chamber:
12. No. replicate chambers
per concentration:
13. No. organisms
per concentration:
14. Feeding regime:
Static non-renewal, static-renewal, or flow-
through
24, 48, or 96 h
20°C ± 1°C; or 25°C ± 1°C
Ambient laboratory illumination
10-20 /iE/m2/s (50-100 ft-c)
(ambient laboratory levels)
16 h light, 8 h darkness
250 mL (minimum)
200 mL (minimum)
Minimum, after 48 h
1-14 days; 24-h range in age
Minimum, 10 for effluent and receiving water
tests
Minimum, 2 for effluent tests
Minimum, 4 for receiving water tests
Minimum, 20 for effluent tests
Minimum, 40 for receiving water tests
Artemia nauplii are made available while
holding prior to the test; add 0.2 mL
Artemia nauplii concentrate 2 h prior to
test solution renewal at 48 h
Cyprinella leedsi (Bannerfin shiner, formerly Notropis leedsi) can be
used with the test conditions in this table, where it is the required
test organism in discharge permits.
Acute and chronic toxicity tests performed simultaneously to obtain
acute/chronic ratios must use the same temperature and water hardness.
5

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TABLE 13. SUMMARY OF TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA FOR
FATHEAD MINNOW, PIMEPHALES PROHELAS, ACUTE TOXICITY TESTS WITH
EFFLUENTS AND RECEIVING WATERS (CONTINUED)
15.	Test chamber cleaning:
16.	Test solution aeration:
17.	Dilution water:
18. Test concentrations:
19. Dilution series:
20. Endpoint:
21. Sampling and sample
holding requirements:
. Cleaning not required
None, unless DO concentration falls below
4.0 mg/L; rate should not exceed 100
bubbles/min
Moderately hard synthetic water prepared
using MILLIPORE MILLI-Q® or equivalent
deionized water and reagent grade chemicals
or 20% DMW (see Section 7, Dilution Water),
receiving water, ground water, or synthetic
water, modified to reflect receiving water
hardness.
Effluents: Minimum of five effluent
concentrations and a control
Receiving Waters: 100% receiving water and a
control
Effluents: >0.5 dilution series
Receiving Waters: None, or >0.5 dilution
series
Effluents: Mortality (LC50 or NOAEC)
Receiving Waters: Mortality (Significant
difference from control)
Effluents and Receiving Waters: Grab or
composite samples are used within 36 h of
completion of the sampling period.
22. Sample volume required:. 2 L for effluents and receiving waters
23. Test acceptability
criterion:
90% or greater survival in controls
6

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APPENDIX B
SUPPLEMENTAL LIST OF ACUTE TOXICITY TEST SPECIES
TEST LIFE
TEST ORGANISM	TEMP STAGE
(°C)
FRESHWATER SPECIES:
VERTEBRATES - WARMWATER

Cyprinella leedsi^
Bannerfin shiner
25
1-14 days
Lepomis macrochirus
Bluegill sunfish
20,25
if ii
Ictalurus punctatus
Channel catfish
II
n if
FRESHWATER SPECIES:
INVERTEBRATES - COLDWATER

Pteronarcys spp.
Stoneflies*
12
1arvae
Pacifastacus



leniusculus
Crayfish*
II
juveniles
Baetis spp.
Mayflies*
II
nymphs
Ephemerella spp.
if
II
n
FRESHWATER SPECIES:
INVERTEBRATES - WARMWATER

Hyalella spp.
Amphipods
20,25
juveniles
Gamarus lacustris
If
n
n
G. fasciatus
ii
f!
n
G. pseudolimnaeus
ll
It
n
Hexagenia limbata
Mayflies
n
nymphs
H. bilineata
(t
n
U
Chironomus spp.
Midges
it
larvae
~Stoneflies, crayfish, and mayflies may have to be field collected
and acclimated for a period of time to ensure the health of the
organisms and that stress from collection is past. Species
1 identification must be verified.
Test conditions for Cyprinella leedsi are found in Table 13.
7

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SUPPLEMENTAL LIST OF ACUTE TOXICITY TEST SPECIES (CONTINUED)
TEST ORGANISM
TEST
TEMP
(6C)
SALIN-
ITY
(*»)
LIFE
STAGE
MARINE AND ESTUARINE SPECIES: VERTEBRATES - COLDWATER
Parophrys vetulus	English sole 12 32-34
Citharichys
sitigmaeus	Sanddab "	"
Pseudopleuronectes
americanus	Winter flounder
MARINE AND ESTUARINE	SPECIES: VERTEBRATES - WARMWATER
Paralichthys
dentatus
P. lethostigma
Fundulus simillis
Fundulus
heteroclitus
Lagodon rhomboides
Orthipristis
chrysoptera
Leostomus xanthurus
Gasterosteus
aculeatus
Atherinops affinis
Flounder
ll
Killifish
Mummichog
Pinfish
Pigfish
Spot
Threespine
stickleback
Topsmelt
21
20-32
25-32
20-32
15-30
10-30
20-32
10-30
1-90 days
n	n
post meta-
morphosis
20,25 32-34 1-90 days
n fi
1-30 days
n	n
1-90 days
n	ii
ft	ii
1-30 days
7-15 days
8

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SUPPLEMENTAL LIST OF ACUTE TOXICITY TEST SPECIES (CONTINUED)
TEST ORGANISM
TEST SALIN- LIFE
TEMP ITY	STAGE
(°C)	(%o)
MARINE AND ESTUARINE SPECIES: INVERTEBRATES - COLDWATER
Panda!us jordani
Strongylocentrotus
droebachiensis
Strongylocentrotus
purpuratus
Dendraster
excentricus
Cancer magister
Holmesimysis
costata
Oceanic shrimp
Green sea urchin
Purple sea urchin
«
Sand dollar
Dungeness crab
Mysid
12
25-32
32-34
MARINE AND ESTUARINE SPECIES: INVERTEBRATES - WARMWATER
Callinectes sapidus
Palaemonetes pugio,
P. vulgaris,
P. intermedius
Penaeus setiferus
Penaeus duorarum
Penaeus aztecus
Crangon
septemspinosa
Mysidopsis almyra
Neomysis americana
Metamysidopsis
elongata
Crassostrea
virginica
Crassostrea gigas
Arbacia punctulata
Blue crab
Grass shrimp
White shrimp
Pink shrimp
Brown shrimp
Sand shrimp
Mysid
American oyster
Pacific oyster
Purple sea urchin
20,25
10-30
10-32
20-32
n
25-32
10-32
20-32
25-32
32-34
juvenile
gametes/embryo
It	It
it	n
juvenile
1-5 days
juvenile
1-10 days
•? It
n n
post-larval
1-5 days
It ft
n n
embryo
n
gametes/embryo
zTest conditions for Homesimysis costata are found in Table 15.
9

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MEMORANDUM
April 10. 1996
SUBJECT: Clarifications Regarding Flexibility in 40 CFR Part 136 Whole Effluent
Toxicity (WET) Test Methods
FROM: Tudor T. Davies, Director
Office of Science and Technology
TO:	Water Management Division Directors, Regions I-X
Environmental Services Division Directors, Regions I-X
The purpose of this memorandum is to provide clarifications regarding the flexibility
in the regulations promulgated at 60 Fed. Reg. 53529 (Oct. 16, 1995), which establish
analytical test methods for the determination of whole effluent toxicity (WET). This
information is important to the conduct of the WET test methods, and I ask that you share
this memorandum with the States in your Region approved to administer the National
Pollutant Discharge Elimination System (NPDES) permitting program. In addition, I would
encourage States to make the information available to their NPDES permittees and
appropriate test analysts.
The WET test methods are codified at 40 CFR part 136, including three test method
manuals which are incorporated by reference. Because they are incorporated by reference,
the test method manuals themselves constitute Federal regulations. The test method manuals
do not, however, strictly prescribe every aspect of method conduct, and it is important to
think carefully about how the WET test data will be used when making decisions on both the
selection of the test species as well as the selection of individual test parameters where
flexibility is allowed.
In many instances, the manuals use discretionary terms such as "may" or "should,"
with the understanding that the laboratory analyst must have flexibility to optimize successful
test completion. In other instances, the manuals use stronger, compulsory terms such as
"shall" or "must" to prescribe procedures necessary for nationwide standardization
(specifically those procedures that assure the predictability of the methods to provide accurate
results). When situations arise that are not addressed by the manuals, questions will become
apparent ~ where the method manuals neither authorize nor prohibit a procedure in question.
In the majority of these instances, we anticipate that silence in the manual will indicate
flexibility for the individual analyst. The remainder of this memorandum addresses issues
that have already arisen in the areas of pH, ammonia, temperature, water hardness, test
dilution concentrations, and a definition of "mean" number of cystocarps in the Champia
parvula test.

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2
1) pH and Ammonia Control
On p. 40 in the freshwater chronic manual (Sections 8.8.6 and 8.8.8) and p. 42 of the
marine chronic manual (Sections 8.8.6), the manuals contain some discussion of the influence
that pH and temperature have on ammonia toxicity. In this instance, the manuals do provide
flexibility to the analyst to control artificial toxicity caused by pH drift provided that the
analyst verifies the source of toxicity is, in fact, artificial. How to determine this "artificial
toxicity is described in Section 8.8.8. As the chronic manuals discuss the use of acids/bases
to adjust the pH, likewise the use of C02 in the headspace to control the pH is comparable.
The following are example techniques where pH can be controlled so that ammonia
toxicity can be assessed. The pH can be controlled using appropriate procedures which do
not significantly alter the nature of the effluent. For example, any procedure which
removes ammonia, but does not remove other toxicants would be allowed. However,
treatment with zeolite, would not routinely be allowed because it removes other toxicants.
Controlling the carbon dioxide (C02) environment, however, would be acceptable if carbon
dioxide can be delivered directly to test chambers with airline tubing and a pipette or by
using a complex solenoid system (on demand only). Another alternative is to maintain a
closed carbon dioxide environment, delivering a solution of C02 in oxygen to the closed
system.
2) Temperature
On p. 53 in the acute manual (Section 9.12.1) and in Tables 11-17, pages 57-70, the
manual describes test temperatures for each acute test. The chronic test methods lists
temperatures for each discrete species test protocol. A concern that has been raised is that,
due to seasonal temperature fluctuations in receiving waters, the predictive capacity of the
test methods does not reflect actual receiving water effects, and thus the methods should
allow for seasonal variation in test temperatures. The acute manual does provide protocols
for a wide variety of test species that encompass a broad range of test temperatures which
could approximate the seasonal temperatures found in the receiving waters. Thus, to
accommodate permittee concerns about the predictive capacity of the standardized'test species
to predict toxicity on a seasonal basis, permits may be written to include different approved
test species for different seasons of the year. In this instance, however, the acute and
chronic manuals do prescribe test temperatures in order to standardize the methods and limit
test variability. Therefore, where variations in test temperature need to be taken into
account, NPDES permits should include different approved test species, rather than
authorizing seasonal variations in test temperatures for a given test species.

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3
3)	Hardness
For the acute manual, the freshwater chronic manual, and the marine/estuarine
chronic manual, Section 7 in each manual describes the use of dilution waters. In Appendix
A of the acute manual, and m each specific test Section of the chronic manuals, the manuals
describe how to culture or obtain the test organisms. Hardness of dilution water, as well as
hardness of water used to culture test organisms, may have an effect on successful
completion of the tests. In some cases, the relative hardness of the dilution water compared
to the organism culture water) may affect the expression of toxicity in the conduct of the
tests, i.e., the accuracy of the tests at predicting toxicity. In this regard, the analyst has
flexibility in performing the tests to optimize successful test completion. The type of the
dilution water used in the effluent toxicity tests depends on the objective of the study. Tests
can be conducted in the standard reconstituted dilution water to assess the absolute toxicity of
the effluent. To evaluate whether or not the toxicity is present in the receiving water, the
test can be conducted with a single grab sample of receiving water, or the hardness of the
dilution water can be adjusted to match that of the receiving water (while taking care not to
exceed the water hardness tolerance of the test organisms, which would cause stress to the
test organisms, and affect the toxicity test results). In any case, if the dilution water is
different than the culture water, then a second set of controls should be tested.
4)	Test Dilution Concentrations
The acute manual on p. 47 (Section 9 3.2), freshwater chronic manual on p. 42
(Section 8.10.2-3), and the manne manual on p. 45 (Section 8.10.2-3) describes whole
effluent toxicity test dilution concentrations. Although permitting agencies might assume that
a 100% effluent concentration must be included in the sample series, the manuals do not
require this. The language "such as" is used to show an example of a concentration series.
In some instances, such as manne tests, using 100% effluent as the highest effluent
concentration is not realistic. In other instances 100% effluent may be unnecessary if the
effluent is not highly variable; or the higher effluent concentrations are not of concern. The
language in the manuals does, however, recommend bracketing the concentration of concern
(such as the WET permit limit, or the receiving water concentration of concern). For
example, if the effluent concentrauon in the receiving water is 12 percent, the dilution series
could be 50 percent, 25 percent, 12 5 percent, 6.25 percent and 3.12 percent effluent.

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4
5) Acceptance Criteria for Champia parviila
The control acceptance criterion for the effluent sexual reproduction test using
Champia parvula is an average of 10 or more cystocarps for the control treatment (p. 359 of
the marine short-term chronic manual, #17). This means that some replicates may have
fewer than 10 cystocarps, and some more than 10 cystocarps. This is acceptable as long as
the overall mean number of cystocarps per replicate is at least 10 cystocarps.
The analyst may on occasion find that a single control replicate will have a greatly
reduced or enhanced number of cystocarps relative to the remaining replicates. As with any
toxicity test the analyst may have to determine the single "odd" replicate is an outlier. If the
overall mean number of cystocarps is at least 10, with the low or high "odd" replicate
excluded, then whether or not a single outlier is present does not effect the determination of
control acceptability. If the remaining three replicates do not average 10 cystocarps, then the
test should be repeated An outlier, however, may effect the analyst's conclusions
concerning the level of toxicity in the samples tested along with that control.
In conclusion, I realize that additional Questions may arise, and I encourage you to
contact Teresa Norberg-King in the Duluth laboratory with these questions. She may be
reached at (218) 720-5529. Over the summer EPA intends to publish a technical correction
notice in the Federal Register for the three manuals and the part 136 rule. These corrections
will not substantively change the content of the rule or manuals. During the next few
months, EPA will be preparing that notice, and the list of corrections. Therefore, if you, or
your staff are aware of mistakes or errors, please contact Teresa Norberg-King in Duluth'on
or before June 30, 1996. In addition, the Office of Wastewater Management is working with
the Regions and labs to provide training on the conduct of these methods, as well as permit
wriung. For more information on this training effort, please contact Jim Taft at (202) 260-
9296. If you have any additional questions, or wish to discuss this further, please contact me
at (202) 260-5400.

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QUALITY ASSURANCE
AND QUALITY CONTROL
(QA/QC)
INTRODUCTION
The development and maintenance of a toxicity testing laboratory,
whether it is part of a NPDES permit holder's facility or a private
supporting lab, requires a myriad of measurements and practices.
Constant attention to producing quality data is critical. The
objective of any data collection activity, and in particular with EPA
regulatory action, is to assure that the data is of known and
documented quality that is adequate for its intended use. Quality
Assurance and Quality Control (QA/QC) practices are fundamental
to demonstrating satisfactory performance of a laboratory. So what
is QA? What is QC? What is the difference?
1

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Topics to be Discussed
¦	QA/QC procedures within the test
methods
¦	Overview of EPA Performance Audit
Inspection for WET labs
¦	EPA DMR/QA Program
¦	Update of National Environmental
Laboratory Accreditation Program
(NELAP)

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¦ Quality Assurance: reliability of data
¦ Quality Control: procedures within QA
program
Talking Points:
•	Quality assurance (QA) is the program that assures the
reliability of data.
•	Quality control (QC) is the day to day activities that focus on
actual procedures within the QA program.
•	Quality assurance includes the policies, objectives, principles,
programs and plans.
•	The importance of laboratory's management and staff
commitment is to producing reliable data elements of a QA
program plan.

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THE IMPORTANCE
OF QUALITY ASSURANCE
AND QUALITY CONTROL
WHEN CONDUCTING
WET TESTS

-------
Elements of a QA/QC Program
¦	Appoint a QA/QC officer
¦	Written QA plan with DQOs
¦	Standard operating procedures (SOPs)
¦	Record Keeping
¦	Qualified staff
¦	Suitable space and equipment
¦	Dilution/culture waters
Talking Points:
List of EPA references on QA/QC:
•	Manual for the evaluation of laboratories performing aquatic
toxicity tests, EMSL-Ci EPA/600/6-90/031.
•	Handbook for analytical quality control in water and
wastewater laboratories, EPA/600/4-79/019
•	Methods for chemical analysis of water and wastes,
EPA/600/4-79/020.
•	Quality assurance guidance for laboratories performing aquatic
toxicity tests, Harrass, M.C. And Klemm, D.J., ASTM STP
1306, in press.
5

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Elements of a QA/QC Program
(cont.)
¦	Effluent sampling and handling
¦	Organism health and performance
¦	Test organisms
¦	Test conditions
¦	Test acceptability criteria
¦	Data evaluation
¦	Reference toxicants

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Importance of Appointing QA
Officer
Talking Points:
•	Single focus of responsibility for QA/QC activities
•	May or may not be the person performing the QC activities
•	Depends on size and other activities of laboratory
•	For small labs, the analysts may wear many hats.
•	For large labs, may have regional and corporate QA officers.
•	Permittee labs
7

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Internal Audits
Talking Points:
•	Performed by the QA officer for the facility.
•	One of the most important practices that keeps the lab on
track for producing valid data.
•	Phased approach-audit parts of the analysis system.
•	System wide approach-audit all parts.
8

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Data Quality Objectives
(DQO):
Help ensure that the data collected are
sufficient and of adequate quality for
their intended use.
Talking Points:
•	DQOs are qualitative and quantitative statements specifying
the quality of data needed to support specific decisions.
•	Constraints to acquiring data
•	Examples

-------
Data Quality can be Described
by the Following:
¦	Precision
¦	Accuracy
¦	Representativeness
¦	Comparability
¦	Completeness
Talking Points:
•	Precision is a measure of mutual agreement among individual
measurements or enumerated values of the same property of
the sample; can be described by the mean, standard deviation
and coefficient of variation.
•	Accuracy refers to the degree of difference between observed
values and known or actual values. While this is appropriate
for the chemical and physical measurements, it is not
applicable in biological systems. Toxicity is relative rather than
absolute. Only organisms can "measure" toxicity and there is
no absolute reference organism.
•	Representativeness is a measure of the extent to which the
data collected accurately reflect the population or group being
sampled.
•	Comparability is a measure of the confidence with which one
data set can be compared to another.
•	Completeness is the percentage of measurements made that
are judged to be valid according to specific criteria.
10

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Written Description of
Laboratory SOPs
Talking Points:
•	Standard operating procedures (SOPs) need to reflect actual
lab methods and not just be photocopied from EPA's manuals.
•	SOPs can be changed to reflect evolving procedures.
•	SOPs are needed for all data acquiring activities and conditions
necessary for meeting test requirements and producing valid
data:
-culturing and holding organisms
-toxicity test methods
-	instrument calibration
-chemical analyses
-	sample chain-of-custody (COC)
-sample collection, handling, preservation and preparation
-	data analyses
n

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Record Keeping and
Preparation
Talking Points:
•	Many records are generated from chain of custody records to
final report generation.
•	What type of information are recorded on QA practices,
organism culturing, sample condition, additional chemistry
measurements; i.e., hardness, TRC, ammonia, etc.
•	Where are the records kept? Are they secure? Are electronic
files backed up?
•	For additional information, see the section on "Report
Preparation" of a toxicity test manual.

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Qualified Staff
Talking Points:
•	Qualified staff is an important element due to lack of
instrumentation for producing final toxicity results.
•	Great reliance is dependent on the skill and judgment of the
analyst.
•	Qualifications, training and experience should be documented.

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Lab Space and Equipment
Talking Points:
•	The facility must be suitable for conducting tests.
•	Should not be collocated with chemistry lab.
•	There should be separate rooms for culturing and toxicity
testing areas.
•	Adequate space is needed to reduce crowding.
•	The facility must be well ventilated and free of fumes.
•	For additional information, see the section on "Facilities,
Equipment, and Supplies" of a toxicity test manual.
14

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Instrumentation, Equipment
and Supplies
•Myriad of instruments are involved (e.g., pH meters, DO meters,
conductivity meters, refractometer, thermometers, photometer,
weighing balance, drying ovens, environmental chambers,
refrigerators, mechanical shakers, spectrophotometers, centrifuge,
waterbath, air pumps and compressors, water purification system,
titrators).
•	Some are used daily; others occasionally depending on the actual
test procedure.
•	Is the instrument calibrated? For example with each use, pH
meters need to be calibrated with at least two buffers; buffers
need to be periodically replaced with fresh solutions; records on
the calibration, maintenance need to be kept.
•	Another example is thermometers. Depending on the type of
thermometer, they should be calibrated two to four times a year.
QC on thermometers are frequently found to be deficient. Either
instruments are of poor quality or calibrated wrong.
•	Plastic or glass for transferring, measuring, and holding solutions,
and test containers; materials must be made of non toxic
materials; if reused must be thoroughly cleaned; avoid all materials
that may absorb potential toxicants

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Condition of Equipment
Talking Points:
•How is equipment maintained and serviced?
•	How frequently? Who does the servicing?
•	For additional information, see the section on "Facilities,
Equipment and Supplies" of a toxicity test manual.

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Instrument Calibration
Talking Points:
•	Instruments used for routine measurements of chemical and
physical parameters, such as pH, DO, temperature, etc. must
be calibrated and standardized according to instrument
manufactures procedures.
•	Calibration data are recorded in a permanent
•	Frequency, at what values, who performs it,
reagents.
•	Major item that is commonly lacking is what
out of specifications?
log book,
with what
happens when it's

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Laboratory Waters
¦	Culture water
¦	Diluent reconstituted water - fresh or
saline
¦	Reagent water
Talking Points:
•	Wide variety of source waters are used depending on its
intended use, may be city water, well waters, natural waters
(salt or fresh).
•	Waters used for reconstituted water must be deionized
•	Culture water may be reconstituted water, natural water or
dechlorinated city water.
•	QC practices on lab pure waters: TRC, conductance (or
resistivity), pH, periodic metal analyses and organic chemicals;
most parameters are performed on each batch; metals and
organics (e.g. organochlorine pesticides and PCBs) at least
yearly or whenever problems are encountered.
•	For additional information, see the section "Dilution Water" of
a toxicity test manual.
18

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Receiving Waters
Talking Points:
•	Some states/regions require upstream uncontaminated water
for diluent water. This depends on the objectives of the test.
•	These need to be non toxic and able to meet the minimum test
acceptability criteria specified for that method.

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Effluent Sampling and
Handling
Talking Points:
•	Location of sampling points
•	Type of sample, grab or composite
•	Type of container
•	Sample preservation
•	Sample shipping or transportation
•	Sample holding times
•	Minimum amount of information required for sample receipt
•	Chain of custody requirements
•	Criteria for rejecting samples
•	For additional information, see the section on "Effluent and
Receiving Water Sampling, Sample Handling, and Sample
Preparation for Toxicity Tests" of a toxicity test manual.
20

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Source and Condition of
Organisms
Talking Points:
•	How and where are organisms cultured? Instructions for
culturing, holding and/or handling the recommended test
organisms are included in specified test method sections.
•	Taxonomic identification is paramount.
•	Must use the age that is specified in the test method.
•	A group of organisms must not be used for a test if the
organisms appear to be unhealthy, discolored, or otherwise
stressed, or if mortality appears to exceed 10% preceding the
test.
•	For additional information, see the section on "Test
Organisms" of a toxicity test manual.
21

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Organism Health and
Performance
Talking Points:
•	Since the organism is the detector in these tests, we need to
know if the organisms are performing as expected.
•	There are a variety of test endpoints depending on the type of
test and species (e.g., survival, growth).
•Test organisms should appear healthy, behave normally, feed
well, have low mortality in culturing, holding and in test
controls.
•	Types of test endpoints: in culture type waters need to know
acceptability such as mortality, growth, reproduction, percent
fertilization.
•	One common requirement is that the organism must achieve
acceptable survival, growth etc. in the controls.

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Test Conditions
Talking Points:
•	While overall conditions are specified, there are different
approaches to achieving those conditions.
•	For example in temperature control, while 20° C may be
specified test condition, it may be achieved by incubators,
environmental chambers, waterbaths, or environmental
controlled rooms.
•	Water temperature and salinity must be maintained within the
limits specified for each test.
•	Specifying EPA method is not good enough.
•	Each test method specifies the required number of replicates.
•	Randomization is an important component of testing.
Randomization techniques should be employed at the start of
the test, including the randomization of the placement of test
organisms in the test chambers and randomization of test
chambers location within the array of chambers.
23

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Test Acceptability Criteria:
Performance of the Control
Organisms
¦ Acute and chronic test endpoints
Talking Points:
•	Acute tests must have 90% or greater survival in the controls.
If there is not 3 90% survival, test is invalid and must be
repeated.
•	Chronic test endpoints depends on the test species and
endpoint for the test to be acceptable:
-fathead minnow must achieve 3 80%survival & >.25 mg
growth
—	Ceriodapnia must achieve 3 80%survival & 3 15 young per
female
-mysid 3 80% survival & >.20 mg & if >50% females in
controls then fecundity (egg development)
-	red abalone 3 80% shell development; must have a
statistical significant effect at 56 ug/L zinc; and must
achieve a %MSD of <20%.
24

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Data Evaluation
Talking Points:
•	Flowcharts are detailed in the test manuals.
•	Requirements for the different statistical programs; i.e.,
Spearman Karber vs. Trimmed Spearman Karber are described.
•	Some states guidelines may differ from the EPA procedures.
•	For additional information, see the section on "Toxicity Test
Endpoints and Data Analysis" of a toxicity test manual.

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Reference Toxicants
Talking Points:
•	Reference toxicant tests indicate the sensitivity of the test
organisms being used and demonstrate a lab's ability to obtain
consistent test results with the test method.
•	Reference toxicants such as zinc sulfate, cadmium chloride,
copper sulfate and copper chloride, sodium or potassium
chloride are suitable reference toxicants. Note, the west coast
chronic test manual specifies the reference toxicant and
dilutions to be tested for each method.
•	It is the lab's responsibility to demonstrate its ability to obtain
consistent, precise results with reference toxicants before the
lab performs toxicity tests with effluents for permit compliance
purposes.
26

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To Demonstrate Ongoing
Laboratory Performance,
Reference Toxicant Tests are
Conducted
Talking Points:
•	Reference toxicant tests are multi-dilution tests with a known chemical that
gauges the sensitivity of the organism
•	Prior to the test or concurrent with the compliance test, reference toxicant
tests are performed.
•	Reference toxicants are performed for each species and test condition.
•	Types of reference toxicants: NaCI, KCI, CuS04,SDS (prepared internally by the
lab; using the same chemical formulation used for each control chart).
•	The reference toxicant is tested using the same concentrations from test to
test under the same test conditions (i.e., the same test duration, type of
dilution water, age of test organisms, feeding) and the same statistical analysis
as the effluent test.
•	The frequency of reference toxicant testing depends on whether the organisms
are cultured in-house or obtained from an outside source. If the lab obtains the
test organisms from an outside source (e.g., organism supplier) then a
reference toxicant test must be conducted concurrently with the effluent test.
If the lab maintains in-house cultures, a reference toxicant test must be
conducted at least once a month. It is preferred, that this reference toxicant
test be performed concurrently with an effluent toxicity test.
•	In-house cultured organisms and outside supplier.
•	Preparation of control charts should be prepared for each combination of
reference toxicant, test species, test conditions and endpoints.
•	What is to be done when things are not in control.
27

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Control Charts are Developed
for Each Test Condition and
Test Species
Talking Points:
•	For example, if permittee was required to perform acute and
chronic Ceriodaphnia tests, the lab would have a control chart
for both types of tests.
•	For a laboratory that performs a wide variety tests for many
permittees, each test condition and species requires a control
chart.
•	However, many permittees' test results (at the same lab) that
have the same test conditions as specified in the permits can
utilize the same control chart.

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Control Charts
OOW7HOL 
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Take Home Points
Labs must have a QA plan that
addresses all data
If QA/QC practices show something
that is not correct, corrective actions
must take place
Invalid tests must be rerun
To keep the lab on track, lab
management should conduct routine
internal audits

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States with Certification
Programs for WET or that
Include WET
Talking Points:
•	Region 1
No States have a certification program that includes WET
•	Region 2
New Jersey
Contact: Betty Boros-Russo
Bureau of Watershed Permitting New Jersey
Dept. of Environmental Protection CN-029
Trenton, NJ 08625
Phone: 609/633-3869
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• Region 3
Virginia
Contact: Deborah Debiasi
Toxics Management Program
Office of Water Permits Support
Virginia Dept. of Environmental Quality
P.O. Box 1009
Richmond, VA 23240-0009
Phone: 804/698-4028
{Virginia does not have a "true" certification program. However, the State
regulation requires that the permittee have approved protocols before WET test
results are acceptable. The permittees contract laboratory must submit SOP's,
reference toxicant test results, etc. to State for approval. If approved, the
contract lab must submit at a minimum, quarterly reference toxicant results. The
State does not conduct inspections.)
• Region 4
North Carolina
Contact: Matt Matthews
Data Assessment & Certification
Water Quality Section
Division of Environmental Management
NC Dept. OF Environmental Health & Natural Resources
4401 Reedy Creek Road
Raleigh, NC 27607
Phone: 919/733-2136
South Carolina
Contact: Nelson Roberts, Jr.
Bureau of EQC Laboratories
SC Dept. of Health & Environmental Control
Building #9, P.O. Box 72
State Park, SC 29147
Phone: 803/935-6857
32

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Florida
Contact: Karl Kircher
Office of Laboratory Services
State of Florida
Dept. of Health & Rehabilitative Services
P.O. Box 210
Jacksonville, FL 32231
Phone: 904/791-1500
Region 5
Wisconsin
Contact: Debra Piper
Office of Technical Services
Wl Dept. of Natural Resources
101 South Webster Street
Madison, Wl 53707-7921
Phone: 608/264-89850
Region 6
Arkansas
Contact: Jeff Ruehr
Arkansas Dept. of Pollution Control & Ecology
P.O. Box 8913
Little Rock, AR 72219
Phone: 501/682-0955

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Louisiana
Contact: Louis "R.C." Johnson
LA Dept. of Environmental Quality
P.O. Box 82215
Baton Rouge, LA 70882
Phone: 504/765-2953
This certification program is tentative at present. The regulations for certification
are to go to Public Notice in October 1996 and the State hopes to begin their
certification program in July 1997. Louisiana's certification program will only
include commercial labs.
Oklahoma
Contact: Anthony Bright
Environmental Laboratory
OK Dept. of Environmental Quality
1000 NE 10th Street
Oklahoma City, OK 73117-1212
Phone: 405/271-5240
• Region 7
Iowa
Contact: Jack Kennedy
University Hygienic Laboratory
H.A. Wallace Building
University of Iowa
900 East Grand Ave.
Des Moines, Iowa 50319
Phone: 515/281-5371
(The University of Iowa has been contracted by the Iowa Department of Natural
Resources to conduct the certification program. The program is pending
legislative decision.)
34

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Kansas
Contact: Aurora Shields
Kansas Health & Environmental Laboratories
Laboratory Improvement Section
Forbes Field, Building 740
Topoeka, KS 66620
Phone: 913/296-6198
Region 8
Utah
Contact: Craig Odekirk
Laboratory Improvement Branch
Utah Dept. of Health
46 North Medical Dr.
Salt Lake City, Utah 84113
Phone: 801/584-8468
Region 9
Arizona
Contact: Gary Brussels
Arizona Dept. of Health Services
Office of Environmental Laboratory
Licensure & Certification
3443 N. Central, Suite 810
Phoenix, AZ 8501 2
Phone: 602/255-3454
California
Contact: Steve Boggs
California Dept. of Health Services
Environmental Laboratory Accreditation Program
601 North 7th St.
Sacramento, CA 94234
Phone: 510/540-2800 or 916/323-4769

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• Region 10
Washington
Contact: Dale VanDonsel
Washington State Dept. Of Ecology
Environmental Laboratory Accreditation Program
P.O. Box 488
Manchester, WA 98366
(360) 895-4649
36

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Performance Audit Inspections
at Laboratories Performing
WET Tests
¦	Objective is to ensure that lab is
providing valid self monitoring data
consistent with NPDES permit
requirements.
¦	Regulatory authority
- 40 CFR Part 1 22.41 (i)(3) and 123.26
Talking Points:
•	Objective of performance audit inspections is to ensure that the laboratory is
providing valid self-monitoring data and performing analyses in a matter consistent
with the NPDES permit requirements.
•	Laboratory OA is required by 40 CFR Section 1 22.41(e) (conditions applicable to all
permits), which states that adequate laboratory and process controls, including QA
procedures, must be provided. Each permittee's laboratory should have a QA/QC
program.
•	The authority to inspect a laboratory can be found in these two documents:
40 CFR Part 112.41 (i) discuss inspection and entry. Part 3 states "Inspect at
reasonable times any facilities, equipment (including monitoring and control
equipment), practices, or operations regulated or required under this permit..."
Section 308(a) Part(B) of the Clean Water Act states that "the Administrator or
his authorized representative..., upon presentation of his credentials-(i) shall have
a right of entry to, upon, or through any premises in which an effluent source is
located in or which any records required to be maintained under clause (A) of this
subsection are located...
37

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While Section 308 and 40 CFR 122.41 provides inspection authority, 40 CFR
123.26 specifies three objectives that should be met during a routine compliance
inspection. According to this section, the inspection should be performed in a
manner which:
1)	Determines compliance or noncompliance with issued permit
conditions and other program requirements
2)	Verifies the accuracy of information submitted by permittees
3)	Verifies the adequacy of sampling and monitoring.
In the absence of unusual circumstances, routine compliance inspections should be
conducted only with reference to a permit.
References:
•	"EPA Manual for the Evaluation of Laboratories Performing Aquatic Toxicity Tests"
EPA/600/4-90/031, January 1991
•	"NPDES Compliance Inspection Manual" EPA/300/B-94/014, September 1994
•	"NPDES Compliance Monitoring Inspector Training" Modules
"Laboratory Analysis", August 1990
"Biomonitoring", August 1990
"Legal Issues", August 1990/600/4-79/020, Revised March 1983
•	"Methods for Chemical Analysis of Water & Wastes" EPA
•	"Handbook for Analytical Quality Control in Water & Wastewater Laboratories"
EPA/600/4-79/019, March 1979
•	"Technical Support Document for Water Quality-based Toxics Control" EPA/505/2-90-
001, March 1991, Appendix B-4
•	"Compliance Monitonng and Enforcement Strategy for Toxics Control"
•	Quality Assurance Guidance for Compliance Monitonng in Effluent Biological
Toxicity Testing", Memorandum from David Lyons to Compliance Branch Chiefs
3/7/90
•	"Conducting a Performance Audit Inspection of a Laboratory Performing Toxicity
Testing", Videotape, Produced 9/94
38

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Elements of the Inspection
Regulatory authority
Notification of inspection
Opening conference
Records review
Tour laboratory
Closing conference

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Notification of Inspection
¦	Have permittee contact their
contract laboratory
¦	This can be done by phone or
308 letter
Talking Points:
•Approximately two weeks before the inspection, the permittee
can be notified by a Section 308 letter or phone that the
facility's contract laboratory is scheduled for a Performance
Audit Inspection on a specific date, if applicable. Explain to
the permittee that they are responsible for the data that their
contract laboratory generates.
•	If the exact date of the inspection is given to the permittee, it
is a good idea to have the permittee tell their contract
laboratory to call the inspector. This will allow the inspector to
tell the laboratory what records are needed and will help
expedite the inspection.
•	The EPA inspector must be certain that the appropriate State
regulatory agency is notified in a timely manner of inspections
to be conducted in its jurisdiction.
40

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Opening Conference
¦	Present credentials
¦	Explain right of entry if necessary
¦	Discuss purpose and order of
inspection
¦	Explain that EPA is only inspecting
those test(s) required in the permit
Talking Points:
•	Outline the objectives and inform the laboratory personnel of
the purpose and scope of the inspection. Discuss the order of
the inspection, (e.g. records review first then a laboratory
tour).
•	This is a good time to get a list of employees and job titles,
involved in the toxicity testing program. This is also a good
time to find out if the lab has a QA/QC officer.
41

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Records Review
¦	Copy of NPDES permit and current
method manuals
¦	All SOP's and QA/QC manual
¦	Reference toxicant control charts
¦	All logbooks - equipment, organism
¦	Chain-of-custody records
Talking Points:
•	A review of facility records should determine that record keeping requirements are
being met. Make sure the laboratory has a current copy of the NPDES permit for their
client. This will ensure that the proper testing requirements are being met.
•	All laboratory data should be recorded on a real-time basis and original data sheets
should be signed and dated by the personnel performing the tests. Records should be
kept in bound notebooks. It s a good idea to randomly pick a completed test report
and trace the data back through the records. For example, you can check the
equipment calibration logbooks (DO, pH, conductivity, etc.) to determine if the
equipment was calibrated on the days the test was conducted. You can cross
reference the animal receipt logbooks with age of test organisms at start of test to
determine if organisms were within the required ages.
•	A review of the chain-of-custody records will indicate if the test was started within
the 36-hour holding time requirement and will usually indicate if the arrival
temperature was greater than 4° C. Chain-of-custody records should also be
complete with no break in the custody signatures.
•	A review of the reference toxicant control data will indicate if any outliers occurred
with the batch of organisms used in the test.
•	SOP's and QA manuals should be reviewed for completeness and accuracy. The
QA/QC manual should identify clearly the individuals involved in the QA programs and
their responsibilities and should document the laboratory's Standard Operating
Procedures that meet user requirements in terms of specificity, completeness,
precision, accuracy, representativeness, and comparability. There should be an SOP
for every operation associated with the toxicity testing lab. Compare SOP's with
what is actually done in the laboratory.
42

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Records Review (cont.)
Evaluate raw data
Taxonomic ID of organisms
Personnel training records and
resumes
DMR/QA study results
Statistical programs

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Tour Laboratory
¦	Inspect sample tracking and storage
¦	Inspect facilities and equipment
¦	Observe lab personnel conduct a test,
if possible
¦	Inspect organism cultures
Talking Points:
•	It is helpful if the inspector pretends they are a sample that has
just entered the laboratory. Have the lab personnel "process"
you through their laboratory. This is a good way to observe
sample check-in and sample storage. The inspector should
observe all equipment used for toxicity testing to ensure it is in
good working order. If possible, it would be good to observe a
technician performing a test. Make note of where testing
occurs as well as where culturing takes place. Observe all
cultures and how lab analysts handle test organisms.
•	Some questions to ask: Is proper light intensity maintained in
testing areas? Is water temperature recorded continuously or
twice daily? How are organisms fed during testing? Does
randomization occur during testing? Are pH buffers used once
or many times for calibration? Are brood boards or mass
cultures used to obtain Ceriodaphnia for a chronic test? What
type of dilution water is used? How is glassware cleaned?
44

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Closing Conference
¦	Discuss major and minor deficiencies
¦	Give recommendations and technical
assistance
¦	Give general content of final report
¦	Discuss how and when final report
will be disseminated
Talking Points:
•	The inspector should use the closing conference to
communicate results of the inspection promptly to the
laboratory management. The discussion should be limited to
specific findings of the visit.
•	Discuss that the final report will be narrative, documenting
deviations from the regulatory requirements as well as
commendations. You can include recommendations that would
improve lab operations. Make it clear to the lab that the report
is sent to the permittee and the permittee is responsible for
sending it to the contract laboratory. Ultimately, the permittee
is responsible for the data submitted to the regulatory agency.
45

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DMR QA
INTRODUCTION
QA/QC activities are ongoing activities at WET laboratories. If there
is a state certification program, state personnel may visit the lab on
an infrequent basis. Performance Audit Inspections (PAI) occur less
frequently. One program whose goal is to evaluate performance on
a yearly basis is the Discharge Monitoring Report Quality Assurance
program (DMR QA). The Discharge Monitoring Report (DMR) is the
actual report form that permittees use to report permit testing
results. The validity of the NPDES program depends on the quality
of the self-monitoring program. The DMR QA is an important tool
to ensure the quality of self-monitoring data.
46

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DMR QA Purpose:
A program to evaluate NPDES
permittees' ability to analyze and
report accurate self monitoring
data.
Talking Points:
•The program begin in 1991 for WET and has evolved using
various organisms and test endpoints
•	Organizations involved:
-EPA Office of Wastewater Enforcement and Compliance
-	EPA Environmental Monitoring Systems Laboratory
-	Regional EPA and State DMR/QA Coordinators
*	Direct contractor support
47

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Participation is Required
¦	Unknown reference toxicant sent to
testing labs
¦	Designed to cover as many permittees
and test conditions as possible
¦	Generalized process
Talking Points:
•	Participation is required based on authority under Section 308
CWA
•	In 1994 over 3500 permittees and 300 labs participated
•	In 1994 19 different tests involving 7 different species
•	The generalized process is as follows: EPA announces plans for
the study and requests permittees to identify the types of WET
tests in their permit and the lab conducting their WET analyses
(for major permittees only)
48

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¦	Contract labs that service multiple
permittees need only conduct one test
for each test type requested
¦	WET labs receive samples and
conduct the required tests
Talking Points:
•	For example: if a lab performs Ceriodaphnia acute tests for 8
permittees, the lab can perform 1 acute test for all 8
permittees.
•Test conditions in the permits must be identical, i.e.,
temperature, duration, dilution water source, etc.
•	Mandatory test conditions are followed.
•	Typically have 45 days to complete tests after sample receipt.
•	Must submit test results by closing date.
49

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Program Requires Biological,
Physical and Chemical Data
Reporting
Talking Points:
•	Biological data depends on test; endpoints may include
mortality, growth, and reproduction.
•	Chemical/physical data are recorded such as, dissolved
oxygen, pH, alkalinity, hardness, conductivity, and
temperature.
•	Statistical data analyses are performed using EPA's flow charts
as specified.
•	Sources of assistance for technical, policy, and study schedule
are identified in package.

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¦
After test completion, lab completes

report form and signs certification

statement
¦
Copy sent to EMSL-Cincinnati and NPDES

permittee
¦
At EPA, reported endpoints are

statistically processed
¦
Acceptance limits are derived for each test

method
¦
Labs falling outside the limits are judged

unacceptable
¦
Results mailed to participants
Talking Points:
•	For DMR QA Study 14, unreasonable values removed for data
set.
•	For point estimates, values were weighted and robust
estimates of the mean and standard deviation calculated for
NOECs; determine median values
•	Acceptance limits are median +/- one test concentration

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¦	Follow up on unacceptable results are
conducted by permitting authority
¦	Unacceptable results can be reporting
errors or analytical errors or both
¦	Need corrective actions at support lab
or permittee
¦	Need for experienced permitting
authority personnel to judge the
adequacy of the labs response to
unacceptable test results
Talking Points:
•	Request explanation of unacceptable test results from
permittee.
•	Reporting errors involve improper filling out of report form.
•	Typically, the labs did not follow instructions or failed to send
report.
•	Analytical errors result from not following instructions, not
preparing concentration properly, or where test organisms
were too sensitive or not sensitive enough.
•	Additional follow up needed if initial lab/permittees response is
inadequate.
52

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Future of the DMR QA Program:
¦	May be privatized
¦	Downsized
¦	Combination of solutions
¦	Remain the same
Talking Points:
•	EPA does not want to be financially involved.
•	Next year's study will be similar to this year's study.
•	Will not change for a year or two.
•	Options are currently being evaluated.
53

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NATIONAL ENVIRONMENTAL
LABORATORY ACCREDITATION
PROGRAM (NELAP)

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Overview of first conference,
February 1995
Structure
Talking Points:
•	Objective of NELAP is to establish a uniform standard to resolve problems of costly
duplicative assessments and redundant evaluations facing the regulated
community.
•	Draft standards and the constitution can be found in the Federal Register, Volume
59, No. 231, Friday December 2, 1994.
•	All conferees voted to support the national accreditation program. Many issues
that need to be addressed were: reciprocity, private sector input, fees,
supplemental requirements and a variety of analytical issues. The program is
voluntary but states will still have to receive legislative approval to join the
program. As an added incentive, states that join NELAP are given wide authority
over fees and can add supplemental requirements.
•	The constitution and by-laws establish a permanent federal oversight program
staffed by a director and executive secretary.
•	A federal advisory commission-the Environmental Laboratory Advisory Board-has
been established to provide input on the process and work with the NELAP director
to develop a national plan. This is the mechanism by which private sector and
local government consensus advice can be solicited by NELAP and EPA.
•	Under the NELAP adopted at the conference, a national conference will serve as a
standards-setting body supervised by a board of directors. The conference is
modeled after the National Conference on Weights and Measures with voting
authority delegated to state and federal officials. At the first conference, five
additional non-voting positions to be selected from private industry have been
added to each committee.
55

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Committees
¦	Policy & structure
¦	Accrediting authority
¦	Quality systems
¦	Proficiency testing
¦	On-site assessment
¦	Accreditation
¦	Regulatory
¦	Education & outreach
Talking Points:
•The NELAP establishes eight standing committees with the following
missions:
-	Policy & Structure-Develop modifications to structure and requirements
of fields of testing.
-Accrediting Authority-Provide standards used by EPA to approve state
authorities.
-Quality Systems-Establish uniform quality system including detailing
personnel and recording keeping requirements
-Proficiency Testing-Determine requirements for performance evaluation
programs specifying scoring, kinds of samples and frequency of
samples under the program.
-On-Site Assessment-Develop training and education requirements for
assessors and procedures for conducting audits.
-Accreditation-Consider reciprocity issues and institute requirements for
accreditation, suspension, and revocation.
-	Regulatory-Develop a fee structure, address reciprocity and statutory
authority issues for participating states.
-	Education and Outreach-Liaison with states and media and develop
cost benefit analysis for each participating state.
56

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Future of NELAP
¦ Second annual meeting July 22-24,
1996, Washington, DC
Talking Points:
•	An interim meeting was held December 6-8, 1995 as a
working session to discuss proposed changes and additions
prior to this annual meeting.
•	NELAP standing committees are regularly meeting to develop
the structure and implementation of the proposed program.
These efforts will be the basis of the next annual meeting.
57

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QUALITY ASSURANCE PROGRAMS STATUTORY AND REGULATORY CONSIDERATIONS
Discharge Monitoring Report-Quality Assurance (DMR-OA1
The DMR-QA program evaluates the ability of a major NPDES permittee to analyze and report data
This program is intended to improve overall analytical performance for self-monitoring data.
Authority for requiring participation is granted under section 308(a) of the Clean Water Act. In the
DMR-QA program, major permittees who have effluent toxicity limits or effluent toxicity self-
monitoring requirements are required to analyze "blind" reference toxicant samples with the type of
toxicity test required in their NPDES permit. The permittees' results are compared to the true value
and an evaluation of the reported data is sent to the permittees. Permittees are expected to use the
same personnel and methods employed for reporting NPDES data to analyze the samples. Permittees
are required to follow the instructions for reporting results and include a signed certification statement
in accordance with 40 CFR Part 122.22.
Toxicity samples, unlike the chemistry samples, are shipped direcdy to the laboratory performing the
tests, either an in-house laboratory or a commercial laboratory. The list of toxicity support
laboratories is generated from the information received from the announcement letters sent to the
permittees. It is the permittee's responsibility to notify the laboratory that they will be receiving the
toxicity samples. The laboratories are only required to perform the type of tests required in the
permit, not all of the tests available.
Both the permittee and the support laboratory are responsible for submitting the toxicity test results
by the designated due date. Support laboratories must submit results to the permittee and the EPA
contractor coordinating the DMR-QA study. Permittees that perform their own toxicity tests are
required to submit their data twice, once on the toxicity data report form and once on the permittees
data report form. Instruction packages received by both the permittee and laboratory contain the data
report forms and further instructions on reporting requirements.
WET testing DMR-QA results are compiled annually by the EPA contractor coordinating the study.
Permittees, EPA Regional Offices, and State coordinators receive performance evaluation reports on
the DMR-QA study results approximately 5 months after the data is reported. Regulatory agencies
(states and EPA) can conduct follow-up investigations to address poor or incomplete DMR-QA
results, failure to participate, or late submittal of DMR-QA results.
Permittees (or contract support laboratories) that receive reports evaluating their results as "not
acceptable" or "unusable" must submit a written response explaining the reason(s) for these results.
This letter should be submitted to the state and/or regional DMR-QA coordinator.
The general schedule for the DMR-QA study is outlined below. Tasks in italics indicate those tasks
to be conducted by the EPA contractor coordinating the DMR-QA study; those in normal format are
those tasks required by the permittee. Since the study schedule spans two fiscal years, years one and
two are labeled as FY 1 and FY 2.
1

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TABLE H. DMR-QA STUDY MILESTONES
ACTION ITEM:
DUE DATE:
Study announcement letter sent to participants
November FY 1
Name & address of toxicity laboratory performing tests
submitted to EPA contractor
Late December FY 1
Samples shipped to participants
January - February FY 2
Analyses performed
Approximately 7 weeks
Results from participants due to EPA contractor
March - April FY 2
Report mailed to participants and DMR-QA Coordinators
August - September FY 2
Corrective action letters (written response) due to study
coordinator
October FY 2
Contacts. Technical assistance with toxicity test conditions, data reporting, and instructions
assistance should be addressed to John Helm, the EPA Headquarters contact for the toxicity testing
DMR-QA program, at (202) 564-4144 (EST).
The EPA contractor coordinating the DMR-QA study from September 29, 1994 to September 30,
1999 is ManTech Environmental Technology, Inc. The contractor should be contacted for study'
schedule, sample shipment, and the availability of additional reference toxicants. The ManTech
contact for regional and state coordinators is Terry Bundy at (919) 818-5743 (EST). The ManTech
contact for permittees is Stewart Nicholson at (919) 406-2164 (EST).
The Regional coordinator or state coordinator should be contacted for the study schedule, corrections
in permittee information, and technical assistance. The state and EPA Region contacts are listed
below.
2

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North Carolina
Biological Laboratory Certification/Criteria Procedures Document
North Carolina Department of Environment,
Health, and Natural Resources
Division of Environmental Management
Water Quality Se9tion ] (
This document has been approved for release
A. Preston SowarS, Jr., Director
	^
Date

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NORTH CAROLINA DIVISION OF ENVIRONMENTAL MANAGEMENT
BIOLOGICAL LABORATORY CERTIFICATION/CRITERIA PROCEDURES DOCUMENT
These procedures are part of the State of North Carolina's response to requirements set forth by the
National Pollutant Discharge Elimination System (NPDES). This document supports the Department
of Environment, Health, and Natural Resources' Administrative Code Section 15A NCAC 2H.1100.
Specific laboratory facility and equipment requirements, quality assurance requirements, standard test
methods/procedures, standard toxicity test reporting forms, and standard scientific reporting units
pertaining to Biological Laboratory certification are described here. Procedures presented here and in
subsequent versions are approved by the Ddirector before being released to the public.
METHODS AND PROCEDURES
The following documents describing NPDES test methods and procedures are recognized as
standard and shall be used to measure the reporting units listed below:
(1)	"Short Term Methods for Estimating the Chronic Toxicity of Effluents and Receiving
Waters to Freshwater Organisms," Second Edition, EPA/600/4-89/001 or subsequent
versions.
(2)	"Methods for Measuring the Acute Toxicity of Effluents to Freshwater and Marine
Organisms," Fourth Edition, EPA/600/4-90/027 or subsequent versions.
(3)	"North Carolina Ceriodaphnia Chronic Effluent Toxicity Procedure," December 1985,
Revised November 1995 or subsequent versions.
(4)	"North Carolina Pass/Fail Methodology for Determining Acute Toxicity in a Single
Effluent Concentration, Revised September 1994 or subsequent versions.
(5)	"North Carolina Phase II Chronic Whole Effluent Toxicity Test Procedure," Revised
November 1995, or subsequent versions.
(6)	Any other toxicological monitoring methods approved by the Director under 15 NCAC
2B.0211 or any additional methods approved and published by the Environmental
Protection Agency.
(7)	"Standard Operating Procedures - Biological Monitoring - Environmental Sciences Branch
- Ecosystems Analysis Unit - Biological Assessment Group," February 1990 or
subsequent versions.
(8)	Any biological field survey analyses which either quantify or enumerate resident aquanc
populations and used to evaluate attainment of Water Quality Standards as defined in 15
NCAC 2B.0211 or 15 NCAC 2B.0212.
2

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LABORATORY FACILITY ANjp EQUIPMENT RFnTTTPPA/fnxrrg
Laboratory facilities and equipment considered as minimum laboratory resources are as follows
(1) Aquatic Toxicology Laboratory
(a)	A minimum of 200 square feet of laboratory space.
(b)	A minimum of 20 linear feet of laboratory bench space.
(c)	A drained sink with hot and cold running water.
(d)	Adequate control of culture environment (i.e. lighting, cooling and heating) to maintain
appropriate organism requirements.
(e)	A refrigerator of adequate size which will maintain sample temperatures between 0°C
and 4°C.
(f)	Current copies of the procedures documents written by EPA and North Carolina's
Water Quality Section (see Methods section for references).
(g)	Glassware, chemicals, supplies and equipment to perform any procedures included in
requested certification.
(h)	Instrumentation capable of measuring dissolved oxygen, pH and temperature directly
from test vessels of any procedure included in certification application. Equivalent
surrogate vessels may be utilized for physical measurements if injury to test organisms
may result.
(i)	Instrumentation or analytical capabilities to perform measurements of total
chlorine to a level at least as low as 0.1 mgfl and total hardness to a level at least as low
as 1 mg/1.
0) A dissecting microscope-and a compound microscope with a minimum magnification of
lOOOx for those laboratories maintaining either of the categories of Acute Toxicity
Testing/Invertebrate or Chronic Toxicity Testing/Invertebrate.
(k) A balance capable of accurately weighing fish larvae to 0.00001 g and Class "S" or
equivalent reference weights for those laboratories maintaining certification for the
category Chronic Toxicity Testing/Vertebrate.
(1) Viable reproducing laboratory cultures of any test organisms included in the
certification application. Use of test organisms for regulatory purposes not maintained
as a viable laboratory culture may be accepted on a case by case basis upon receipt of
written permission from the State Laboratory.
(m) Appropriate dilution water for use in whole effluent toxicity testing with chemical
characteristics such that the pH is between 6.5 and 8.5 S.U. and total hardness as
calcium carbonate is between 30 and 50 ppm. Should receiving waters have
characteristics outside of staled ranges then alternate pH and hardness ranges may be
3

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accepted upon demonstration that the alternate ranges axe better suited to testing
objectives and quality assurance standards have teen met
(n) Appropriate Chain-of-Custody documentation forms and seals.
(2) Aquatic Population Survey and Analysis Laboratory
(a)	A minimum of 150 square feet of laboratory space.
(b)	A minimum of 8 linear feet of laboratory bench space.
(c)	Binocular dissecting microscopes and compound microscopes suitable for survey type.
(d)	Vials, preservatives, and space to maintain representative sample collections for at least
one year after collection.
(e)	Current taxonomic guides and references specified by the Division.
(f)	Appropriate chain of custody documentation, laboratory records and seals are to be
available.
(g)	Sampling equipment to support collection of appropriate biological organisms.
(h)	Settling tubes and an inverted microscope with a minimum magnification of 300x for
those laboratories maintaining certification for die parameter Algae.
QUALITY ASSURANCE REQUIREMENTS
Emphasis is placed on good laboratory practices and proper documentation. Additional quality
assurance requirements to those found in the previously cited documents are as follows:
(1)	All instruments used in or associated with toxicity testing are to be calibrated daily or with
each use and recorded in a designated notebook (i.e.. automatic sampling equipment, pH
meter, D.O. meter, conductivity meter, etc.)
(2)	A minimum of five valid reference toxicant tests must be performed and entered on a
control chart for each organism and test type for which a lab is certified. A maximum of 30
datapoints are to be entered on the control chart
(3)	A reference toxicant test should be performed every two weeks for each organism used in
acute whole effluent toxicity testing, or alternatively, acute reference toxicant tests may
performed such that NC NPDES acute tests are performed within one week of an acute
reference toxicant test for the organism in question. In the case of the latter, to maintain
acute certification for an organism, acute reference toxicant tests must be performed, at
minimum, on a quarterly frequency.
(4)	A reference toxicant test should be performed once per month for each organism used in
chronic whole effluent toxicity testing, or alternatively, tests may be performed such that
NC NPDES chronic tests are performed within two weeks of a chronic reference toxicant
test for the organism in question. In the case of the latter, to maintain chronic certification
4

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for an organism, chronic reference toxicant tests must be performed, at minimum on a
quarterly frequency.
(5) Acceptable alternative culture media utilized to culture tfc algae Stlensmm, cipncomumm
for use as Ceriodaphnia food are:
(a)	Tie MBL medium as described by HandhooV of Phvrniopira,	r,.,...
Methods and r.rnwth 1973. j.Slein ed University
Cambridge, Mass.
(b)	Additional nutrients may be used in the preparation of algae medium described in
Section 12, subsection 8.2.5 of EPA/600/4-89/001. Specifically, the volume of
nutrient stock solutions found in Table 1 on page 115 of that document may be
adjusted so that solutions 1,2, and 5 are added at a rate of 2 ml/1 and solutions 3 and 4
are added at a rate of 6 ml/1
(6)	A representative of each test organism cultured shall be taonomically identified to the
species level at a minimum frequency of once per quarter. The specimen shall be preserved
and held for a minimum of one year.
(7)	If closed incubators are utilized for toxicity testing and/or test organism culturing purposes
cultunng and testing activities may not be contained within the same incubator.
(8)	Effluent samples collected for chronic tests are to be used within 72 hours of collection.
TTie beginning of this period is defined as the time of the collection of a grab sample or the
time of collection of the last subsample of a composite sample, to the time that the
organisms are introduced to the test solution or the last renewal of the test solution.
(9)	A single sample log will be maintained for all samples entering the laboratory which lists,
at a minimum, sample identity including any sample numbers), sample temperature at
receipt, time and date of receipt, name of person received from, and name of person
receiving sample.
PROCEDURE MODIFTrATTDMC
Modifications from test protocols from the cited EPA documents Mow. These modifications are
in addition to those specified in individual procedures documents. References to the EPA
manuals are given to provide context to the modification being made to the EPA method.
(1)	Acute toxicity tests using Ceriodaphnia dubia, Daphnia pulex, and Pimephales promelas
will be conducted at 25±1°C. (Ref. EPA/600/4-90/027, pp. 56-61, Tables 11-13.)'
(2)	Organisms used in acute toxicity tests will have food made available for a minimum of two
hours pnor to initiation of testing. For cladoceran species, this feeding will be a minimum
of 0.05 ml of YCT and 0.05 ml of a solution of the algae Selenastrum capricornutum (with
5

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a cell concentration of 1.71 X 107 cells/ml) per 15 milliliters of culture solution. (Ref
EPA/600/4-90/027, pp. 56-61, Tables 11-13.)
(3)	Fathead minnows used in acute toxicity tests will be 1 to 14 days in age, and 72 hour range
in age. (Ref. EPA/600/4-90/027, pp. 60-61, Table 13.)
(4)	For each sample used in a toxicity test, pH, specific conductance, and total residual
chlorine will be measured and recorded from an undiluted aliquot the day of the test.
Dissolved oxygen and pH will be measured in the control and the highest toxicant
concentration tested at the beginning of the test, prior io and following each renewal, and at
the termination of the test (Ref. EPA/600/4-90/027, p. 71, 10.2.1.4.)
(5)	Ceriodaphnia dubia used in toxicity tests will be obtained from individual cultures, from
third or subsequent broods of adults not being more than 14 days in age, containing eight
or more neonates, with a average adult mortality not exceeding 20% per culture board.
(Ref. EPA/600/4-90/027, p. 138, 3.7.6.)
(6)	Chronic Ceriodaphnia dubia analyses will have an additional test acceptability criterion of
complete third brood neonate production by at least 80% of the surviving control
organisms. (Ref EPA/600/4-89/001, p. 124, Table 3.)
(7)	Ceriodaphnia dubia neonate reproduction totals from chronic tests shall include only
organisms produced in the first through third broods.
(8)	The percentage of male Ceriodaphnia control organisms may not exceed 20% in chronic
Ceriodaphnia tests.
(9)	The Ceriodaphnia control organism reproduction coefficient of variation (CV) must be less
than 40% for a chronic Ceriodaphnia test to be considered acceptable.
(10) "Observed-effect" as referred to in NC DEM chronic Ceriodaphnia procedures documents
will be defined as either
a)	A statistically significant decrease in survival of the treatment organisms as compared to
the control organisms or
b)	A twenty percent or greater decrease in treatment organism reproduction as compared to
control organism reproduction which is also determined to be statistically different from
control organism reproduction.
FORMS
The forms for reporting whole effluent toxicity test results (see attachments) are as follows:
(1)	AT-1 form, entitled Effluent Toxicity Report Form - Chronic Pass/Fail and Acute LC50, is
used for reporting chronic pass/fail toxicity test results or acute LC30S.
(2)	AT-2 form, entitled Effluent Aquatic Toxicity Report Form - Acute Pass/Fail, is used for
reporting acute pass/fail toxicity test results.
6

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(3) AT-3 form, enutled Effluent Aquatic Toxicity Report Form/Phase n Chrome
Cenodaphua. is used for reporting Phase II chronic toxic,ty test results or chrome pass/fail
results.
REPORTING IINTTS
The list of reporting units considered as standard are defined as:
(1)	LC50 - The toxicant concentration killing 50% of exposed organisms at a specific time of
observation.
(2)	NOEC-(No Observed Effect Concentration) The highest or single concentration of
toxicant to which organisms are exposed in a life cycle or partial life-cycle test, which
causes no statistically significant adverse effect on the observed parameters (usually
hatchability, survival, growth, and/or reproduction).
(3)	LOEC-( Lowest Observed Effect Concentration) The lowest concentration of toxicant to
which organisms are exposed in a life cycle or partial life cycle test, which causes a
statistically significant adverse effect on the observed parameters (usually hatchability,
survival, growth, and/or reproduction).
(4)	Chronic V alue (ChV) - A numeric value representing the geometric mean of the numeric
values of concentrations analyzed as the No Observed Effect Concentration (NOEC) and
the Lowest Observed Effect (LOEQ by chronic toxicity testing. The chronic value is an
estimate of the toxicant concentration that will be the actual no effect concentration based on
the chronic effect tested. ChV = Antilog [LoglO LOEC -r LoglO NOEQ/2.
(5)	Biological Water Quality Rating - A rating, ranging from Excellent to Poor, which gives
an indication of water quality based on the composition of the biological community, using
standardized techniques as specified by the Division of Environmental Management
(6)	Total Taxa Richness - The total number of different tax- collected, taken to the lowest
practical taxonomic level.
(7)	EPT Taxa Richness - The total number of different taxa collected belonging to the orders
Ephemeroptera (mayflies), Plecoptera (stoneflies), and Tricoptera (caddisflies), taken to the
lowest practical taxonomic level.
(8)	Diversity - The number and abundance of taxa in a specified location summarized using a
mathematical formula to allow comparisons of community structure.
(9)	ET(x) The relative toxicity of a toxicant measured in terms of the time it takes to elicit a
given response from a given percentage (x) of the exposed test organisms.
(10)	TLM - Median tolerance limit - The toxicant concentration at which 50% of test organisms
survive for a specified exposure time. The term has been superseded by median lethal
concentration (LC50).
7

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(11)	LC(x), EC(x) - Lethal concentration (LC) or effective concentration (EC). A point
estimate of the toxicant concentration that would adversely affect a given percent(x) of the
test organisms.
(12)	Maximum Acceptable Concentration (MATC) - Concentration to be determined within
the interval bounded by the LOEC and NOEC which is used as the concentration of
toxicant predicted to have no detrimental impacts on the test population.
(13)	Toxic Unit Acute - The reciprocal of the toxicant dilution that causes the acute effect by
the end of the acute exposure period, for example: I/LC50.
(14)	Toxic Unit Chronic - The reciprocal of the toxicant dilution that causes no unacceptable
effect on the test organisms by the end of the chronic exposure period, for example:
1/ChV.
8

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State of North Carolina
Department of Environment,
Health and Natural Resources
Division of Environmental Management
James B Hunt, Jr., Governor
Jonathan B Howes, Secretary
A Preston Howard, Jr, P E., Director
8/24/95
Biological Laboratory Certification Application
Prior to submission please attach:
1.	Check for fees.
2.	Personnel Resumes.
3.	Standard Operating Procedures Document
4.	Reference Toxicant Data and Control Charts.

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North Carolina Biological Laboratory Certification Application
Company Name.
Phone
Fax
Physical Street Address
Street	
City.
State.
Zip.
Mailing Address (if different from physical street address)
Street	
City.
State.
Zip.
Contact person from this laboratory to receive correspondence from North Carolina's Water Quality Section
Please list any NC certifications for wastewater chemical analyses or any non-NCDEM biological certifications maintain^ by the
laboratory:
Has this laboratory ever been denied certification or decertified for performing toxicity testing or aquatic population survey and
analysis? Which staie(s) and why?
The following information is necessary for shipment of certification performance evaluation samples:
Preferred Overnight Carrier	 Account#		
(Must be capable of picking up packages in Raleigh, N.C.)

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North Carolina Biological Laboratory Certification Application
Personnel Attach resume for each person listed.
Laboratory Supervisor-Aquatic toxicity testing/Biological survey
Name/Title	
Academic Training.
Professional Certifications (ex. Ecological Society of America)
Substitute Supervisor
Name/Title	
Academic Traming_
Professional Certifications
Other personnel involved with toxicity testing and/or biological survey;
Name/Title
Academic Trammg_
Professional Certifications
Name/Tide
Academic Traming_
Professional Certifications
Name/Title	
Academic Training.
Professional Certifications
Name/Tide
Academic Traimng_
Professional Certifications
Laboratory Specifications
Total laboratory area in square feet _
Total linear bench space m feet _
What is the culture water source for organisms to be nvd in N.C. tests?
What is the dilution water source for North Carolina tests?	

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North Carolina Biological Laboratory Certification Application
Categories Desired for Certification-List parameters(organisms) desired under each category.
Active cultures are required for certification. Regulatory testing performed with organisms not maintained as a viable reproducing
culture may be considered on a case by case basis by the Data Assessment and Certification Group. Currently, riarfr»^»rans must be
cultured 111-house.
	(1) Acute Toxicity Testing/Invertebrate
	(2) Acute Toxicity Testing/Vertebrate
	(3) Chronic Toxicity Testing/In vertebrate
	(4) Chronic Toxicity Testing/Vertebrate
	(5) Aquatic Population Survey and Analysis-Check desired parameters
	 fish
	 maaoinvertebrates
	 algae
	 aquancmacrophytes
	 zoopiankton
	(6) Algal and Aquatic Plant Toxicity Testing
Total number of categories desired for certification:.

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North Carolina Biological Laboratory Certification Application
Certification Rate Schedule
Number of categories	Fee
1	$500.00
2	S900.00
3	S1300 00
4	S 1700.00
5	S2100.00
6	S2500.00
Checks should be made payable to: North Carolina Department of Environment, Health, and Natural Resources.
Total cost of certification renewal:	Check number	
Attach Check here:
Carotaa as ner Nr?r r!P^?^mneiTl'ynins
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North Carolina Biological Laboratory Certification Application
Cultunng These pages may be duplicated as necessary for multiple cultures. Attach additional sheets as required.
Organism	
Original Source	Date(s) obtained	
Describe the cultunng system, including vessels and environmental controls:
Describe cultunng strategy including population control, culture water replacement, food type, feeding frequency and amount, special
substrates, etc.
How is the age of the organisms determined for testing purposes?
What is the source of culture water?	
How is the culture water prepared (treated) before use?
What is the average hardness of culture water when used in the culture?
before being treated?
How is the culture water stored? 	

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	W°"b C'r0li" Biological Labors,pry C.r,iflc„,0n Appli-...-
Wtaanal^ are perfomedon the cjlme wafcr, m wtat 0 4e	of fflese			
Descnbe any additional quality assurance procedures utilized:
What reference toxicant is used to determine population health?
What is the source of this toxicant?	
How often are reference toxicant tests performed?	
What dilution water is utilized for these tests?	
How is each reference toxicant test evaluated graphically?	
What are the consequences of out-of-range values?	
Are EPA certified reference toxicant samples analyzed?.	How often?
Which personnel are responsible for taxonomic identification of the organism7
How often are these identifications accomplished?
What references are used for this identification?
Are representative specimens preserved and/or mounted?.
If so, at what intervals?	

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North Carolina Biological Laboratory Certification Application
Testing These pages should be duplicated for each test type/orgamsm combination applied for.
Category.	Organism 	Test Type	
Describe all environmental systems employed in testing for the control of temperature, light intensity, photoperiod, etc.
List the manufacturer, composition, and volume of testing vessels employed.
List types and composition of dosing equipmentfdiluters, pipettes, etc) employed.
How is dilution water treated prior to use?.
What analyses arc performed on the water, and how frequendy are these conducted?.
How is the diluuon water stored?.
What is the average hardness of the dilution water before treatment?.
after treatment?.

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North Carolina Biological Laboratory Certification Application
List any replicate, blank, or blind analyses performed as part of in-lab quality assurance of toxicity testing (the analyses and response;,
to be detailed in the Standard Operating Procedures Document):

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North Carolina Biological Laboratory Certification Application
Testing Equipment
Any changes m this equipment list which affect the ability to perform testing should be reported to the State Laboratory within 30
days of such change. Failure to do so could result in categorical decertification.
Make Model	Callb. method
NIST traceable thermometer	
D.O meter	
pH meter					
chlorine analysis			
comlucuvity meter			
hardness analysis
refngcraior					
incubator			
dissecung microscope							
compound microscope							
light meter		
Hemacytometer						
autoclave					
centrifuge
shaker table	
spectrophotometer
fluororncter
Additional equipment
List any replicate, blank, or blind analyses performed as part of ui-lab quality assurance of water quality measurements performed in
toxicity testing (the analyses and responses to be detailed in the Standard Operating Procedures Document):

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North Carolina Biological Laboratory Certification Application
Sampling
Will your facility provide sample collection equipment or conduct sampling for North Carolina clients?
Does your facility employ automated sampling equipment7	
If so, list equipment used:
What type of collection vessels and shipping containers will be provided to clients?
What commercial carriers are used for sample shipment?.
Outline sampling procedures, including instructions to sampling personnel, and sample identification:
Are sample temperatures recorded on receipt of sample?	
Are total residual chlonne measurements recorded on receipt of sample?
Is a sample log maintained?	
Chain-of-Custody
Will adhesive seals and/or lockable shipping containers be provided to clients?	
Outline the chain-of-custody instructions provided to clients and the steps taken at your lab upon receipt of the sample:

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North Carolina Biological Laboratory Certification Application
Data Analyses
Lisi data analysis techniques employed for each test endpoint (These should be described in detail in the Standard Operation Procedures
document)	
Describe how data calculations, data entry, and «an«irai analyses are quality a«^airp/f-

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North Carolina Biological Laboratory Certification Application
Aquatic Population Survey and Analysis
General Equipment List
List general sampling and sample identification equipment:
Make	Model	Calib. method (if appropriate)
thermometer
D.O meter
pH meter
conductivity meter
dissecting microscope
compound microscope
Fish
Sample Collection
Will your facility provide sample collection equipment or conduct sampling for North Carolina clients?.
If so what type of collection equipment and shipping containers will be provided to clients?	
What commercial carriers are used to sample shipment?	
List sampling and sample identification equipment:
Make Model	Calib. method (if appropriate)
multi-parameter meter 		
scales			
length measuring device		
backpack shocker		
generator	
nets (list)	

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North Carolina Biological Laboratory Certification Application
Additional equipment:
Describe collection techniques employed, including the purpose of each technique and sample identification (attach additional pages if
necessary):		
Describe sample preservation techniques used:.
Chain-of-Custody
Will adhesive seals and/or lockable shipping containers be provided to clients?.

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North Carolina Biological Laboratory Certification Application
Describe chain-of-custody procedures and the steps taken at your lab upon receipt of the sample:
Is a sample log maintained?	
Identification
Are reference organism collections available for taxonomic review'
Are reference or whole samples from population surveys maintained for at least one year after collection ?_	
Are copies of all taxonomic guides and references specified by the Division of Environmental Management located in the laboratory
(see Appendix)?	
List or append other references used for taxonomy or population analyses:	
Data Analyses
Are you using an Index of Biological Integrity (IBI)? If so, list metrics:
		
2			8._
3"					9._
4"				10.
5"				
6	12.

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North Carolina Biological Laboratory Certification Application
Describe data analysis techniques employed (these should be described in detail in the Standard Operanon Procedures document):
Describe how data calculations, data entry, and statistical analyses are quality assured:
In-lab Quality Assurance
List any replicate, blank, or blind analyses performed as part of in-lab quality assurance of population survey (the analyses to be
detailed in the Standard Operating Procedures Document):

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North Carolina Biological Laboratory Certification Application
Macroinvertebrates
Sample Collection
Will your facility provide sample collection equipment or conduct sampling for North Carolina clients'
If so, what type of collection equipment and shipping containers will be provided to clients'	
What commercial earners are used for sample shipment?.
List sampling and sample identification equipment:
Mate Model	Calib. method (if appropriate)
trawls (list)	
dredges (list)				
box sampler	
Hess sampler		
Surber sampler
sweep net, please list type	
(A-frame, D-frame, etc.)
Additional equipment
Describe collection^,ckmg techniques employed, including the purpose of each technique and sample identification (attach additional
pages if necessary):

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North Carolina Biological Laboratory Certification Application
Collection/picking techniques (continued):
Describe sample preservation techniques used:.
Chain-of-Custody
Will adhesive seals and/or lockable shipping containers be provided to clients?		
Describe chain-of-custody procedures and the steps taken at your lab upon receipt of the sample:
Is a sample log maintained?

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Identification
Are reference organism collections available for taxonomic review''
Are reference or whole samples from popuianon surveys maintained for at least one year after collection?	
to copte of all axonamc gdfa art ataes spewed by the D,vision of Envtamem, Ma^emem locaKl m to labour.
List or append other references used for taxonomy or population analyses:
Data Analyses
Descrik dm aMysis mploy«d (tee shouZd be to dcM m Smiard Opemo, documm):
Describe how data calculations, data entry, and statistical analyses are quality assured;

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North Carolina Biological Laboratory Certification Application
In-Iab Quality Assurance
Discuss analyses performed as part of in—lab quality assurance of population survey (tbe analyses to be detailed in the Standard
Operating Procedures Document):

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North Carolina Biological Laboratory Certification Application
TAXONOMY
The: following list specifies those genera that can normally be taken to the species level using readily available and accented
taxonomic keys Many other genera can be taken to the species level depending on the taxonomic expert! oT^SS usmo
kSroPraaSPSl	UDPUbllSlied manUSCnptS 311(10ther such sources ** differentiate species, but may not be'widefy
is not odSS^™11 5110111(1 ^ UDdenaken Whenever	and the reference used for identification listed, if the genus
EPHEMEROPTERA
Baetis
Ephemerella
SerrcaeUa
PLECOPTERA
Acroneuna
Helopicus
Paragnetma
TRICHOPTERA
Brachycenmis
Hydropsyche
Neophylax
Rhyacophila
ODONATA
Baensca
Eurylophella
Stenacron
Agnetma
Isogenoides
Perlmella
Ceraclea
Micrasema
Neaopsyche
Symphitopsyche
DruneUa
Neoephemera
Stenonema
Dtploperia
Isoperla
Diplecirona
Molama
Psychomyia
Tnaenodes
Boyena
MEGALOPTERA
Neurocordulia
Chauliodes
COLEOPTERA
Nigroma
CRUSTACEA
OUCOCHAETA (if mature specimens)
Allonms
Bratislava
Stylana
Slavina
Uncwus
Bothnoneurum
llyodnlus
Potamothrix
Amphichaeta
Paramos
Ophidonais
Specana
Vejdovskyella
Brandmim
Isochaetides
Quistadrilus
Araeonats
Haemonais
Tubifex
Spirospema
Aulodrdus
Haber
Limnodnlus
DIPTERA
Ablabesmyia
Orthocladius
Cncotopus
Polypedilwn
Labnmdwia

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North Carolina Biological Laboratory Certification Application
Algae
Sample Collection
Will your facility provide sample collection equipment or conduct sampling for North Carolina clients?	
If so what type of collection equipment and shipping containers will be provided to clients?	
What commercial carriers are used for sample shipment?	
List sampling and identification equipment:
Make	Model	Calib. method
light meter					
centrifuge			
Additional equipment:
Describe collection techniques employed, including the purpose of each technique:.

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		N°rth Carolina Biological Laboratory Certification Application
Describe sample preservation techniques used;	
Describe identification and enumeration techniques employed;
Ch ain-of- C us tody
Will adhesive seals and/or lockable shipping containers be provided to clients?
Describe chain-of-custody procedures and the steps taken at your lab upon receipt of the sample:
Is a sample log maintained?

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North Carolina Biological Laboratory Certification Application
Identification
Are reference algae collections available for taxonomic review? 	
Are reference or whole samples from population surveys maintained for at one year after collection?	
Are copies of all taxonomic guides and references specified by the Division of Environmental Management located in the laboratory
(see Appendix)7	
List or append other references used for taxonomy or population analyses:			
Data Analyses
Describe data analysis techniques employed (these should be described in detail in the Standard Operation Procedures document):
Describe bow data calculations, data entry, and statistical analyses are quality

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North Carolina Biological Laboratory Certification Application
In-Iab Quality Assurance
List any replicate, blank, or blind analyses performed as pan of in-lab quality assurance of population survey (the analyses to be
detailed in the Standard Operating Procedures Document):

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North Carolina Biological Laboratory Certification Application
Listed below are taxa 'bar can be taken to genus using readily available and accepted taxonomic keys. Pbytoplankton
genera should be taken to the species level whenever possible. Depending on taxonomic expertise, many other genera
may be taken to species level using papers, in house keys, unpublished manuscripts and other such sources that allow differentiation of
species, but which may not be widely known or accepted. Documentation of references used to identify genera not listed below should
be readily available.
PHYTOPLANKTON TAXA IDENTIFIED TO GENUS BY DEM
BACILLARIOPHYCEAE
ACHNANTHES
AMPHORA
AMPHIPRORA
ANOMOEONE1S
ASTERIONELLA
ATTHEYA
BACILLARIA
CAPARTOGRAMMA
CHAETOCERAS
COCCONEIS
COSCINODISCUS
CYCLOTELLA
CYLINDROCYSTIS
CYMATOPLEURA
CYM BELLA
DIATOMA
DIPLONEIS
EVNOT1A
FRAGILAR1A
FRUSTUUA
GOMPHONEMA
GYROSIGMA
LEPTOCYL1NDRUS
MELOSIRA
MERIDION
NAVICULA
NEIDTUM
NITZSCHIA
PINNVLAR1A
PLEUROSIGMA
RHIZOSOLENIA
RHOICOSPHENIA
RHOPALODIA
SKELOTONEMA
STEPHANODISCUS
STAURONEIS
SURIRELLA
SYNEDRA
TABELLARIA
THALASSIONEMA
CHLOROPHYCEAE
ACTINASTRUM
ANKISTRODESMUS
ARTHRODESMUS
ASKENASYELLA
ASTEROCOCCUS
BOTRYOCOCCUS
CARTER1A
CHARACIUM
CHLAMYDOMONAS
CHLORELLA
CHLOROCOCCALES
CHLOHORMID1UM
CHLOROGONIUM
CHODATELLA
CL0STER1UM
CLOSTERIOPSIS
COELASTRUM
COSMARIUM
CRUCIGEN1A
CYLINDROCYSTIS
DESMIDIUM
DICTYOSPHAERIUM
DIMORPHOCOCCUS
ELAKATOTHRIX
EUASTRUM
EUDORINA
FRANCEIA
GLOEOCYSTIS
GOLENKINIA
GONIUM
HAEMATOCOCCUS
HYDRODICTYON
K1RCHNERIELLA
LAGERHEIMIA
MES0TAEN1UM
MICRA CTINIUM
MOUGEOTIA
MO UGEOTIOPSIS
NANNOCHLORIS
NEPHROCYTIUM
OEDOGONIUM
OOCYSTIS
PANDORINA
PEDIASTRUM
PHACOTVS
PHEASTER
PLEUROTAENIUM
POC1LLOM ON AS
P0LYBLEPHAR1DES
POLYEDRIOPSIS

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North Carolina Biological Laboratory Certification Application
CHLOROPHYCEAE - continued
PROTOCOCCUS
PSEUDOKEPHRYON
PTEROMONAS
QUADRICULA
SCENEDESMUS
SCHR0DER1A
SELENASTRUM
SPHAEROCYSTIS
SPHAERELL0PS1S
SPIROGYRA
SPONDYLOSIUM
STAURASTRUM
TETRASPORA
TETRASTRUM
TETRASELM1S
TETRAEDRON
THORAKOMONAS
TREUBARIA
ULOTHRIX
VOLVOX
WESTELLA
XANTHIDIUM
CHRYSOPHYCEAE
APEDINELLA
B1C0ECA
CENTRITRACTUS
CHROMULINA
CHR YSIDIASTR UM
CHRYSOCOCCUS
CHR YSOSPHAERELLA
CYCL0NEX1S
DESMARELLA
DINOBRYON
HETEROMASHX
KEPHRY10N
MALLOMONAS
OCHROMONAS
0PHI0CYT1UM
PAVLOVA
PSEUDOPEDINELLA
SPHAEROECA
SYNURA
CRYPTOPHYCEAE
CALYCOMONAS	CRYPTOMONAS
CHROOMONAS	RHODOMONAS
CYANOPHYCEAE
ANABAENA
ANABAENOPSIS
ANACYSTIS
APHANIZOMENON
APHANOCAPSA
APHANOTHECE
ARTHROSPIRA
CHROOCOCCUS
COCCOCHLOR1S
DACTYLOCOCCOPSIS
CLOEOCAPSA
GLOEOTHECE
GOMPHOSPHAERIA
J0HANNESBAPT1SA
LYNGBYA
MERISMOPEDIA
MICROCYSTIS
NODULARIA
OSCILLATORIA
PHORMIDIUM
POLYCYSTIS
RAPHIDIOPSIS
SPIRUL1NA

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North Carolina Biological Laboratory Certification Application
DINOPHYCEAE
AMPHIDINIUM
CERATIUM
CYSTODINIUM
EXUVIAELLA
GLENODINIUM
GYMN0DIN1UM
GYRODINIUM
HEJEROCAPSA
KATODINIUM
PERIDINIUM
POLYRIKOS
PROROCENTRUM
EUGLENOPHYCEAE
EUGLENA	PHACUS
EUTREPTIA	TRACHELOMONAS
LEPOCINCUS
PRASINOPHYCEAE
P YRAM1M ON AS
PRYMNES10PHYCEAE
CHR YSOCHROMULINA
XANTHOPHYCEAE
OLISTHODISCUS
TRIBONEMA

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North Carolina Biological Laboratory Certification Application
Aquatic Macrophytes
Descnbe specimen collection techniques:
If quantitative sampling is conducted, please descnbe List sampling techniques and equipment:
Describe sample preservanon techniques used:.
Are reference plant collections available for taxononuc review'	
Are reference or whole samples from population surveys maintained for at least one year after collection?.
Descnbe charn-of-cusiody procedures:	

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North Carolina Biological Laboratory Certification Application
Are copies of all taxonranic guides and references specified by the Division of Environmental Management located in the laboratory
(see Appendix)?	
List or append other references used for taxonomy or population analyses:	
Describe data analysis techniques employed:.

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North Carolina Biological Laboratory Certification Application
Zooplankton
Describe collection techniques employed, including the purpose of each technique-
List sampling equipment:
Describe sample preservation techniques used.
Are reference organism collections available for taxonomic review? 	
Are reference or whole samples from population surveys maintained for at least one year after collection?.
Describe chain-of-custody procedures: 			

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North Carolina Biological Laboratory Certification Application
Are copies of all taxonomic guides and references specified by the Division of Environmental Management located in the laboratory
(see Appendix)?	
List or append other references used for taxonomy or population analyses:		
Describe data analysis techniques employed:.

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iNorth Carolina Biological Laboratory Certification Application
Appendix: Required and Recommended References
FISH REFERENCES
REQUIRED
Dahlberg, Michael D. 1975. Guide to Coastal Fishes of Georgia and Nearby States. University of Georgia
Press. Athens, GA. 187 p.
Menhinick, Edward F. 1991. The Freshwater Fishes of North Carolina. North Carolina Wildlife Resources
Commission, Raleigh, N.C. 227 p.
PHYTOPLANKTON REFERENCES
REQUIRED
Whitford, L.A. and G J. Schumacher. 1984. A Manual of Fresh-water Algae. Sparks Press, Raleigh N C 337
pp.
Whitford, L.A. 1974. Additions to the Freshwater Algae in North Carolina, VIIL Jour. Elisha Mitchell Scl Coc
OfM 11_1 1*7
Whitford, L.A. 1982. Additions to the freshwater algae in North Carolina, X. Jour. Elisha Mitchell Sci Soc
98(l):32-36.
Patrick, Ruth and Charles W. Reimer. 1966. The Diatoms of the United States, Vol. 1. Academy of Nat Sci. of
Philadelphia, Philadelphia, Pa. 688 pp.
Patrick, Ruth and Charles W. Reimer. 1975. The Diatoms of the United States. Vol. 2, Part 1. Academy of Nat
Scl of Philadelphia, Philadelphia, Pa. 213 pp.
Prescott, G.W. 1973. Algae of the Western Great Lakes Area. Wm. C. Brown Co. Pub., Dubuque, Iowa. 977
pp.
RECOMMENDED
Tiffany, L.H. 1934. The Plankton Algae of the West End of Lake Erie. Ohio State University Contribution No.
6, Ohio State Univ. Press, Columbus, Ohio 77 pp.
Weber, Cornelius I. 1971. A Guide to the Common Diatoms at Water Pollution Surveillance System Stations.
U.S. EPA, Cincinnati, Ohio. 101 pp.

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North Carolina Biological Laboratory Certification Application
AQUATIC PLANT REFERENCES
REQUIRED
Radford, A.E., H.E. Ahles and C.R. Bell. 1968. Manual of the Vascular Flora of the Carolinas. The University
of North Carolina Press, Chapel Hill, NC. 1184 pp.
Although Radford et al. is outdated, it is still a superb reference text and the only one that covers all
vascular plants for North Carolina
RECOMMENDED
Aulbach-Sraith, C.A. and S.J. deKozlowski. 1990. Aquatic and Wetland Plants of South Carolina. South
Carolina Water Resources Commission, Columbia SC. 123 pp.
Copies can be obtained from: Publications Coordinator, SC Water Resources Commission, 1201 Main
Sl, Suite 1100, Columbia, SC 29201. (803) 737- 0800; Cost $10.00
Beal, Ernest O. 1977. A Manual of Marsh and Aquatic Vascular Plants of North Carolina, with Habitat para
The North Carolina Agriculture Research Service, Raleigh, NC.
Copies can be obtained from: North Carolina Agriculture Research Service, NC State University, Raleigh, NC 27695;
Gleason, H.A. and A. CronquisL 1991. Manual of Vascular Plants of Northeastern United States and Adjacent
Canada, 2nd edition The New York Botanical Garden, The Bronx, NY, NY. 910 pp.
Gleason and Cronqist's second edition includes the recent changes in plant nomenclature.
Godfrey. R.K. and J.W. Wooten. 1979. Aquatic and Wetland Plants of Southeastern United States:
Monocotyledons. University of Georgia Press. Athens, GA.
Godfrey. R.K. and J.W. Wooten. 1981. Aquatic and Wetland Plants of Southeastern United States:
Dicotyledons. University of Georgia Press. Athens, GA.
Cost $3.00

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STATISTICAL ENDPOINTS
AND DATA EVALUATION
MODULE

-------
Three kind of lies
Lies,
Damn Lies,
and Statistics
Mark Twain

-------
Module Objectives
Describe hypothesis testing
procedures
Describe point estimate techniques
Evaluate acute test results
Evaluate chronic test results

-------
Statistical Endpoints
¦	Acute tests
LC50, NOEC, or pass/fail
¦	Chronic tests
NOEC, IC25 or EC25
Talking Points:
•	This statistical section will highlight some of the statistical
discussions covered in the EPA acute [USEPA 1993] and chronic
test methods [USEPA 1994a, 1994b, 1995]. The objective of a
toxicity test is to estimate the highest "safe" or "no-effect
concentration" of wastewaters.
•	When a single WET test is conducted, the observed toxicological
measurement endpoint, such as survival, growth or reproduction is
recorded. At the end of a test, the data are subjected to an array
of statistical analyses to quantify the effects observed during the
test.
•	Acute tests are reported as a lethal concentration (LC). Typically,
a LC 50 is reported. LC 50 is the toxicant concentration that
would cause death in 50 percent of the tested organisms.
Sometimes, acute tests are reported as either a pass or fail when
conducting a t-test. This is the case, when only the instream
waste concentration {IWC) is compared to a control.
•	Chronic tests are reported as either a NOEC or as a point estimate
value (ECp). The no observable effect concentration (NOEC) is
determined by hypothesis testing procedures.
4

-------
Statistical Approaches
¦	Hypothesis testing
NOEC
¦	Point estimate techniques
LCp, ECp or ICp
Talking Points.
•	The NOEC is the highest concentration tested of a toxicant
that causes no observable adverse effects, but the use of the
NOEC does not mean that there was "no toxic effect", but
only no statistically significant effect. The lowest observed
effect concentration is the lowest concentration of toxicant to
which organisms are exposed in a test, which causes
statistically significant adverse effects on the test organisms
(i.e., where the values for the observed endpoints are
statistically different from the control).
•	For point estimates, typically the results can be reported as
the effective concentration (EC), the lethal concentration (LC)
or the inhibition concentration (ICp). LCp is used for the
measurement of lethality. ECp is used for the measurement of
quanta! data, such as fertilization, shell development, and
germination. ICp is used for the measurement of continuous
data, such as reproduction, growth and length.
•	To use a point estimate such as an LC, EC or IC to determine
a "safe" concentration requires a biological judgment of what
constitutes an acceptable level or adverse effect.
5

-------
Toxicity Units
= 100/LC50 or 100/NOEC
= 100/NOEC or 100/ECp

-------
Hypothesis Testing Facts
¦	NOEC are not point estimates
¦	Cannot calculate coefficients of
variation or confidence intervals
¦	NOEC may represent a different
amount of effect from test to test
¦	NOEC is a lower concentration level
than the LOEC
Talking Points:
•	The NOEC and LOEC values are the results of hypothesis testing. They are not point
estimates.
•	NOEC and LOEC are limited to the concentrations selected for the test. The width of
the NOEC-LOEC interval is a function of the dilution series and differs greatly depending
on whether a dilution factor of 0.3 or 0.5 is used in the test design. Therefore, USEPA
recommends the use of the > 0.5 deletion factor. It is not possible to place confidence
limits on the NOEC and LOEC derived from a given test and it is difficult to quantify the
precision of the NOEC and LOEC endpoints between tests. Coefficient of variation
(CV) cannot be calculated for NOEC endpoints determined using an analysis of variance
(hypothesis testing). If the data from a series of tests performed with the same
toxicant, toxicant concentrations, and test species, were analyzed with hypothesis
tests, precision could only be assessed by a qualitative comparison of the NOEC-LOEC
intervals, with the understanding that maximum precision would be attained if all tests
yielded the same NOEC-LOEC interval.
•	The NOEC will vary from test to test because of test variability, variability of the
toxicant(s) with time, sensitivity of the test organisms and/or laboratory technique.
•	The definitions of NOEC and LOEC in the method manuals assume a strict dose-
response. If this assumption were always the case, there would be no issue
concerning the endpoint definitions because the NOEC would always be a lower
concentration level than the LOEC. However, this strict dose-response relationship
does not exist with all toxicants. When this occurs the test must be repeated or the
lowest NOEC should be reported for compliance purposes. For example, a test is
conducted with the following test concentrations 0, 6.25, 12.5, 25, 50 and 100
percent effluent. The concentrations of 12.5, 50 and 100 percent were statistically
different from the control. What are the LOEC and NOEC7 The LOEC is 12.5,
therefore, the NOEC is 6.25.
7

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STATISTICAL ANALYSIS OF FATHEAD MINNOW LARVAL
SURVIVAL AND GROWTH TEST
SURVIVAL HYPOTHESIS TESTING
NON-NORMAL OlSTVUBUTIOtf
NORMAL DISTRIBUTION
HOMOGENEOUS
VARIANCE
HETEROGENEOUS1
VARIANCE
NO
YES
NO
ENDPOUfTESTlMATESr
NOEC.LOEC
BARTLEITS TEST
SHAPWO-WHJCS TEST
EQUAL NUMBER OF
REPLICATES?"
EQUAL NUMBS) OF
REPLICATES?
ARC SINE
TRANSFORMATION:
SURVIVAL DATA
PROPORTION SURVIVING
STEELS MANY-ONE
RANK TEST
WOJCOXONRANKSUI*
TEST WITH
BONFBWONIADJUSTMBfT
Figure 5, Flowchart for statistical analysis of the fathead minnow, Piaephales
promelas, larval survival data by hypothesis testing.

-------
Hypothesis Tests
¦	Parametric
¦	Non-parametric
Talking Points:
•	Parametric tests must meet data assumptions, such as normal
data and equal variance. If these assumptions are not met
then nonparametric tests must be performed.
•	Dunnett's procedure consists of an analysis of variance
(ANOVA) to determine the error term, which is then used in a
multiple comparison procedure for comparing each of the
treatment means wih the control mean, in a series of paired
tests. Use of Dunnett's procedure requires at least three
replicates per treatment to check the assumptions of the test.
•	In cases where the number of replicates for each concentration
are not equal, a t test may be performed with Bonferroni's
adjustment for multiple comparisons, instead of Dunnett's
procedure.
9

-------
Parametric Tests
¦	Must test assumptions:
-	normally distributed data
-	tested by Sharpiro-Wilks
-	variance is equal
-	tested by Bartlett's test
¦	Tests
-	Dunnetts
-	T-test with Bonferroni adjustment
Talking Points:
•	The assumptions for parametric tests are that the data are
distributed normally as tested by Sharpiro-Wilks Test and that
the group variances are homogeneous as tested by Bartlett's
Test.
•	The first step in these analyses is to transform the quantal
responses (e.g., survival, fertilization) expressed as the
proportion surviving, by the arc-sine-square-root
transformation. This transformation is commonly used on
proportionality data to stabilize the variance and satisfy the
normality requirement.
•	If either of these statistical assumptions (normality or equal
variances) fail, then a nonparametric test must be performed.

-------
Nonparametric Tests
¦	Nonparametric tests
- based on ranks
¦	Steel's Many-one Rank Test
¦	Wilcoxon Rank Sum
Talking Points:
•	Steel's test is fairly insensitive to heterogeneity of variance.
Steel's test is a nonparametric multiple comparison procedure
for comparing several treatments with a control. This method
is similar to Dunnett's procedure, except that it is not
necessary to meet the assumption of normality. The data are
ranked, and the analysis is performed on the ranks rather than
on the data themselves.
•	It is necessary to have at least four replicates per toxicant
concentration to use Steel's test. Unlike Dunnett's procedure,
the sensitivity of this test cannot be stated in terms of the
minimum difference between treatment means and the control
mean that can be detected as statistically significant.
•	Wilcoxon Rank Sum is another nonparametric test to be used
when there are unequal number of replicates. The data are
ranked and the analysis proceeds exactly as in Steel's Test
except that Bonferroni's adjustment for multiple comparisons
is used instead of Steel's tables.
11

-------
Point Estimates Facts
¦	Can calculate coefficients of variation
and confidence intervals
¦	Always estimating same effect
¦	Need specification of a biological
effect - what value of p
Talking points
•	Point estimation techniques have the advantage of providing a
point estimate of the toxicant concentration causing a given
amount of adverse effect, the precision of which can be
quantitatively assessed (1) within tests by calculation of 95%
confidence limits and (2) across tests by calculating a
standard deviation and coefficient of variation.
•	Point estimates always estimates the same effect, such as an
IC 25 from one test to another test result using an IC 25.
However, when using a point estimate technique, it does not
mean that at the endpoint concentration that there was "no
toxic effect".
•	Regulators must decide which "value of p" to use for
compliance purposes.

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Methods to Calculate LC or
EC Endpoints
¦	Pro bit
¦	Spearman-Karber
¦	Trimmed Spearman-Karber
¦	Graphical
Talking Points:
• Each model has different assumptions.
•The Spearman-Karber, Trimmed Spearman-Karber, and
Graphical models can only calculate an EC or LC50 effect.
13

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DETERMINATION OF THE LC50
FROM A MULTI-EFFLUENT-CONCENTRATION
ACUTE TOXICITY TEST
NO
YES
NO
NO
~
YES
YES
NO
YES
PROBIT METHOD
GRAPHICAL METHOD
LCSO
MORTALITY DATA
# DEAD
ONE OR MORE
PARTIAL MORTALITIES?
SPEARMAN-KARBER
METHOD
TRIMMED SPEARMAN
KAR8ER METHOD
TWO OR MORE
PARTIAL MORTALITIES?
LCSO AND 95%
CONFIDENCE
INTERVAL
IS PROBIT MODEL
APPROPRIATE?
(SIGNIFICANT X* TEST)
ZERO MORTALITY IN THE
LOWEST EFFLUENT CONC.
AND 100% MORTALITY IN THE
HIGHEST EFFLUENT CONC.?
Figure 6. Flowchart for determination of the LC50 for multi-effluent-
concentration acute toxicity tests.

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Probit
¦	Assumptions
¦	Two partial mortalities
- chi-square test for heterogeneity
Talking Points:
•	Probit analysis is used to estimate an LC or EC value and the
associated 95% confidence interval. The analysis consists of
adjusting the data for mortality in the control, and then using a
maximum likelihood technique to estimate the parameters of
the underlying log tolerance distribution, which is assumed to
have a particular shape.
•	If the normality assumption is not met, and at least two partial
mortalities are not obtained, Probit analysis should not be
used.
•	Chi-square test for heterogeneity provides a good test of
appropriateness of the analysis.
15

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Spearman-Karber
¦	A symmetric distribution about the
mean is assumed
¦	Assumptions
-	zero response in the lowest toxicant
concentration
-	100% response in the highest toxicant
concentration
Talking Points:
•	This model only calculates a LC or EC 50. So, in cases where
Probit analysis is not appropriate, then use Spearman-Karber
for acute tests. However, chronic tests should use the Linear
Interpolation method, since the effect to be detected is less
than a 50% effect.
•	If these assumptions are not met, then use trimmed
Spearman-Karber. The trimmed Spearman-Karber procedure
was designed to use in instances where multiple
concentrations within a test give a 100% response (or 0%
response).
16

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17

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Inhibition Concentration (ICp)
¦	Assumptions of ICp
¦	Responses are monotonically non-
decreasing
Talking Points:
•	The Linear Interpolation Method is a procedure to calculate a
point estimate of the effluent concentration that causes a
given percent (p) reduction (e.g., 15%, 25%, etc.) in
response, such as growth or reproduction from the control.
•	Use of the Linear Interpolation Method is based on the
assumption that the responses are monotonically non-
increasing (the mean response for each higher concentration is
less than or equal to the mean response for the previous
concentration). There is no defined statistical procedure
provided to test the assumption of monotonicity.
•	Where the observed means are not strictly monotonic by
examination, they are adjusted by smoothing. In cases, where
the responses at the low toxicant concentrations are much
higher than in the controls, the smoothing process may result
in a large upward adjustment in the control mean.

-------
Benefits of Hypothesis
Testing
Results provide information regarding
test variability (MSD)
Results inform regulator of the no-
observed effect level
The researcher can test just the IWC
vs. the control

-------
Concerns with Hypothesis
Testing
¦	The true effect level may lie
somewhere in between the NOEC and
LOEC
¦	Poor or excessive statistical power
¦	Cannot generate intra or inter-lab
precision results
Talking points:
See next slide.
20

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Recommendations to
Hypothesis Testing Concerns
¦	Better spacing of the effluent
concentrations - bracket IWC
¦	Establish a test sensitivity criterion -
MSD limit
Talking points:
• To alleviate some of these concerns, the spacing of the
dilution series should be controlled and ideally the
concentrations should bracket the instream waste
concentration (IWC) as one of the test concentrations. A way
to address the concern over test variability is to establish a
test sensitivity criterion, such as an minimum significant
difference (MSD) that must be met when using hypothesis
tests.
21

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MSD
The following formula is used to calculate MSO (as
recommended by USEPA 1995)
MSD = d sw 
-------
Benefits of Point Estimate
Techniques
¦	Can quantify intra and inter-lab
precision
¦	Uses all the information from a dose-
response relationship
¦	Minimize the importance of the effects
at the IWC
Talking Points:
*	Precision is a measure of reproducibility both within a lab
(intra lab} and among several labs {inter lab) using the same
test method and reference toxicant. Precision can be
quantified by a variety of measures including the coefficient of
variation (CV = standard deviation/mean * 100) of point
estimates (e.g., LC50 for acute endpoints and EC/IC25 for
chronic endpoints) from multiple tests conducted with the
same test method and reference toxicant.
•	The selection of test concentrations is less important for point
estimate techniques, because the test uses all the dose-
response information.
23

-------
Concerns with Point Estimate
Techniques
Point estimate is model dependent,
especially for small levels of p
Appropriate model may vary with
effluent sample, species, amount of
toxicant
Selection of appropriate p value to
regulate
Talking Points:
•	For simple linear curve fitting models for point estimation,
typical data can depart from the models for several reasons.
A hormesis like-effect can occur where the response is greater
at the higher concentration than the control.
•	The primary question in applying the point estimation
techniques has been what effect level {e.g., ECp) should be
reported for compliance purposes? In 1991, the USEPA
evaluated existing data for two freshwater test species
methods, C. dubia and P. promelas and three east coast
marine test methods, Arbacia punctulata, Cyprinodon
variegatus, and C. parvula. In the comparisons of both types
of data, EPA indicated that an NOEC derived using the IC25 is
approximately the analogue of an NOEC derived using
hypothesis testing [USEPA 1991].
•	With the development of the standardized west coast marine
toxicity test methods, an evaluation was conducted to
evaluate what "p" value is approximately equivalent to the
NOEC. For all the test methods analyzed, the approximate "p"
value was below an EC25.
24

-------
Evaluation of Toxicity Tests
Verify that appropriate
method/species is used
Verify test results meet the required
TAC
Examine chemical and physical
parameters of test
Examine statistical results
Compare test result with permit limit

-------
Take Home Points
The statistical approaches are
described in the manuals
Incorporate the MSD evaluation for
hypothesis testing
Verify that test meet the required TAC

-------
Evaluation of Toxicity Data
Chronic Toxicity Data
1 Examine the test results to verify that the laboratory is using the test method and dilution
series as required in the NPDES permit. Note: This may only need to be performed after a
permit has been first issued
2. Evaluate the test results to verify that the laboratory met the test acceptability criteria (TAC)
as specified in the test method
Example: A laboratory conducts the chronic reproduction and survival water flea,
Ceriodaphrda dubia test. The following criteria must be achieved for both the reference
toxicant and effluent test:
a)	Survival in the controls must be at least 80%,
b)	Reproduction in the controls must average 15 or more young per surviving female;
c)	The laboratory must report the MSD value.
3	Examine the chemical and physical parameters of the test
Minimum and maximum pH, temperature and dissolved oxygen for the test. Note:
The test method specifies that the temperature should be 20 + 1 °C. The data
reviewer should evaluate these parameters on best professional judgement. For
example, the test met the required TAC, and the data demonstrates a normal dose
response curve, but the temperature minimum was 18.5 °C and maximum was 20.0
°C. This should be an acceptable test result.
4	Examine the statistical results to verify the following:
a)	Did the laboratory use the correct statistical programs (see Appendix B, [USEPA
1994a, 1994b. 1995]^
b)	Did the laboratory perform the necessary number of replicates?
c)	Do the data indicate a good dose response curve? Note: Reference toxicant tests
should have good dose response curves, but this may not be the case with effluent
tests
5	Calculate the TUc and compare with permit limit.
NOEC = 50% effluent
TUc = 100/50 = 2 0 TUc
Acute Toxicity Data
1 Examine the test results to verify that the laboratory is using the test method and dilution
1

-------
series required in the NPDES permit. Note: This may only need to be performed after a
permit has been first issued
2.	Evaluate the test results to verify that the laboratory met the TAC as specified in the test
method.
The only TAC for al! acute test methods is the following for both the reference toxicant and
the effluent test:
a) Survival in the controls must be at least 90%.
3.	Examine the chemical and physical parameters of the test:
Minimum and maximum pH, temperature and dissolved oxygen for the test. Note:
The test method specifies that the temperature should be 25 + 1 °C. The data
reviewer should evaluate these parameter on a best professional judgement. For
example, the test met the required TAC, and the data demonstrates a normal dose
response curve, but the temperature minimum was 23.5 °C and maximum was 25.0
°C. This should be an acceptable test result.
4.	Examine the statistical results to verify the following:
a)	Did the laboratory use the correct statistical programs (see Appendix B, [USEPA
1993])7
b)	Did the laboratory perform the necessary number of replicates?
c)	Do the data indicate a good dose response curve? Note: Reference toxicant tests
should have good dose response curves, but this may not be the case with effluent
tests
5.	Calculate the TUa and compare with permit limit
LC50 = 67% effluent
TUa = 100/67 = 1 49 TUa
2

-------
DETERMINATION OF PASS OR FAIL
FROM A SINGLE -EFFLUENT-CONCENTRATION
ACUTE TOXICTY TEST
NO
YES
NO
NO
YES
FAIL
PASS
T-TEST
MODIFIED
T-TEST
WILCOXON RANK
SUM TEST
SIGNIFICANT DIFF.
IN SURVIVAL?
ARC SINE
TRANSFORMATION
NORMALITY?
(SHAPIRO-WILK'S TEST)
SURVIVAL DATA
PROPORTION SURVIVING
HOMOGENEITY OF VARIANCE
(F-TEST)
Figure 12. Flowchart for analysis of single-effluent-concentration test data.
3

-------
DETERMINATION OF THE NOAEC
FROM A MULTI-EFFLUENT-CONCENTRATION
ACUTE TOXICITY TEST
NO
YES
YES
NO
NO
NO
YES
YES
DUNNETTS
TEST
EQUAL NUMBER OF
REPLICATES?
EQUAL NUMBER OF
REPUCATES?
T-TESTWITH
BONFERRONI
ADJUSTMENT
NORMALITY?
(SHAPIRO-WIUCS TEST)
ARC SINE
TRANSFORMATION
SURVIVAL DATA
PROPORTION SURVIVING
STEEL'S MANY-ONE
RANK TEST
HOMOGENEITY OF VARIANCE
(BARTLETTS TEST)
W1LCOXON RANK SUM
TEST WITH
BONFERRONI ADJUSTMENT
ENDPOINT ESTIMATES
NOAEC
Figure 13. Flowchart for analysis of multi-effluent-concentration test data.
4

-------
DETERMINATION OF THE LC50
FROM A MULTI-EFFLUENT-CONCENTRATION
ACUTE TOXICITY TEST
NO
YES
NO
NO
YES
YES
NO
YES
PROB1T METHOD
MORTALITY DATA
M DEAD
GRAPHICAL METHOD
LC50
ONE OR MORE
PARTIAL MORTALITIES?
SPEAR MAN-KARBER
METHOD
TRIMMED SPEARMAN
KARBER METHOD
TWO OR MORE
PARTIAL MORTALITIES?
LC50AND 95%
CONFIDENCE
INTERVAL
IS PROBIT MODEL
APPROPRIATE?
(SIGNIFICANT Xs TEST)
ZERO MORTALITY IN THE
LOWEST EFFLUENT CONC.
AND 100% MORTALITY IN THE
HIGHEST EFFLUENT CONC.?
Figure 6. Flowchart for determination of the LC50 for mu"Iti-effluent-
concentration acute toxicity tests.
5

-------
STATISTICAL ANALYSIS OF FATHEAD MINNOW LARVAL
SURVIVAL AND GROWTH TEST
SURVIVAL POINT ESTIMATION
NO
YES
NO
NO
YES
YES
NO
YES
PROBIT METHOD
MORTALITY DATA
# DEAD
GRAPHICAL METHOD
LC50
SPEARMAN-KARBER
METHOD
ONE OR MORE
PARTIAL MORTALITIES?
TRIMMED SPEARMAN
KARBER METHOD
TWO OR MORE
PARTIAL MORTALITIES?
LC50 AND 95%
CONFIDENCE
INTERVAL
IS PROBrr MODEL
APPROPRIATE?
(SIGNIFICANT X1 TEST)
ZERO MORTALITY IN THE
LOWEST EFFLUENT CONC.
AND 100% MORTALITY IN THE
HIGHEST EFFLUENT CONC.?
Figure 6. Flowchart for statistical analysis of the fathead minnow, Pimephales
promelas, larval survival data by point estimation.
6

-------
STATISTICAL ANALYSIS OF FATHEAD MINNOW LARVAL
SURVIVAL AND GROWTH TEST
SURVIVAL HYPOTHESIS TESTING
NON-NORMAL DISTRIBUTION
NORMAL DISTRIBUTION
HOMOGENEOUS
VARIANCE
HETEROGENEOUS
VARIANCE
NO
YES
YES
NO
BARTLETTS TEST
SHAPIRO-WIUCS TEST
DUNNETTS
TEST
EQUAL NUMBER OF
REPLICATES?
EQUAL NUMBER OF
REPLICATES?
T-TESTWTTH
BONFERRONI
ADJUSTMENT
ARC SINE
TRANSFORMATION
SURVIVAL DATA
PROPORTION SURVIVING
STEEL'S MANY-ONE
RANK TEST
W1LCOXON RANK SUM
TEST WITH
BONFERRONI ADJUSTMENT
ENDPOINT ESTIMATES
NOEC, LOEC
Figure 5. Flowchart for statistical analysis of the fathead minnow, Pimephales
promelas, larval survival data by hypothesis testing.
7

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PERMIT DEVELOPMENT
Purpose of Module and Topics to be Covered:
•	How to determine whether there is a reasonable potential for
an excursion above a narrative or numeric standard
•	Techniques for developing a wasteload allocation where a
reasonable potential exists.
•	Approaches for developing permit limits from the wasteload
allocation
•	Expression of permit limits and development of special
conditions
1

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The Three Approaches
¦	Chemical-specific approach
¦	Whole effluent toxicity approach
¦	Biological criteria approach
Talking Points:
•	Section 101 (a) of the CWA states: "The objective of the Act is
to restore and maintain the chemical, physical and biological
integrity of the Nation's waters". Taken together, chemical,
physical, and biological integrity define the overall ecological
integrity of an ecosystem.
•	For the protection of aquatic life, the integrated strategy
involves the use of three control approaches: chemical-specific
control, WET control, and biological criteria/bioassessments
and biosurvey. Each of these approaches has capabilities and
limitations [TSD, page 21].
•	Regulatory agencies should strive to fully integrate all three
approaches since each has its respective capabilities and
limitations.
•	Caution should be exercised where biosurvey information is
employed as a screening tool; the results of such-analysis
should be used to complement, rather than override WET
testing information.
2

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WET Criteria
¦	Narrative - no toxics in toxic amounts
¦	Numeric
Talking Points:
•	The use of toxicity testing and whole effluent toxicity limits is
based upon a State's narrative water quality criterion and/or in
some cases, a State numeric criterion for toxicity.
•	In the Water Quality Standards regulation, Section
131.11(a)(2) requires States to develop implementation
procedures that explain how the State will ensure that
narrative toxics criteria are met.
3

-------
Toxicity Criteria
¦ Acute 0.3 TUa =100/LC50

-------
Toxicity Criteria
Chronic 1.0 TUc =100/NOEC
100/ECp

-------
Where Do Criteria Apply:
Mixing Zones
¦ A mixing zone is an "allocated impact
zone" where water quality criteria can
be exceeded as effluent mixes with
ambient.
- constraints:
•	can not impair integrity of the water body
•	no significant health risks
•	no lethality to organisms passing through
mixing zone
Talking Points:
•The analyst must determine the volume of water (if any} that is
available to mix with effluent
•This decision has both policy and technical dimensions.
6

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Mixing Zone
Outfall
Shoreli
C > CCC
nixing Zona
C > QIC
Flow
C x CCC

Talking Points:
•	Mixing zone is a limited area or volume of water where initial
dilution of a discharge takes place and where water quality
criteria can be exceeded but acutely toxic conditions are
prevented.
•	Mixing zones must ensure an adequate "zone of passage" for
mobile aquatic organisms.
•	EPA recommends that States have a definitive statement in
their standards on whether or not mixing zones are allowed.
Where mixing zones provisions are part of the State standards,
the State should describe the procedures for defining mixing
zones.
•	CWA does not require attainment of water quality at the end-
of-pipe.
•	In the zone immediately surrounding the outfall, neither the
acute nor the chronic criterion is met. The acute criterion is
met at the edge of this zone. This zone is sometimes called
the "zone of initial dilution" (ZID). In the next mixing zone, the
acute, but not the chronic, criterion is met. The chronic
criterion is met at the edge of the second mixing zone.
7

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State Mixing Zone Criteria
¦	21 States: no specific criteria
¦	9 States: a specific distance
¦	29 States: % of cross-sectional area/flow
¦	4 States: a given surface area
Talking Points:
• In many States, determination of the size of the mixing zones
is primarily a policy decision, while in other States no specific
approach is specified and it may determined on a case-by-case
basis.

-------
Determination of Mixing Zone
Dilution
¦	Field studies
-	actual measurement of instream
contaminant concentrations
-	dye studies
¦	Modeling
-	calibrated to actual observations
-	simulate critical conditions
Talking Points:
•	Where mixing zones are developed on a case-by-case basis,
they may be modeled, based on theoretical inputs that attempt
to simulate actual conditions, or they may be determined using
actual studies.
•	In any case, once the mixing zone has been determined, the
analyst has a critical piece of information for use in a water
quality model (for either determinations of projected effluent
quality in the reasonable potential determination or when
developing final permit limits).
9

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Steps in Developing WET

Permit Limits:
(1)
Conduct reasonable potential

analysis
(2)
Develop wasteload allocation (WLA)
(3)
Translate the WLA into permit limits
(4)
Specify all necessary permit

conditions
Talking Points:
• There are four general steps that a permit writer should follow in developing WET
permit limits and conditions.
-	The first step is that a reasonable potential analysis must be conducted to
determine if a WET limit is needed based on the factors required by 40 CFR
§122.44(d){1)(ii). The permit writer/permitting authority has flexibility in
developing a methodology to assess reasonable potential. Decisions must be
made concerning the mix of actual WET data and effluent/discharger
characteristics used to assess whether reasonable potential to exceed state
numeric/narrative criteria exists. The methodology may range from a simple
procedure based on limited WET data to a more complicated statistical approach
as discussed in the TSD. In order to write defensible permit limits, the permit
writer/permitting authority should develop and use a consistent approach to
assess reasonable potential.
-	Once reasonable potential is determined to exist, the second step requires that
the permit writer develop the appropriate wasteload allocation (WLA). The
WLA provides a definition of effluent quality that is necessary to meet the
applicable numeric/narrative water quality criterion. There is flexibility in how
the WLA is derived.
-	The third step for the permit writer involves translating the WLA into WET
permit limits — flexibility also exists in how to express the WET permit limit.
-The final step involves specifying the permit conditions regarding the test
species, test frequency, sample type, dilution series, etc.

-------
40 CFR §122.44(d)(1) --
Regulatory Authority for:
¦	Determining "reasonable potential" to
exceed water quality criterion
(narrative or numeric)
¦	Development of permit limits where
reasonable potential exists
Talking Points:
•	Where state numeric water quality criteria for either specific
chemicals or WET exist and where reasonable potential to
exceed that numeric criterion is determined to exist, the permit
must contain numeric limits for the parameter(s) in question.
•	Where only a state narrative water quality criterion for WET
exists and where a reasonable potential to exceed that
narrative criterion exists, the permit must contain either a limit
on WET or a limit on the causative toxicant (if this can be
determined). The permit writer has the flexibility to include a
chemical-specific limit in lieu of the WET numeric limit if the
fact sheet is documented to show that the chemical-specific
limit is sufficient to maintain the narrative water quality
criterion.
11

-------
Factors that Must be Considered in
Evaluating WET Reasonable
Potential (122.44(d)(1)(ii))
¦	Existing controls on point and nonpoint
sources of pollution
¦	Variability of the pollutant or pollutant
parameter in the effluent
¦	Sensitivity of the species to toxicity testing
¦	Dilution of the effluent in the receiving
water
Talking Points:
•	Required under 40 CFR Part 122.44(d) (1)(ii), the permit writer must consider a number
of factors in establishing reasonable potential.
•	The regulatory authority may have effluent toxicity data available from previous
monitoring or the authority may decide to require the permittee to generate effluent
monitoring data prior to permit issuance or as a condition of the issued permit.
•	If the regulatory authority so chooses, or if the circumstances dictate, the authority
may decide to develop and impose a permit limit for WET or for individual toxicants
without facility-specific effluent monitoring data, or prior to generation of effluent data.
However, in doing so, the regulatory authority must satisfy all requirements of 40 CFR
122.44(d)(1)(n). In such cases, the fact sheet for the permit should clearly outline the
basis for the best professional judgment decision and the information used to establish
the permit, in accordance with the requirements of 40 CFR Section 124.8.
•	The regulatory authority can use a variety of factors and information where facility-
specific effluent data are unavailable. These factors should also be considered with
available effluent monitoring data.
- In order to write defensible WET permit limits and achieve consistent application,
EPA recommends that permitting authorities develop clear procedures to assess
WET reasonable potential. At a minimum, such procedures should take into
account the elements specified at 40 CFR §122.44(d)(1)(ii). At a minimum. EPA
recommends that all major discharges be assessed and all minors, on a case-by-
case basis.
12

-------

Determining Reasonable

Potential without Effluent Data
¦
Size of discharge/dilution available
¦
History of fish kills
¦
Compliance/enforcement history
¦
Raw materials used
¦
Products produced
¦
Existence of local Pretreatment program
¦
Presence of categorical industries
¦
Chlorination/ammonia problems
¦
Number of commercial and industrial users
Talking Points:
•	There are a host of factors that should be considered in making
reasonable potential determinations.
•	It is possible to make a reasonable potential finding in the
absence of actual effluent WET data.
•	The permit writer should exercise caution and discretion in
considering these and other factors. Some of these factors
may or may not actually have a bearing on the whether an
effluent is likely to be toxic, under certain circumstances. All
appropriate and relevant information relating to the non-
effluent data information should be considered.
13

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Effluent Characterization for
Reasonable Potential Analysis:
¦	Require toxicity data collection 12-18
months in advance of permit
application
¦	Recommend quarterly toxicity tests
with three species for a year
¦	Conduct acute or chronic based on
dilution at edge of mixing zones
Talking Points:
•	EPA recommends, as a minimum, that three species (plant, fish, and
invertebrate) to be tested quarterly for a minimum of 1 year.
•	Where effluent WET data are to be collected, the permit writer must
decide what type of WET data are most appropriate for assessment
of a given discharge. Based on the TSD (pg. 59), EPA recommends
that:
-Dilution at the edge of the mixing zone is determined by
modeling using either the steady-state or dynamic approaches
(discussed in more detail below).
-EPA recommends that a discharger conduct acute toxicity
testing if the dilution of the effluent is greater than 1000:1 at
the edge of the mixing zone.
-EPA recommends that a discharger conduct either acute or
chronic toxicity testing if the dilution of the effluent falls
between 100:1 and 1000:1 at the edge of the mixing zone.
- EPA recommends that a discharger conduct chronic toxicity
testing if dilution of the effluent falls below 100:1 at the edge of
the mixing zone [TSD, page 59],
14

-------
Reasonable Potential
Procedures Based on WET
Data:
Must consider uncertainty associated
with sparse data sets and effluent
variability
Talking Points:
•	The permitting authority has the flexibility to base its
reasonable potential procedures and determinations solely on
historical WET data. However, such procedures which assess
the potential to exceed applicable numeric/narrative water
quality criteria must be based on the uncertainty associated
with limited data and effluent variability.
•	Such procedures may be simple and range from a finding of
any WET failure in the historical database at the receiving
water concentration (RWC) to a certain percentage of WET
test failures at the RWC based on the historical database.
•The procedures may also be more complicated -- the TSD
procedure (pg. 56) is an example of a statistical approach
which combines knowledge of effluent variability (the
coefficient of variation (CV)) with a desired level of confidence
and probability.
•	Regardless of the complexity of the methodology, if reasonable
potential is determined to exist, the 40 CFR § 122.44(d)(1)
regulations require that the NPDES permit contain a WET limit.
15

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Possible Reasonable Potential

Outcomes
(1)
Excursions above CMC or CCC

- establish WET limit
(2)
Reasonable potential for excursions above

CMC or CCC

- establish WET limit
(3)
No reasonable potential for excursion above

CMC or CCC

- conduct WET tests at least once every 5 years
(4)
Inadequate information

- WET monitoring with reopener clause
Talking Points:
• There are 4 basic outcomes to a permitting authority's assessment of
reasonable potential as to whether a WET permit limit is required.
1)	If historical data show excursions above the applicable numeric
criterion for WET or narrative "no toxics" water quality criterion, the
permit must contain a limit. The analysis of reasonable potential is
not required.
2)	If historical data do not show excursions, but the reasonable potential
procedure does show the potential for such excursions, the permit
must contain a WET limit.
3)	If the reasonable potential procedures do not indicate such excursions,
WET monitoring should be required at time of permit reapplication
(assuming discharger is a major or significant minor).
4)	If there is inadequate information with which to assess reasonable
potential, the permit should require some frequency of WET monitoring
with a reopener clause to include a WET limit if reasonable potential is
found to exist after analysis of an adequate database. In some cases,
the regulatory authority may make a preliminary "no reasonable
potential" finding based on limited available data, but conclude that,
due to uncertainties concerning effluent variability, more data are
needed. In such cases, the regulatory authority may install a
monitoring requirement in the permit to better characterize the
effluent.
16

-------
Why Use Models?
Models can help determine pollutant loadings
that will not violate water quality criteria.
Environmental
Conditions
Receiving Water
Quality
Pollutant Load
Reduce Load +
¦No-
Compliance
with criteria"'
MODEL
Yes
I
Done
Talking Points:
•	Once reasonable potential to exceed the applicable
numeric/narrative criterion is determined to exist, the
permitting authority has flexibility in choosing the method for
developing a wasteload allocation (WLA) that is designed to
ensure that the water quality criterion of the receiving water is
attained or maintained.
•	Models are used to project receiving water quality and to
determine, based on that projection, where there is a
"reasonable potential."
•	In addition, where a reasonable potential is projected, models
can be used to help establish permit conditions that are
necessary to avoid causing an excursion above a water quality
criterion.

-------
Water Quality Models
¦	Used to calculate WLAs
¦	Different types:
-	single value steady state
-	two-value steady state
-	continuous simulation dynamic
-	Monte Carlo dynamic
-	log normal probabilistic dynamic
Talking Points:
•	Analyst needs to decide which model to use; This workshop
will focus on steady state models, as they're more commonly
used, but the reader is referred to Chapter 4 of the Technical
Support Document for more detailed information.
•	Wasteload allocation (WLA) is the portion of a receiving
water's total maximum daily load that is allocated to one of its
existing or future point sources of pollution.
18

-------
Steady State Models
¦	One or two value WQS
¦	Worst case assumptions
¦	When correctly done, more
conservative
Talking Points:
•	Steady state models assumes all conditions are static; they're
usually, but not always, based on worst case conditions.
•	There are two types of steady-state models: single-value
(chronic) or two-value (acute and chronic). Typical single- or
two-value steady-state WLAs are calculated at critical
conditions using worst-case assumptions for flow, effluent,
and environmental effect and are more conservative than
dynamic WLAs.
•	The permitting authority has the flexibility to develop seasonal
single- or two-value WLAs based on seasonal critical stream
flows. EPA recommends that steady-state WLAs be used
where few WET data are available and where daily water flow
records are not available.
19

-------
Dynamic Models
¦	Account for variability
¦	Determine probability that WQS are
exceeded
¦	Need considerable data
¦	Generally, result in higher WLAs
Talking Points:
•	Dynamic models tend to be more data intensive and
sophisticated, but may provide a more accurate picture of
what's happening in a receiving stream.
•	There is no need to develop or assess worst-case conditions
and results are not likely to be over conservative. However,
large amounts of appropriate data and resources are required
Use of dynamic models is recommended where adequate
receiving water and effluent data are available to estimate
frequency distributions.
20

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Water Quality Permit Limit
Goals
¦	Account for effluent variability
¦	Achieve water quality protection
-	protect WLA and WQS
-	prevent acute and chronic impacts
-	consider receiving water dilution
¦	Accountability
-	measure compliance
-	be fully enforceable
Talking Points:
•	Effluents may be variable (due to human activity for POTWs,
production cycles, treatment system performance, climate,
etc.). Certain permit limit derivation procedures specifically
account for effluent variability (the statistically-based approach
described in more detail below); other approaches do not
specifically account for it, but utilize conservative assumptions
with enough safety factors "built-in" to account for it
indirectly.
•	Permit writers also need to ensure that permit limit derivation
procedures are protective of the wasteload allocation (designed
to ensure that water quality standards are met -- both acute
and chronic, if so specified by the States). The wasteload
allocation should also be based upon allowable dilution.
•	A critical, but often overlooked consideration, is the need to
express permit limitations and conditions in a manner that can
be clearly understood by both the permittee and the regulatory
authority and which is fully enforceable.
21

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We Can Characterize the Desired
Effluent by a LTA and CV
PROB.
CV
WLA
LTA
CONCENTRATION
Talking Points:
•	Effluent behavior, if plotted as a function of probability of
occurrence typically exhibits a lognormal appearance, with
values clustered around a long term average, but skewed such
that extreme values are often seen on the high end of the
scale.
•	This is because treatment efficiency at the low end of
concentration scale is limited, while effluent concentrations
may vary widely at the high end of the scale, reflecting various
degrees of treatment system performance and loadings.
22

-------
Goal is to Reduce Effluent to
be Below WLA
Actual
PROB.
Desired
WLA
CONCENTRATION
Talking Points:
• The goal of the permit is to establish conditions that will "drive
behavior" so that compliance with the permit limit ensures that
the wasteload allocation is achieved.
23

-------
Because There are Two WLA, We
Need to Use the More Stringent
LTAaLTAc WLAe WLA^
CONCENTRATION

-------
Comparison of WLA to Permit
Limit
WLA:
Acute	1 hour
Chronic	4 days
Human health	up to 30 days
Limit:
AML	30 days
MDL	24 hours
Talking Points:
• Ultimately, the permit writer will need to "translate" between
the outputs of a wasteload allocation model and a permit limit.

-------
Permit Limit Derivation
Procedures
¦	Direct incorporation of 1-value model
as one limit expression; use of BPJ
factor for other limit
¦	Direct incorporation of WLAs from 2-
value steady model as permit limits
¦	Statistical translation of WLAs from 2-
value steady state model
Talking Points:
•	The permit writer can derive WET permit limits from the WLA
in several ways. Each approach has its own advantages and
disadvantages. The permit writer should carefully evaluate
each approach in light of its defensibility and resources and
data needed to implement it.
•	One widely used approach is the direct use of the WLA as the
permit limit. This approach is simple to implement. However,
it does not take effluent variability into account statistically
and may yield overly conservative permit limits. If compliance
monitoring is infrequent, permit limits based on this approach
may not be protective.
•	Another way to derive permit limits from the WLA is the TSD
statistical approach (pg. 98), where the permit writer accounts
for effluent variability through use of the coefficient of
variation (CV), converts the WLA to a long-term average, and
uses an acute-to-chronic ratio (ACR) where appropriate to
convert between acute and chronic WET data. Although more
complicated and data-intensive, WET permit limits based on
the TSD statistical approach are defensible, toxicologically
protective, and take effluent variability into account.

-------
Direct Incorporation of 1-
Value Steady State WLA to
Derive Permit Limits
Set WLA =MDL
Set AML =50% MDL
-	MDL may be overly stringent
-	50% factor may not be protective
Talking Points:
• The permit writer must take the WLA results and translate
them into the appropriate permit limits. One widely used,
relatively simple, approach is to take the WLA and set the WET
permit limit directly equal to it. Where a single value WLA has
been derived, it can directly implemented as the daily
maximum WET permit limit. The monthly average WET permit
limit can be subsequently derived by dividing the daily
maximum limit by a factor of 1.5/2.0 (using best professional
judgment).
27

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Direct Incorporation of Outputs of
2-Value Steady State WLA to
Derive Permit Limits:
¦	Set WLAa =MDL
¦	Set WLAc = AML
-	not always protective against chronic
effects
-	may unnecessarily lead to both acute and
chronic tests
Talking Points:
• Where two-value WLAs have been derived, the acute WLA
value can be directly implemented as the daily maximum WET
permit limit and the chronic WLA value can be directly
implemented as the monthly average WET permit limit. This
approach may result in overly conservative permit limits and
does not take effluent variability into account.
28

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Statistical Derivation from Two
Value Steady State Process
I	Acute and Chronic Wa&teload Allocations	H
I Step 1 Convert Acute WLA into Chronic WLA ^
	(WET only)	¦
I	Step 2 Calculate LTA for Both	H
Step 3 Select Lowest LTA
Step 4- Calculate Max and Avg Limits
Talking Points:
•The statistical approach to derive WET permit limits from the WLA is more
complex and data intensive, but it does take effluent variability into account.
•	In order to later compare the results from a two-value WLA to determine whether
acute or chronic impacts are more stringent, the permit writer must first convert
the acute WLA to a chronic WLA using an acute-chronic ratio (ACR). The value
of the ACR can be discharge-specific or a value may be assumed (typically 10).
•	Once the single- or two-value WLA is determined, it must be converted to a
single- or two-value long-term average (LTA). The LTA ensures that the WLA is
met under critical conditions over the long-term. The LTA is derived from an
estimate of effluent variability (the coefficient of variation (CV)) and a statistical
multiplier which represents the upper bound occurrence percentile (TSD, pg.102).
The smaller of the two-value LTAs is the more stringent and should be used to
derive the permit limits.
•The NPDES regulations require that POTW permit limits be expressed as a
monthly and weekly average (AML/AWL) and that industrial permit limits be
expressed as a monthly average and daily maximum (AML/MDL). For the final
step, the permit writer must then convert the more stringent LTA to the
AML/AWL or AML/MDL, as appropriate. The conversion is based again on an
estimate of effluent variability (the coefficient of variation (CV)), a statistical
multiplier which represents the upper bound occurrence percentile, and the
number of monthly compliance samples (TSD, pg. 103). The conversion from
LTA permit limit is analogous to the WLA LTA conversion discussed above.
29

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Acute to Chronic Ratio
ACR = LC50/NOEC
or
ACR = TUc/TUa
Talking Points:
•	Acute-to-chronic ration (ACR) is the ratio of the acute toxicity of an effluent or a
toxicant to its chronic toxicity. It is used as a factor for estimating chronic
toxicity on the basis of acute toxicity data or for estimating acute toxicity on the
basis of chronic toxicity data.
•	ACR varies between species for a given chemical and for any one species,
between different toxicants.
•	With the exception of a small number of "outliers" for which confirmation is not
possible, ACRs above 20 for effluents discharged by NPDES permitteess have not
been observed by EPA. The majority of observed ACRs are very seldom above
10. However, higher ACRs may be found for selected facilities. Where acute and
chronic toxicity data are available, the ACR should be calculated directly for that
specific effluent. (See Appendix A-3 of the Technical Support Document).
•	For Example, toxicity data (fathead minnow) from DMR:
LC50 (% effluent)
58.0
50.0
3.0
10.0
30.0
25.0
1.0
19.8
NO EC (% effluent)
ACR
1.16
8.40
5.50
1.54
1.79
5.90
4.05
25.2
55.0
46.3
44.8
5.9
Average 39.2
30

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Statistical Approach Derives Permit
Limits from the WLA
¦ Advantages of statistical treatment of
two-value steady state outputs
-	reflects effluent variability
-	compares acute and chronic WLAs to
determine the more limiting
-factors actual number of samples into
limit derivation

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Permits Must Specify
¦	Test species and method
¦	Testing frequency
¦	Type of sample
¦	Selection of dilution water/series
¦	Test duration and type
¦	Quality assurance procedures
¦	Statistical endpoint
¦	Steps to address toxicity
Talking Points:
•	It is critical that the permit writer be clear as to which tests,
species, endpoints, dilution and diluent are required.
•	Note - see acute and chronic permit sample language.
•	Section 122.43(c) requires all permit conditions to be
incorporated expressly or by reference to a specific citation to
the applicable regulations or requirements.
•	Section 122.48(a) requires permits to specify proper use of
methods.
•	Section 122.48(c) requires permits to specify monitoring type,
intervals, and frequency.
32

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Units of Expression and
Detection Levels
Talking Points:
•	The permit limit for toxicity and detection levels associated with the
various types of toxicity tests determine the type of monitoring
requirement which should be specified.
•	Either acute or chronic test might apply to a given situation, depending
upon the test detection levels or test sensitivity.
•	The acute toxicity test, when using an LC50 as the test endpoint, has
an upper sensitivity level of 100% effluent, or 1.0 TUa. If the permit
limit was 0.3 TUa, the endpoint would need to be changed to a greater
level of sensitivity, such as "no statistically significant difference
between 100% effluent and the control." Alternatively, the limit
expressed as acute toxicity could be multiplied by the acute-to-chronic
ratio and chronic toxicity could be used for compliance monitoring
purposes.
•	Similarly, an effluent with a NOEC of greater than 100% presents a
similar test sensitivity problem. However, an effluent with a NOEC of
greater than 100% contains less than 1.0 TUc and would meet the EPA
recommended criterion for toxicity at either the edge of the mixing zone
(if dilution is allowed) or at the end of the pipe.
33

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Specifications for
Acute/Chronic Toxicity Tests:
¦	Acute toxicity
-	endpoint - mortality
-	test duration - either 24, 48 or 96 hours
¦	Chronic toxicity
-endpoint - growth, reproduction and
sometimes mortality
-	test duration - 7 days or less
Talking Points:
•	Acute toxicity tests are used to determine the concentration of effluent or ambient
water that produces an adverse effect on a group of test organisms during either a 24,
48 or 96 hour exposure. The endpoint measured is mortality. In an acute toxicity test,
an effluent sample is collected, diluted and placed in test chamber with the chosen test
species. After 24, 48 or 96 hours, the number of live organisms remaining in each test
concentration and a control is recorded.
•	A chronic toxicity test is defined as a short-test in which sublethal effects, such as
growth, length, reproduction, fertilization, or larval development are measured in
addition to sometimes mortality. The short-term chronic toxicity test methods
developed range from 1 hour to 7 day exposure.
t Another aspect to consider when testing for acute toxicity testing is whether the
permittee is currently conducting a chronic toxicity test which also includes a survival
endpoint, such as the Pimephales promelas 7 day growth and survival test. In this
situation, compliance with acute and chronic requirements can be jointly evaluated; the
chronic toxicity at the end of the 7 day test and acute toxicity at either 48 or 96 hours
into the 7 day test. This is also known as a "dual endpoint" test; this is an effective
use of both time and financial resources.
•	The chronic test methods that could be evaluated for both acute and chronic
requirements are the topsmelt, the silverside, the Pacific mysid and the Atlantic mysid.
The chronic water flea test method, Ceriodaphnia dubia, is not typically analyzed for
both acute and chronic requirements because the test design is not amenable to
calculation of a lethal concentration (LC50) value as needed for the acute requirement.
•	The first decision for a permit writer to make in selecting the appropriate toxicity tests
is whether to measure acute or chronic effects. The next question to answer is
whether to test with freshwater or marine species. Once that decision has been made,
the following parameters should be considered when selecting the appropriate test
species: taxonomic diversity; type of facility and toxicants; and seasonal and temporal
effects.
34

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Selection of Test Methods
SDecies
TvDe of discharaer
Freshwater species
Discharges into freshwater

waterbody
Marine species
Discharges into estuarine

or marine waterbody
Talking Points:
•	The decision of whether to use freshwater or estuarine and marine
test methods is based on salinity of the receiving water. As a
general rule, EPA recommends the following [TSD pg 61]:
1.	Freshwater organisms be used when the receiving water
salinity is less than 1,000 mg/L.
2.	Marine organisms be used when the receiving water equal or
exceeds 1,000 mg/L.
•	Saline effluent discharged to saltwater: The dissolved salts in the
effluent are possible pollutants. These salts may or may not be the
same as those present in the receiving water. The proportion of
dissolved salts in the effluent may be different from that of the
dissolved salts in the receiving water. The toxicity test should
determine if these salts contribute to ambient toxicity. For this
reason, marine organisms are the preferred test species.
35

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Talking Points:
•	Saline effluent discharged to freshwater: The dissolved salts in the
effluent are possible pollutants that are not present in the receiving
water. The toxicity test should determine whether the dissolved salts
are contributing to ambient toxicity. For this reason, freshwater
organisms are the preferred test species.
•	Freshwater Effluent Discharged to Saltwater: The lack of dissolved
salts in the effluent can cause a toxic effect in the marine toxicity test
organisms. In contrast to the scenarios presented above, the toxicity
test does not need to measure this effect as lack of salts is not a
pollutant. The marine toxicity test methods account for this by requiring
the salinity of the effluent be adjusted to approximate the salinity of the
receiving water. For this reason, marine organisms are the preferred
test species.
•	Effluent salinity may be lower than that tolerated by the test species
(see marine test method tables). Salinity adjustment is necessary when
effluent concentrations to be tested are high enough to reduce test
solution salinity below the acceptable range such as 34 + 2%o as
specified in the test method. To maintain acceptable salinity, these
higher test concentrations of effluent must be adjusted by adding
hypersaline brine or artificial sea salts as specified in the toxicity
manual. The toxicity testing laboratory should refer to the section on
hypersaline brine in the chronic marine toxicity test methods.
36

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Species Selection
¦	Approved test methods
¦	Taxonomic diversity
¦	Type of toxicant
¦	Seasonal and temporal components
Talking Points:
•	EPA has recently added new biological testing methods to the list
of approved and standardized analytical methods for testing
wastewater pollutants. This information was published in the
Federal Register as a final rulemaking (amendment) to the 40
CFR Part 136 analytical methods. This rule became effective on
November 15, 1995.
•	The test methods standardized in this rule will replace
unapproved test methods for NPDES permits issued after the
effective date of this rule. Existing NPDES permits will not be re-
opened to include test methods from this rule, unless the
permitting authority wants to re-open existing permits. However,
the NPDES permittee may request to the permitting authority the
need to replace existing methods with the newly promulgated
methods or the west coast chronic marine methods.
•	The permitting authority selects the appropriate species to be
tested based on taxonomic diversity, type of facility, types of
potential toxicants and effluent seasonal and temporal effects.
In addition, the permitting authority should evaluate any existing
toxicity data provided by the permittee.
37

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Talking Points:
•	In the selection of test species, EPA recommends the use of
species from ecologically diverse taxa [TSD p.58]. The
recommendation is to screen an effluent with at least three species
(a fish, an invertebrate, and a plant) for chronic testing and two
species (a fish and an invertebrate) for acute testing. This
recommendation is based upon the fact that there are species
sensitivity differences among different groups of organisms to
different toxicants.
•	It is important to consider the type of toxicants that may be
discharged from a facility and which species would be appropriate
for the such toxicants. For example, if a facility is discharging
waste that primarily consists of herbicides, a plant test method
may be more appropriate. Certain species have been found to be
sensitive to certain toxicants. Invertebrates are more sensitive to
organophosphate pesticides (e.g., diazinon) than fish. Fish are
more sensitive to ammonia than invertebrates. In situations where
multiple species screening is not practical (such as ambient toxicity
testing programs) it may be appropriate to test with the species
with known sensitivity to the toxicants of concern.
•	It may be necessary to consider possible seasonal or temporal
changes in the effluent when selecting the appropriate testing
species. For example, pesticides may be of concern after spring
runoff and typically invertebrates such as water fleas or mysids are
more sensitive.
38

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Testing Frequency
Possible Frequency of testing Size of discharger
Monthly	> 1 MGD
Quarterly	< 1 MGD
Other factors to consider: compliance record,
effluent variability
Talking Points:
•	Monitoring frequency is a compromise between need and cost. All
toxic effects testing and exposure assessment parameters, for both
individual chemicals and effluent toxicity, are associated with some
degree of uncertainty. The more limited the amount of test data,
the larger the statistical uncertainty.
•	An example of uncertainty associated with limited monitoring data
occurs when only one piece of effluent data is available (e.g.,
NOEC = 30%) for a facility. Effluent variability, based on the data
in the TSD, could range from 20 to more than 100 percent. With
only one data point available, it is impossible to determine where in
this range the effluent variability really falls. To be protective, EPA
recommends assuming that variability is at the high end of this
range. Collection of additional data will, in most cases, result in a
less conservative assumption regarding effluent variability.

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Talking Points:
•Tables 3-1 and 3-2 of the TSD show reasonable potential multiplying
factors based on the number of samples and the effluent coefficient of
variation (CV). At the default CV of 0.6 and a probability basis of 99
percent, the multiplying factor is 13.2 with only one sample. With four
samples, the factor decreases to 4.7. Independent of other
considerations, such as a database analysis of individual chemicals and
WET results, EPA has observed that 10 or more samples per month
provides the greatest statistical likelihood that the average of monthly
values will approach the true monthly long-term average value.
•	In some cases, the available effluent data may not actually project an
excursion above the acute or chronic toxicity criterion. Under these
conditions, the regulatory authority may decide to require that toxicity
tests be repeated at a minimum of quarterly for majors and annually for
minors. If no reasonable potential exists for excursions above the
acute or chronic toxicity criterion, the regulatory authority may decide
to require that the toxicity tests be repeated at least once before permit
reissuance. The fact sheet should full describe the basis for all
monitoring requirements.
•	Some regulatory authorities allow reduced frequencies based on test
results. For example, EPA Region IX recommends that frequency be
reduced if no individual toxicity test results in a value greater than the
WET limit or trigger divided by the reasonable potential factor. The
reasonable potential factor, from Table 3-1 of the TSD, is based on the
number of samples and CV. The reasonable potential factor decreases
with increased number of samples. If WET limits are required, though,
the minimum reporting frequency allowed by the regulations at 40 CFR
122.44(i)(2) is annually.
•	In addition, the frequency of testing may be adjusted in accordance
with historical monitoring data for a particular discharge.
40

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Type of Sample
¦	Type of facility
¦	Effluent variability
¦	Sampling logistics
¦	State guidelines
Talking Points:
•	Effluent samples should be collected as either 24-hour
composite or grab samples. The most frequently used sampling
is the 24-hour composite.
•	The decision on whether to collect grab or composite samples
is based on the objectives of the test and an understanding of
the short and long-term operations and schedules of the
discharger. If the effluent quality varies considerably with time,
which can occur where holding times within the treatment
facility are short, grab samples may be preferable because of
the ease of collection and the potential of observing peaks
(spikes) in toxicity.
•	Grab samples may need to be used for stormwater testing and
power plants. However, the sampling duration of a grab
sample is so short that full characterization of an effluent over
a 24-hour period would require a prohibitive number of
separate samples and tests.
41

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Selection of Dilution Water
¦	Standard lab water or receiving water
¦	Depends on the objectives of the test
Talking Points:
•	As stated in the testing manuals the selection of dilution water is based on the
objectives of the test.
•	The use of dilution water is an important part of toxicity testing. Dilution water may be
either standard laboratory water and/or receiving water. The type of dilution water used
in effluent toxicity tests will depend largely on the objectives of the test. These
objectives are:
(1)	If the objective of the test is to estimate the absolute acute or chronic toxicity
of the effluent, which is the primary objective of NPDES permit-related toxicity
testing, a standard laboratory dilution water as defined in each test method is
used.
(2)	If the objective of the test is to estimate the toxicity of the effluent in
uncontaminated receiving water, the test may be conducted using dilution
water consisting of a single grab sample of receiving water (if non-toxic),
collected either upstream and outside the influence of the outfall, or with other
uncontaminated natural water (ground or surface) or standard dilution water
having approximately the same characteristics (hardness and/or salinity) as the
receiving water.
(3)	If the objective of the test is to determine the additive or mitigating effects of
the discharge on already contaminated receiving water, the test is performed
using dilution water consisting of receiving water collected immediately
upstream or outside the influence of the outfall.
Note: If the test organisms have cultured in water which is different from the test
dilution water, a second set of controls, using culture water should be included in the
test.
42

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Selection of Dilution Series for
Testing
¦	Multiple dilution concentration test
¦	Single concentration test
Talking Points:
•	Chronic toxicity tests should be performed with a minimum of
five treatments and a control.
•	One of the five effluent treatments must be a concentration of
effluent mixed with dilution water which corresponds to the
facility's IWC. At least two of the effluent treatments must be
of lesser effluent concentration than the IWC, with one being
at least one-half the concentration of the IWC. No
concentration should be greater than two times that of the
next lower concentration. Example (IWC = 100%), dilution
series should be 100, 75, 50, 25 and 12.5% effluent.
•	It is important to calculate the dilution by dividing the total
stream flow (upstream flow plus effluent flow) by the effluent
flow, assuming complex mix. The instream waste
concentration (IWC) is the inverse of the dilution factor.
•	Sometimes acute and chronic toxicity tests are only conducted
with the IWC and a control.
•	Ambient and stormwater toxicity testing are sometimes
conducted with 100% receiving water and a control.
43

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Selection of Test Duration
¦	Acute duration - either 24, 48 or 96
hours in length
¦	Chronic duration - range from 40
minutes to 7 days
Talking Points:
•	The test duration for the chronic tests range from 40 minutes
to 7 days. The chronic test methods specify the duration of the
test, such as 48 hours for the red abalone larval development
test.
•	The acute test methods can be conducted as either 24, 48 or
96 hours in duration. If the toxicant is fast acting (if this
information is available), then select either a 24 or 48 hour
duration. These tests are usually conducted as static non-
renewal tests. Non-renewal testing is important when it may
be difficult to collect effluent renewals such as stormwater or
overseas samples. If the mode of toxicant is unknown as is
the case with most effluents, then select a 96-hour test with a
renewal at 48 hours.
44

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Selection of Test Type
¦ Tests may be conducted as either:
-	static non-renewal
-	static renewal
-	flow-through
Talking Points:
•	Static non-renewal tests: The test organisms are exposed to the same
test solution for the duration of the test.
•	Static renewal tests: The test organisms are exposed to a fresh test
solution of the same concentration of sample every 24-hours or other
prescribed interval, either by transferring the test organism from one test
chamber to another, or by replacing all or a portion of solution in the test
chambers.
•	Flow-through tests: (1) sample is pumped continuously from the
sampling point directly to the dilutor system; or (2) grab or composite
samples are collected periodically, and then placed in a tank to the dilutor
system.
•	The chronic test methods specifies whether the test is to be conducted
as static non-renewal or as static renewal.
•The acute test methods can be conducted as either static non-renewal,
static renewal or flow-through tests. See Diamond et al., 1995 for a
description of a flow-through system design using larval fish. The acute
test manual highlights some advantages and disadvantages of the test
types to consider when determining whether to use static non-renewal,
static renewal or flow through for acute toxicity testing [USEPA 1993,
p.45].
45

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Quality Assurance Procedures
¦	Reference toxicant testing
-	in house culture, monthly testing
-	outside organisms, concurrent testing
¦	Conduct using the same test
conditions
Talking Points:
•	Reference toxicant tests indicate the sensitivity of the test
organisms and demonstrate the lab's ability to perform the
test.
•	Reference toxicants to be used are specified in the test method
manuals (e.g., copper sulfate, sodium chloride). Frequency of
testing depends on whether the lab is using in-house cultured
organisms or outside sources (e.g., commercial supply houses).
If organisms are not cultured in-house, then concurrent testing
with reference toxicants shall be conducted. For organisms
cultured in-house, then monthly reference toxicant is required.
•	Reference toxicant tests shall be conducted using the same
test conditions as the effluent toxicity test (i.e., same test
duration, etc).

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Statistical Endpoints
¦	Acute:
-LC50
-	Pass/fail
-	NOEC
¦	Chronic:
-	EC/ICp
-NOEC
-	Pass/fail
Talking Points:
•	The recommended statistical analysis of most data from acute
and chronic toxicity tests follow a decision is illustrated in the
test manuals.
•	Acute tests are typically reported as a lethal concentration
(LC). Sometimes, acute tests are reported as either a pass or
fail when conducting a t-test. This is the case, when only the
instream waste concentration (IWC)is compared to a control.
•	Chronic tests are reported as either a NOEC or as a point
estimate (EC or ICp). The no observable effect concentration
(NOEC) is determined by hypothesis testing procedures.
47

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Steps to Address Toxicity
¦	Accelerated testing
¦	Include TRE/TIE language
Talking Points:
•	Some permit conditions require accelerated testing when a
permit limit or monitoring requirement has been triggered.
•	Reference to steps to be taken to identify and control the
toxicant(s) should be included in the permit (TRE and TIE
requirements). Include reference to the TRE documents.
•	Some permits include a requirement that the permittee shall
submit a copy of the permittee's TRE workplan (1-2 pages)
within 90 days of the effective date. See example TRE
workplans.
48

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PERMITTING CASE
EXAMPLES
North Carolina
Talking Points:
•	This discussion will focus on the ways that EPA guidance has been
implemented in a delegated state's NPDES program. Hopefully, this will be an
interactive discussion to resolve problems.
•	Comparisons and contrasts to EPA recommended strategies will be offered to
highlight considerations made in that process.
•	In many cases, regulator resource constraints affect the ways in which
programs are developed. Consideration of categories of discharges versus
case specific evaluations is a recurring theme throughout this discussion: a
situation often predicated by limited regulatory resources.
•	Where possible, allowances are made for permittees to make a showing that
generic assumptions may not hold true on a site specific basis.
To place context on North Carolina's experience:
•	Limits began in permits in 1987
•	Biological Laboratory Certification began in 1988 ( Required for NPDES WET
analyses)
•	Currently >550 permits with WET limits ("75% chronic Ceriodaphnia)
•	Receive and review >2,000 toxicity test reports/year
•	Current compliance with all WET limits >90% at any time
i

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CHRONIC TOXICITY PASS/FAIL PERMIT LIMIT (GRTRlY)
The effluent discharge shell at no time exhibit chrome toxicity using test procedures outlined m
1 J The North Carolina Cenodaphma chrome effluent bioosay procedure (North Carolina Chrome Bioassay
Pr ©cod i*e - Revised * September 1989) or subsequent vctsjotb
The effluent concentration it which there iniy be no observable intabition of reproduction or significant mortality
°	(defined as treatment two in the North Carolina procedure document) The permit holder shall
perform Quarterly morutonng using tha procedure to establish compliance with the permit condition The
first test will be performed after thirty days from the effective date of tho permit during the months of
		 Effluent sampling for this testing shall be performed at the NPOES
permitted final effluent discharge below all treatment processes
All toxicity testing results required « part of tfes permit condition will be entered on the Effluent Discharge
Momtonng form (MR-1) for the month m which it was performed, using the parameter code TGP3B
Additionally, OEM Form AT-1 (original) ts to be tent to the following address
Address
Test data shall be complete and accirate and indude all supporting chemical/physical measurements performed m
association with the toxicity tests, as wet! as all dose/response data Total residual cNonne of the effluent
toxicity sample must be messired and reported if chlorine a employed for d&nfection of the waste stream
Shotid any single ouarterty mentoring indicate a failure to meet specified bmita. t>wn monttty moratory wiO
begm tfnmediatety irttjl such time that a smgle test a passed Upon passing, this monthly test requrement
wtll revert to quarterly m the months speafied above
Should any test data from this momtonng requrement or tests performed by the North Carolina Division of
Environmental Manaoement indicate potential impacts to the receiving stream, tt*s permit may be re-optned
and modified to melude alternate monitoring requirements or bmrts
NOTE Failure to achwve test conditions as specified in the cited document such as miwmi*n control orgemsm
survival and appropriate environmental controls, shall constitute an invalid test and will require immediate
retestmgfwithm 30 days of imtial morutonng event) Fa^ure to submit sutaMe test results will constitute
noncompliance wrth momtonng requirements
Talking Points:
• Permit limits are written such that:
-a limit is clearly defined
-	species and test method are specified
-sampling point, duration and frequency are specified
-acceptability criteria are considered
-	reporting requirements are specified
2

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CHRONIC TOXICITY PERMIT LIMIT (QRTRLYJ
The effluent discharge shall at no time exfobt 48 hoi* acute toxicity as lethality m an effluent concentration of	%
nor measure a quarterly arithmetic average chrome value leaa than tho same percentage of waste The chronic
value will be determined using the geometric mean of the highest concentration having no statistically detectable
rmpairmem of reproduction or survival and the lowest concentration that does have a statistically detectable
impairment of reproduction or survival The presence of 48 hour acute toxicity will be determined using Ftsher's
Exaet Test at 48 hours from test initiation. Collection methods, exposure regimes, and further statistical methods
are defined in The North Carolina Phase II Chrome Whole Effluent Toxicity Test Procedure (July, 1991) or
subsequent versions
The permit holder shall perform at a mintmun. quarterly monitoring using these procedures to establish compliance
with the permit condition The first test will be performed within thirty daya from the effective date of this permit
during the months of	 Efftusni sampling for this testing shall be performed at the NPOES
permitted final effluent discharge below at! treatment processes
If the test procedure performed as the first teat of any single quarter measures 48 hour acute toxicity or a chrome
value less than that specified above, then multiple concentration testirq shall be performed, at a minimum, in
each of the two following months
All toxicity testing results requred as pan of this permit condition will be entered on the Effluent Discharge Monitoring
Form (MR-1) for the months in which tests were performed, using the parameter code THP3B for the Chrome
Value and TGA3B for the 48 hour Acute Toxicity measure (Pass/Fail) Additionally DEM Form AT-3 (original) ts
to be sent to the following address Address
Test data shall be complete and accurate and include all supporting chemical/physical measurements performed m
association with the toxicity tests, as wdl as all dose/response data Total residual chlonne of the effluent
toxicity sample must be measured and reported if chlorine ts employed for disinfection of the waste stream
Should any test data from thrc monitoring reourement or tests performed by the North Carolina Division of
Environmental Management indicate potential impacts to the receiving stream, this permit may be re-opened and
modified to tndude alternate momtonng requremems or limits
NOTE Faikre to actveve test conditions as specified in the cited document, such as mimmifn control organism
survival and appropriate environmental controls shall constitute an invalid test and will require immediate
retestmg (within 30 days of initial momtonng event) Failure to submit swtable test resets will constitute
noncompliance with monitoring requirements
Talking Points:
•	Consistency with the way that other limits are written should
be maintained.
•	In contrast to some recommendations and practices, this limit
makes no mention of how compliance should be achieved
(e.g. conduct TIE/TRE).
•	As would be the case for a conventional parameter, the
expectation of compliance only, is outlined.
•	The limit is thus clear: compliant or non-compliant. It does not
extend itself further into the realm of enforcement.

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NCAC T15A:02B.0103
Analytical Procedures
(b) Biological procedures. Biological tests to
determine conformity or non-conformity with
standards will be based on methods published by
the U.S. Environmental Protection Agency as
outlined in...
...or methods published by the North Carolina
Department of Environment, Health and Natural
Resources, as outlined in...
...or other such methods as approved as the director
Talking Points:
•	Methods development and support is a very time consuming
process but one that is necessary in evolution of best possible
practices.
•	Promulgation of WET methods in 40 CFR part 136 should
greatly assist with implementing standard test practices. There
are still many areas where method interpretation will be
needed, both as policy and in individual cases. SETAC Pellston
WET and other activities should bring closure to many issues
of method validity.
•	NC has developed abbreviated testing procedures (pass/fail)
which use fewer samples/renewals and are therefore cheaper
to perform.
•	Retain local flexibility in implementing methods but base on
existing protocols to extent possible to decrease need for local
defense/support.
4

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NCAC T15A:02B.0202
Definitions
[paraphrasing]
(1) Acute Toxicity
(a)	specific chemicals- one half or less FAV
(b)	or 1 /3 or less lowest LC50
(c)	effluents- no statistically measurable (t-
test at 99%CL)
(d)	or Director may approve case-by-case on
conc.- response curve
Talking Points:
•	Get involved in standards development and definition. WET
does need some specific language but it should compliment
existing standards for specific chemical substances. A large
part of North Carolina's success in WET has been the
involvement of biologists and biology in the entire water
quality program.
•	Definitions should clearly define considerations necessary to
establish compliance with narrative or numeric criteria.
•	Retain flexibility.
5

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NCAC T15A:02B.0202
Definitions
(13) Chronic toxicity to aquatic life means any
harmful effect sustained by either
resident aquatic populations or indicator
species used as test organisms in a
controlled toxicity test due to long-term
exposure (relative to the life cycle of the
organism) or exposure during a
substantial portion of the duration of a
sensitive period of the life cycle to a
specific chemical substance or mixture of
chemicals (as in an effluent).
Talking Points:
•	The way that standards are written (or re-written) will directly
influence the way that permit limits are written.
•	Chronic toxicity is defined here as ...any harmful effect...
That is interpreted as any measurable effect and thus chronic
limits are established as a test of hypothesis rather than a
biological point estimate of acceptable/sustainable reduction
of a population.
6

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NCAC T15A:02B.0208
Standards for Toxic Substances
and Temperature
(a)(1) Aquatic life standards. The concentration of
toxic substances will not result in chronic
toxicity. Any levels in excess of the chronic
value will be considered to result in chronic
toxicity. In the absence of direct measurements
of chronic toxicity...
[paraphrasing]
not> prediction of chronic through A:C ratio
not> 1/20 lowest LC50 non-persistent
not > 1/100 lowest LC50 persistent
Talking Points:
•	With aquatic life standards clearly defined in regulation, their
implementation through permits becomes a matter of testing
the standard: a test that can only be performed by WET
analysis.
•	This linkage with criteria and other chemical specific standards
allows consideration of specific chemicals through existing
data, extrapolation of exiting data, or by direct measurement.
7

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Reasonable Potential
¦	All major
¦	All complex minor
- excludes:
•	100% domestic minor
•	non-contact cooling water
•	aquaculture
•	swimming pools
•	mine dewatering
•	water filtration backwash
•	carwashes
•	rock and gem mines
•	sand dredging
Talking Points:
•	Rather than making individual decisions on the reasonable
potential for wastewater effluents to cause aquatic toxicity
problems based on pre-permit WET data, the Director of the
NCDEM made the generic determination that any any major
discharger and any discharger of complex waste had the
potential to cause those problems and developed the policy
that those discharges would have WET limits included as
permit requirements.
•	Some exclusions, as listed, were provided where the
wastewater would not be considered complex for the
purpose of establishing WET limits. In these cases, specific
chemical limits alone would be deemed acceptable for
controlling the discharge of toxics in toxic amounts.
8

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Limits
¦	IWC >0.25%: Chronic limit at IWC (P/F
or P2)
¦	IWC <0.25: Acute limit at 90% (P/F)
¦	Consideration of diluting flows
-	lakes
-	tidal
¦	Consideration of frequency/duration of
exposure
-	stormwater
-	intermittent cooling water
Talking Points:
•	In considering exposure, limits were established by evaluating IWC as
maximum permitted effluent flow and 7Q10 receiving stream flow (per
regulation).
•	Facilities with discharges to flowing systems with IWC >0.25% receive a
chronic toxicity limit at the diluted IWC. Acute toxicity in a mixing zone is
not separately limited. State evaluation of a broad range of discharges
using both the chronic and acute analyses has shown that the chronic
limitation (using Ceriodaphnia) was nearly always protective of acute
toxicity to T\sh(Pimephales).
•	Facilities with discharges to flowing systems with IWC <0.25% receive an
acute toxicity limit of 90%. At this point of dilution, the effluent is
considered unlikely to cause chronic toxicity.
•	Other significant considerations of exposure include diluting flows where
simple downstream dilution may not occur; e.g., lakes and estuaries. In
these cases, high ultimate dilution is assumed and acute toxicity is
protected using an appropriate species.
•	Frequency and duration of exposure are considerations in intermittent
discharge situations. With stormwater discharges, 7Q10 streamflow
conditions probably do not represent realistic exposure conditions.
9

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Statistical Endpoints
¦	Acute
-	set up as either LC50 or t-test of
significance
¦	Chronic-use ChV
-	established in regulation
-	do not have to justify biological
significance
-	have made functional modifications- PSC
Talking Points:
•	Acute limits as either an LC50 if concentration/response
information is needed or as a t-test of significance if pass/fail.
•	Chronic limits are established/implied as a Chronic Value
(geometric mean of NOEC/LOEC): the predicted point of
observable chronic response.
•	The hypothesis test obviates the need for the regulator to
define a level of biological significance. In practice, statistical
and biological significance may be close.
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Monitoring Frequency
¦	Continuous discharge- quarterly in
specified months
-	reverts to monthly on single fail
-	reverts to quarterly on single pass
¦	Intermittent discharge- more case
specific
-first five discharge events then annually
thereafter
-	each discharge event
Talking Points:
•	Monitoring frequency, like number of species, becomes a
balance between obtaining best possible information and
resistance to requirements.
•	NC has established a monitoring frequency of quarterly;
reverting to monthly on a single failure and back to quarterly
on a single pass .
•	Facilities that are compliant (Currently >90%) thus have
minimized testing costs.
•	Intermittent discharges may require a more case specific
consideration of frequency or even need for limits. These may
frequently start the permit process with monitoring only
requirements.
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Species Selection
¦	Chron\c-Ceriodaphnia dubia
¦	Acute - freshwater - fathead minnow
¦	Acute saltwater - Mysidopsis bah/a
-	saltwater discharges usually tidal, thus
acute
-	if chronic then M. bahia
-	saltwater permittees may use a
freshwater species with no salinity
modification if desired
Talking Points:
•	Ceriodaphnia dubia will be the chronic toxicity test organism
required for freshwater testing. C. dubia is considered by NC
the most sensitive of species commonly used and available for
WET chronic testing to the widest variety of toxicants. While
in certain cases this may not hold true (e.g., ammonia),
chemical specific limits are deemed sufficient to provide
protection. Chronic tests have not yet been required in
saltwater because of considerations of dilution and thus
exposure.
•	Fathead minnows are tested in freshwater to protect against
acute toxicity to fish species in areas of immediate dilution.
•	Freshwater organisms may be allowed to test acute toxicity in
marine waters in consideration of testing costs.
•	Regulators should consider the benefits and drawbacks of
supporting additional test species(e.g., QA/QC, performance
evaluation, cultures) when deciding on use of multiple species
for permit limits.
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Practical Sensitivity Criteria
(PSC)
¦	Sets lower bound of C. dubia reproduction
suppression for compliance purposes
¦	Sets upper bound of maximum statistical
insensitivity for QA purposes
¦	Established by review of thousands of test
results
¦	Reduces disincentive for production of
sensitive data
Talking Points:
•	For chronic tests, NC has established a compliance level of
response (Practical Sensitivity Criteria) using large datasets of
reported data.
These consider both:
-the lower level of response (maximum sensitivity) that
laboratories generally achieve, and
-the upper bound of lack of response (minimum sensitivity)
that labs should be able to achieve.
•	This removes disincentive to produce relatively sensitive (low
variability) data and provides incentive not to produce
insensitive (highly variable) data.
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Quality Assurance
¦	Laboratory Certification/Control
¦	Data Review
-	enforce methods
-	enforce test acceptability criteria
¦	Split sample review
-	data represents reasonable duplication of
analysis
¦	DMR/QA
¦	Important stuff
Talking Points:
•	The importance of assuring data quality, preferably by both
the permittee and the regulator, cannot be overemphasized.
This is true not only for assessment of individual reports, but
also for the credibility of the program. Problems with data
quality must be addressed.
•	Specified methods and acceptability criteria must be enforced.
Neither discharger nor regulator should use invalid data.
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Take Home Points
¦	Establish authority, definitions in regulation
¦	Retain flexibility
¦	Apply appropriate exposure considerations
¦	Write permit language consistent with
chemical specific
¦	Limit the need for site specific consideration
¦	Consider economics of monitoring
¦	Make it enforceable and enforce
¦	Require valid data and enforce

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SAMPLE ACUTE PERMIT LANGUAGE
This appendix contains suggested language and format for including whole effluent toxicity
testing requirements and/or limits in permits. Items marked in redline are individual decisions that
need to be made by the permit writer. Information and guidance on making those decisions are
discussed in the previous sections of this document.
I. ACUTE TOXICITY
WHOLE EFFLUENT TOXICITY TESTING
The permittee shall conduct monthly/quarteriy/semi-ar.nual/annual toxicity tests on
grab/24-hour composite effluent samples. Samples shall be taken at the NPDES
sampling location. If, after one year of testing, the maximum measured toxicity is
less than or equal to {target/reasonable potential factor}' TUa'. then monitoring
frequency shall be reduced to annual/once more before permit issuance. (If a WET
limit is required, monitoring frequency shall be reduced to no less than once per
year.)
1. Test Species and Methods:
NOTE: CHOOSE EITHER FRESHWATER OR MARINE
Freshwater
a.	The permittee shall conduct 48-hour non-renewal /96-hour static renewal tests
with an invertebrate, the water flea. Ceriodaphnia dubia/Dzphma pulex or
Daphnui magna and a vertebrate, the fathead minnow, Pimephales
promelas/cainbow trout, Oncorhynchus mykisf for the first three suites3 of
tests. After this screening period, monitoring shall be conducted on the most
sensitive species.
b.	Every year, the permittee shall re-screen once with the two species listed
above and continue to monitor with the most sensitive species. Rescreening
shall be conducted at a different time of year from the previous year's
screening.
1	"Target" is the trigger for toxicity when a WET limit is not required. If a limit is
required, then the target is the limit. The reasonable potential factor is found in
Table 3-1 of the TSD, page 57. It is based on the CV and number of samples taken.
2	Any freshwater species listed in Appendix B, "Supplemental List of Acute Toxicity
Test Species", may be used in place of the foregoing.
"Suites of tests" means the two or three species used for testing during the permit
term.
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c. The presence of acute toxicity shall be determined as specified in Methods for
Measuring the Acute Toxicity of Effluents to Freshwater and Marine
Organisms, Fourth Edition, EPA/600/4-90/027F, August 1993. Or
subsequent editions
Marine/Estuarine
a.	The permittee shall conduct 48-hour non-renewal/96-hour static renewal tests
with the invertebrate Pacific mysid, Holmesimysis costata/Atlantic mysid,
Mysidopsis bahia and a vertebrate, the topsmelt, Atherinops affinisl inland
silverside, Menidia betyllwa for the first three tests. After this screening
period, monitoring shall be conducted using the most sensitive species.
b.	Every year, the permittee shall re-screen once with the two species listed
above for one month and continue to monitor with the most sensitive species.
Rescreening shall consist of one test conducted at a different time than the
previous year's test.
c.	The presence of acute toxicity will be determined as specified in Methods for
Measuring the Acute Toxicity of Effluents to Freshwater and Marine
Organisms, Fourth Edition, EPA/600/4-90/027F, August 1993 or subsequent
editions.
2. Limits/Definition of Toxicity"
If greater than 3:1 dilution is available, sections 2a & 2b apply: if less than 3:1,
Sections 2c & 2d apply.
a.	For the purposes of this permit, acute toxicity is defined as an LC50 <
{trigger, etc. to be specified} , determined using the test organisms and
statistical procedures required in Part	of the permit. fWhen a limit for
acute toxicity is appropriate, put it in the limits section. If a permit limit is not
appropriate, then this section should be called "Definition of Toxicity".]
b.	Where the LC50 is calculated, results shall be reported in TUa, where TUa
= 100/LC50 (in percent effluent).
c.	Acute toxicity is significantly reduced survival at 100 percent or at the
instrcam waste concentration (TWC) compared to a control, using a t-test.
Hie IWC is the effluent concentration at the edge of the mixing zone.
d.	Results shall be reported as pass (P) or fail (F) when using a t-test.
if a WET I fruit is appropriate, then this section should be contained in the Effluent Limitations section
of the permit, and not in the monitoring requirements sections.
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3. Quality assurance
NOTE: CHOOSE ONE, LC50 or t-test
a. A series of five dilutions and a control will be tested. The series shall include
the instream waste concentration (IWC) (permit writer should insert the actual
value of the IWC), two dilutions above the IWC, and two dilutions below the
IWC.
a.	Two dilutions shall be used, i.e., 100 percent or the IWC and a control pfiii
t-test is used instead of an LC5Q).
b.	If organisms are cultured in-house, reference toxicant tests shall be run
monthly. Otherwise, concurrent testing with reference toxicants shall be
conducted.
c.	If either of the reference toxicant tests or the effluent tests do not meet all test
acceptability criteria as specified in the test methods manual, then the
permittee must re-sample and re-test within 14 days of receiving the results of
the failed test/as soon as possible.
d.	Reference toxicant tests shall be conducted using the same test conditions as
the effluent toxicity test (i.e., same test duration, etc.).
e.	Control and dilution water should be receiving water or lab water, as
appropriate, described in the manual". If the dilution water is different
from the culture water, a second control shall be used, using culture water.
f.	Chemical testing for the parameters for which effluent limitations exist shall
be performed on a split of each sample collected for WET testing. To the
extent that the timing of sample collection coincides with that of the sampling
required in Part	of this permit, chemical analysis of the split sample will
fulfill the requirements of that Part as well.
4. Preparation of Generic TRE Workplan
The permittee shall submit to EPA a copy of the permittee's toxicity reduction
evaluation (TRE) workplan [1-2 pages] within 90 days of the effective date of this
permit. This plan shall describe the steps the permittee intends to follow in the event
that toxicity is detected, and should include at a minimum:
5 The manuals describe various situations in which either receiving water or lab water
should be used for control and dilution water. Depending upon the objective of the
test, either lab water or receiving water may be used.
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(a)	A description of the investigation and evaluation techniques that would
be used to identify potential causes/sources of toxicity, effluent
variability, treatment system efficiency;
(b)	A description of the facility's method of maximizing in-house
treatment efficiency, good housekeeping practices, and a list of all
chemicals used in operation of the facility;
(c)	If a toxicity identification evaluation (TIE) is necessary, who will
conduct it (i.e., in-house or outside consultant)
5. Reporting
a.	The permittee shall submit the results of the toxicity tests in TUs with
the discharge monitoring reports (DMR) for the month in which the
tests are conducted.
b.	The full report shall be submitted by the end of the month in which the
DMR is submitted.
c.	The full report shall consist of: (1) the toxicity test results; (2) the dates
of sample collection and initiation of each toxicity test; (3) the type of
production; (4) the flow rate at the time of sample collection; and (5)
the results of the effluent analyses for chemical/physical parameters
required for the outfalI(s) as defined in Part of the permit.
d.	Test results for acute tests shall be reported according to the acute
methods manual chapter on Report Preparation, and shall be attached to
the DMR. Where possible, the permittee shall submit the data on an
electronic disk (3.5") in the Toxicity Standardized Electronic Reporting
Form (TSERF).
e.	Evaluation results--the permittee shall notify EPA and the State in
writing within fifteen (15) days of receipt of the results of the
exceedance of the iimit/TRE trigger of
(1)	The finding of the TRE or other investigation to identify the
cause(s) of toxicity;
(2)	Actions the permittee has taken or will take to mitigate the
impact of the discharge, to correct the noncompliance and to
prevent the recurrence of toxicity;
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(3)	Where corrective actions including a TRE have not been
completed, an expeditious schedule under which corrective
actions will be implemented; and
(4)	If no actions have been taken, the reason for not taking action.
6.	Accelerated Testing
a.	If acute toxicity is greater than .	 TUa in
any test (i.e., the permit limit or the TRE
trigger when no permit limit], then the permittee
shall conduct six more tests, bi-weekly (every two
weeks), over a twelve-week period, beginning
within two weeks of receipt of the sample results
of the exceedance.
b.	If implementation of the generic TRE workplan
indicates the source of toxicity (for instance, a
temporary plant upset), then only one additional
test is necessary. If toxicity is detected in
this test, then Part 5a. shall apply.
c.	If any of the six additional tests indicate acute
toxicity greater than 		 TUa [i.e., the permit
limit when a limit is used, or the trigger when no
permit limit for WET is used] then, in accordance
with EPA manuals EPA/600/2-88/070 (industrial) or
EPA/600/4-89/001A (municipal) and the permittee's TRE
workplan , the permittee shall initiate a TRE within
fifteen (IS) days of receipt of the sample results
of the exceedance.
d.	If none of the six tests indicates toxicity, then
the permittee may return to the routine testing
frequency.
7.	Toxicity Identification Evaluation (TIE)
a.	If acute toxicity is detected in any two of the
six bi-weekly tests, the permittee shall, in
accordance with EPA acute and chronic manuals
EPA/600/6-91/005F (Phase I), EPA/600/R-92/080
(Phase II), and EPA-600/R-92/081 (Phase III),
initiate a TIE within 15 days.
b.	If a TIE is triggered prior to completion of the accelerated testing, the
accelerated testing schedule may be terminated, or used as necessary in
performing the TIE.
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8. Reopener
This permit may be modified in accordance with the
requirements set forth at 40 CFR Parts 122 and 124, to
include appropriate conditions or limits to address
demonstrated effluent toxicity based on newly available
information, or to implement any EPA-approved new State
water quality standards applicable to effluent
toxicity.
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n. CHRONIC TOXICITY
WHOLE EFFLUENT TOXICITY TESTING
The permittee shall conduct monthiy/quarteriy/semi-anaual/aimual toxicity tests on
grab/24-hour composite effluent samples. Samples shall be taken at the NPDES
sampling iocation. If, after one year of testing, the maximum measured toxicity is
less than or equal to (target/reasonable potential factor} TUe3. then monitoring
frequency shall be reduced to annual/once more before permit issuance. (If a WET
limit is required^ monitoring frequency shall be reduced to no less than once per
year.)	
1. Test Species and Methods:
NOTE: CHOOSE EITHER FRESHWATER OR MARINE LANGUAGE
Freshwater
a.	The permittee shall conduct short-term tests with the water flea, Ceriod.aphn.ia
dubia (survival and reproduction test), the fathead minnow, Pimephales
promelas (larval survival and growth test) and the green alga, Selanastrum
capricornutum (growth test) for the first three suites2 of tests. After this
screening period, monitoring shall be conducted using the most sensitive
species.
b.	Every year, the permittee shall re-screen once with the three species listed
above and continue to monitor with the most sensitive species. Re-screening
shall be conducted at a different time of year from the previous year's re-
screening
c.	The presence of chronic toxicity shall be estimated as specified in Short-Term
Methods for Estimating the Chronic Toxicity of Effluents and Receiving
Waters to Freshwater Organisms, Third Edition, EPA/600-4-91-002, luly
d.	The permittee may also determine compliance with acute fathead minnow test
based on the mortality data from chronic test data.
1	"Target" is the trigger for toxicity when a WET limit is not required. If a limit is
required, then the target is the limit. The reasonable potential factor is found in
Table 3-1 of the TSD, page 57. It is based on the CV and number of samples taken.
2	"Suites of tests" means the two or three species used for testing during the permit
term.
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Marine/Estuarine
Test Species and Methods:
a. The permittee shall conduct tests with a vertebrate, an invertebrate, and a
plant, as follows for the first three suites of tests. For Region 10: Hie
permittee shall conduct tests with a vertebrate and two invertebrates, as
follows for the first three suites of tests. After the screening period,
monitoring shall be conducted using the most sensitive species.
For Region 9 only:
Plant: Giant kelp, Macrocystis pyrifera (germination and germ-tube length
test).
For both Regions 9 and 10:
Vertebrate: Inland sifverside, Menidia beryllina (survival and
growth)}topsmek, Atherinops affinis (survival and growth).
Invertebrate: (select one for Region 9 and two for Region 10)
1.	Atlantic mysid, Mysidopsis hahia (survival, growth and fecundity
test)/Pacific mysid, Holmesimysis costata (survival and growth test),
2.	Bivalve species, mussel, Mytilis spp. or Pacific oyster, Crassostrea
gigas (larval development test),
3.	Purple urchin, Strongylocentrotus purpuratus and sand dollar,
Dendraster excentricus (fertilization test),
4.	Purple urchin, Strongylocentrotus purpuratus (larval development
test), and sand dollar, Dendraster excentricus (larval development
test).
5.	Red abalone, Haliotis rufescens (larval development test).
b.	Every year, the permittee shall re-screen once with the three species listed
above and continue to monitor with the most sensitive species. Re-screening
shall be conducted at a different time of year from the previous year's re-
screening.
c.	The chronic toxicity of the effluent shall be estimated as specified in Short-
Term Methods for Estimating the Chronic Toxicity of Effluents and
Receiving Waters to Marine and Estuarine Organisms3, EPA-600-4-91-
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003, July 1994, to be specified when using Metiidia or Mysidopsis species
and/or Short-Term Methods for Estimating the Chronic Toxicity of
Effluents and Receiving Waters to West Coast Marine and Estuarine
Organisms,3 EPA/600/R-95/136, August 1995. to be specified when using
West Coast marine organisms.
2. Limits/Definition of Toxicity
a.	Chronic toxicity measures a sublethal effect (e.g., reduced growth,
reproduction) to experimental test organisms exposed to an effluent or ambient
waters compared to that of the control organisms. [When a permit limit is
appropriate, the following shall apply]: lie chronic toxicity limitation is: 1
TUc based on any monthly median (where there is not a mixing zone), or the
value calculated using the statistical method, expressed as a monthly average
(where mixing zones are allowed), or any one test result with a daily
maximum greater than 2.0 TUc at the edge of the mixing zone, (or other
language, based on State wqs.) If a permit limit is not appropriate, then this
section should be called "Definition of Toxicity".
b.	Results shall be reported in TUc, where TUc = 100/NOEC or 100/ICp or
ECp (in percent effluent). The no observed effect concentration (NOEC) is
the highest concentration of toxicant to which organisms are exposed in a
chronic test, that causes no observable adverse effect on the test organisms
(e.g., the highest concentration of toxicant to which the values for the
observed responses are not statistically significant different from the
controls)5. The inhibition concentration, IC, is a point estimate of the toxicant
concentration that causes a given percent reduction (p) in a non-quanta!
biological measurement (e.g.. reproduction or growth) calculated from a
continuous model (the EPA Interpolation Method). The effective
concentration, EC, is a point estimate of the toxicant concentration that woiild
cause a given percent reduction (p) in quanta! biological measurement (e.g.,
larval development, survival) calculated from a continuous model (e.g..
Probit).
If a WET limit is appropriate, then this section should be contained in the
Effluent Limitations section of the permit, and not in the monitoring
requirements sections.
If in the calculation of a NOEC, two tested concentrations cause statistically adverse
effects, but an intermediate concentration did not cause statistically significant effects,
the test should be repeated or the lowest concentration must be used. For example:
6.25, 12.5, 25, 50 and 100% effluent concentrations are tested. The 12.5 and 50%
concentrations are statistically significant, but 25% is not significant. If the test is not
repeated, then the NOEC is 6.25%.
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3. Quality assurance
a.	A series of five dilutions and a control shall be tested. The series shall
include the instream waste concentration (IWC) {permit writer should insert
the actual value of the IWC), two dilutions above the IWC. and two dilutions
below the IWC. The IWC is the concentration of effluent at the edge of the
mixing zone. If there is no mixing zone, then the dilution series would be the
following concentrations: for example, 12.5, 25, 50, 75 and 100 percent
effluent.
b.	If organisms are not cultured in-house, concurrent testing with reference
toxicants shall be conducted. Where organisms are cultured in-house,
monthly reference toxicant testing is sufficient.
c.	If either the reference toxicant tests or the effluent tests do not meet all test
acceptability criteria as specified in the test methods manual, then the
permittee must re-sample and re-test within 14 days/as soon as possible.
d.	Reference toxicant tests shall be conducted using the same test conditions as
the effluent toxicity test (i.e., same test duration, etc.).
e.	Control and dilution water should be receiving water or lab water, as
appropriate, as described in the manual'' If the dilution water used is
different from the cuiture water, a second control, using culture water shall
also be used.
f.	Chemical testing for the parameters for which effluent limitations exist shall
be performed on a split of each sample collected for WET testing. To the
extent that the timing of sample collection coincides with that of the sampling
required in Part	of this permit, chemical analysis of the split sample will
fulfill the requirements of that Part as well.
4. Preparation of Generic TRE Workplan
The permittee shall submit to EPA a copy of the permittee's toxicity reduction
evaluation (TRE) workplan [1-2 pages] within 90 days of the effective date of this
permit. This plan shall describe the steps the permittee intends to follow in the event
that toxicity is detected, and should include at a minimum:
(a) A description of the investigation and evaluation techniques that would
be used to identify potential causes/sources of toxicity, effluent
variability, treatment system efficiency;
The manuals describe various situations in which either receiving water or lab water
should be used for control and dilution water. Depending upon the objective of the
test, either lab water or receiving water may be used.
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(b) A description of the facility's method of maximizing in-house
treatment efficiency, good housekeeping practices, and a list of all
chemicals used in operation of the facility;
(c) If a toxicity identification evaluation (TIE) is necessary, who will
conduct it (i.e., in-house or other)
5. Reporting
a.	The permittee shall submit the results of the toxicity tests, including any
accelerated testing conducted during the month, in TUs with the discharge
monitoring reports (DMR) for the month in which the tests are conducted. If
the generic TRE workplan is used to determine that accelerated testing is
unnecessary, then those results shall also be submitted with the DMR for the
month in which the investigation occurred.
b.	The full report shall be submitted by the end of the month in which the DMR
is submitted.
c.	The full report shall consist of: (1) the toxicity test results; (2) the dates of
sample collection and initiation of each toxicity test; (3) the type of
production; (4) the flow rate at the time of sample collection; and (5) the
results of the effluent analyses for chemical/physical parameters required for
the ouitall($) as defined in Part	of the permit.
d.	Test results for chronic tests shall be reported according to the chronic manual
chapter on Report Preparation, and shall be attached to the DMR. Where
possible, the results shall also be submitted on electronic disk (3.5") in the
TSERF format.
e.	Evaluation results-the permittee shall notify EPA and the State in writing
within fifteen {15) days of receipt of the results of the exceedance of the
iimit/TRE trigger of
(1)	The finding of the TRE or other investigation to identify the cause(s)
of toxicity;
(2)	Actions the permittee has taken or will take to mitigate the impact of
the discharge, to correct the noncompliance and to prevent the
recurrence of toxicity;
(3)	Where corrective actions including a TRE have not been completed,
an expeditious schedule under which corrective actions will be
implemented; and
(4)	If no actions have been taken, the reason for not taking action.
6. Accelerated Testing:
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a.	If chronic toxicity as defined [i.e., the permit limit or the TR£ trigger, when
there is no limftj is detected, then the permittee shall conduct six more tests,
bi-weekly (every two weeks), over a twelve-week period. Testing shall
commence within two weeks of receipt of the sample results of the
exceedance.
b.	If implementation of the generic TRE workplan indicates the source of toxicity
(for instance, a temporary plant upset), then only one additional test is
necessary. If toxicity is detected in this test, then Part 5a. shall apply.
c.	If chronic toxicity as defined [i.e., the permit limit or the TRE trigger, when
there is no limit] is detected in any of the six additional tests, then, in
accordance with the permittee's TRE workplan and, at a minimum, EPA
manuals EPA/600/2-88/070 (industrial) or EPA/600/4-89/00IA (municipal),
the permittee shall initiate a TRE within fifteen (15) days of receipt of the
sample results of the exceedance.
d. If none of the six tests indicates toxicity, then the permittee may return to the
normal testing frequency.
7.	Toxicity Identification Evaluation (TIE)
a.	If chronic toxicity is detected in any two of the six bi-weekly tests, then the
permittee shall, in accordance with EPA acute and chronic manuals
EPA/600/6-91/005F (Phase I), EPA/600/R-92/080 (Phase II), and EPA-
600/R-92/081 (Phase III), initiate a TIE within 15 days.
b.	If a TIE is triggered prior to completion of the accelerated testing, the
accelerated testing schedule may be terminated, or used as necessary in
performing the TIE.
8.	Reopener
This permit may be modified in accordance with the requirements set forth at 40 CFR
Parts 122 and 124, to include appropriate conditions or limits to address demonstrated
effluent toxicity based on newly available information, or to implement any EPA-
approved new State water quality standards applicable to effluent toxicity.
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SAMPLE FACT SHEET LANGUAGE
OPTION 1
Under 40 CFR 122.44(d), permits must contain limits on whole effluent toxicity when a discharge has
reasonable potential to cause or contribute to an exceedance of the water quality standard ("reasonable
potential"). Toxicity testing requirements and limits as contained in Item	on Page	and in
Part	have been included to ensure that the effluent from Outfall(s)	conform(s) with
appropriate State water quality standards and/or replatlons, and/or Regional guidance as contained in
the document, "Regions 9 and 10 Guidance for Implementing Whole Effluent Toxicity Testing
Programs", dated May 31,1996, as appropriate. Due to the intermittent nature of the discharges from
this facility, acute whole effluent toxicity (WET) conditions have been included. Because acute WET
limits were in the previous NPDES permit, §402(o)(l) of the CWA is applicable for Outfall 001.
Because no acute WET data are available for Outfall 002, monitoring only will be required. Since it
is possible that a discharge may last more than 4 days, chronic WET monitoring provisions have also
been included for both outfalls. For Outfall 001, if reasonable potential to exceed appropriate State
water quality standards and/or regulations is found to exist, the permit may be reopened to include a
chronic WET limit. For Outfall 002, if reasonable potential to exceed appropriate State water quality
standards and/or regulations is found to exist, the permit may be reopened to include an acute and/or
chronic WET limit, as appropriate. EPA notes that the State has not granted a mixing zone for
chronic WET to this facility. Until such time as a mixing zone is granted for this parameter, EPA
will evaluate the chronic WET monitoring results and base reasonable potential on 100% effluent, at
the end of the pipe. The inclusion of a chronic whole effluent toxicity limit in the permit its
authorized and required by 40 CFR §122.44(d)(i)(v). The inclusion of an acute whole effluent
toxicity limit in the permit is authorized and required by 40 CFR §I22.44(d)(l){iv).
For chronic testing, the permittee is required to perform the following tests: Pimephalespromelas
(fathead minnow), larval survival and growth test and Ceriodaphnh dub'm, three-brood, 7-day
survival and reproduction test. Either static renewal or flow-through testing may be used. For acute
testing, the permit requires a 96-hour LC*, test using Onchorynchus Jtisutch (coho salmon).
The permit allows for a reduction in monitoring frequency to a specified frequency if no
individual test result is greater than the target (or limit) divided by the reasonable potential
factor (based on number of samples required for monitoring). The target is equal to the
criterion times the dilution allowed (in this case, the target equals 1.0 TUc times 100, or 100
TUc). Since quarterly monitoring is required for the first year, the reasonable potential
factor from Table 3-1 in the TSD is 4.7 (at a CV of 0.6 and 4 samples). If no test results
are greater than the value specified above (21.3 TUc), it would be reasonable to assume that
the discharge has low probability of causing an impact to receiving waters. If there are no
significant changes to the facility, a reduced frequency would be appropriate. The TSD
recommends that if no reasonable potential exists, that monitoring be conducted once before
permit reissuance. If there is a limit for WET, the minimum monitoring frequency allowed
is annual.
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STATISTICS
Q: The EPA definition of the NOEC is "highest concentration of toxicant to which organisms are
exposed in a full life-cycle or partial life-cycle test, that causes no observable adverse effects
on the test organisms." How should the NOEC be reported for the following example:
dilution concentrations 6.25, 12.5, 25, 50 and 100 percent effluent. The concentrations 12.5,
50 and 100 percent were statistically different from the control. What is the LOEC?
A: The LOEC is the concentration of 12.5, therefore, the NOEC is 6.25. The definitions of
NOEC and LOEC in the methods manuals assume a strict dose-response relationship between
toxicant concentration and organism response. If this assumption were always the case, there
would be no issue concerning the endpoint definitions because the NOEC would always be a
lower concentration level than the LOEC. However, this strict dose-response relationship
does not exist with all toxicants. When this occurs the test must be repeated or the lowest
NOEC should be reported for compliance purposes.
Q: Is it appropriate to analyze toxicity data for compliance reporting using statistical tools other
than those identified in the EPA flowcharts for statistical analysis?
A: Section 11.1.4 of the most recent edition of the acute manual (1993) states: "The data
analysis methods recommended in the EPA toxicity testing methods manuals were chosen
primarily because they are (1) well-tested and well-documented, (2) applicable to most types
of test data sets for which they are recommended, but still powerful, and (3) most easily
understood by non-statisticians. Many other methods were considered in the selection
process, and it is recognized that the methods selected are not the only possible methods of
analysis." The appropriateness of other methods for use on acute and chronic toxicity test
results, however, must be determined with a careful evaluation of a complete array of possible
toxicity test results on which the method might be used.
Q: How are males in the Ceriodaphnia dubia survival and reproduction test calculated for the
survival endpoint?
A: Males are included for the survival analysis as either dead or alive the same as females.
Q: In the chronic tests with survival endpoints (e.g., Pimephalespromelas survival and growth
test) can the survival be used for acute test results?
A: Yes, it is recommended to report both 7 day survival results, in addition to either the 48 or
96-hour survival results. This reduces the costs of compliance testing for requirements of
acute and chronic testing.
Q: According to the recommended test conditions section the number of replicates per
concentration is four (minimum of three). When a test is conducted with only three replicates
and the data fails the assumptions of parametric testing, what analysis should be performed?
A: If the data fails the assumptions of parametric testing, then non-parametric statistics would be
performed, however a minimum of four replicates are necessary. In the situation described
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above the data could be forced through parametric tests and the results would be interpreted
with caution. Ideally, the test should be repeated and the laboratory should use a minimum of
four replicates.
DATA SUBMISSION
Q: Should test results that foil the required test acceptability criteria (e.g., ^ 90% survival in the
controls for the acute toxicity methods) be reported to the permitting authority?
A: It is the permittee's responsibility to determine if the results of toxicity tests fulfill test
requirements and, therefore, should be submitted. The permitting authority will reject data
that do not meet test method specifications.
TEST ORGANISMS
Q: What type of documentation and level of effort is appropriate to demonstrate a laboratory's
effort to obtain organisms for a test?
A: A laboratory should make best effort to obtain spawnable test organisms from two organism
suppliers. Documentation should consist of order forms or verification of order placed by
phone (signed and dated entries in a bound notebook).
SALINITY ISSUES
Q: Should salinities of effluent be matched to ambient salinity or to a "typical" ambient salinity?
A: The test must be conducted at a salinity that is acceptable for the particular test species (e.g.,
the red abalone test must be conducted at 34 + 2%o). However, when conducting ambient
toxicity tests the salinities should be matched to ambient salinities, not to a "typical" ambient
salinity.
Q: If there are difficulties with commercial brine, what is the preferred source of salt?
A: Brine such as commerical salts or hypersaline brine are used to achieve the required method
salinity. The preferred source of brine is to use clean seawater that has been concentrated by
evaporation or freezing procedures. See the section on hypersaline brine additions in the
marine chronic test method manuals.
TESTING CONDITIONS
Q: Should temperature be held constant during testing if the test temperature is higher than
ambient temperature?
A: The test must be conducted at the test temperature as specified in the toxicity test manual for
that specific test species.
ENFORCEMENT
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Q: If conducting tests with two or more species, how is compliance determined? Looking at all
test results together, regardless of species, or looking at results on a species by species basis?
A: Look at species by species basis: compliance would be based on the endpoint per species
with the lowest NOEC value or point estimate value (EC 25) as specified in the permit per
test endpoint per test species.
Q: The laboratory reports the NOEC and LOEC as percent effluent for both survival and growth
with the chronic fathead minnow and both survival and reproduction with the chronic
Ceriodaphnia dubia. What should be entered onto the DMR?
A: Report the lowest NOEC value of either the survival or growth for the fathead minnow test
and the lowest NOEC value of either the survival or reproduction for Ceriodaphnia dubia
test.
Q: When both a brine and dilution water control are used for the marine toxicity test methods,
which control should be used to compare to the treatments?
A: First, a t-test is conducted to compare the brine control to the dilution water control. If there
is no statistical difference between the controls, then use the dilution water control for all the
treatments. If there is a statistical difference between the controls, then use the dilution water
control for the treatments without brine addition and the brine control for the treatment with
brine addition.
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ACUTE FRESHWATER LANGUAGE - 1/22/96
PART IV
Whole Effluent Toxicity Testing Program
As required by Part _L of this permit, the permittee shall initiate the series of tests described below
beginning in month, year to evaluate whole effluent toxicity of the discharge from outfall	. Ail test
species, procedures and quality assurance criteria used shall be in accordance with Methods for Measuring
the Acute Toxicity of Effluents to Freshwater and Marine Organisms. EPA/600/4-90/027F, or the most
current edition. The dilution/control water used will be moderately hard water as described in
EPA/600/4-90/027F, Section 7, or the most current edition. A standard reference toxicant quality
assurance test shall be conducted concurrently with each species used in the toxicity tests and the results
submitted with the discharge monitoring report (DMR). Alternatively, if monthly QA/QC reference
toxicant tests are conducted, these results must be submitted with the DMR.
1.	a. The permittee shall conduct 96-hour acute static-renewal multi-concentration toxicity tests
using the daphnid fCeriodanhnia dubial and the fathead minnow fPimephales promelasl. All
tests shall be conducted on four separate grab samples collected at evenly-spaced (6-hr)
intervals over a 24-hour period and used in four separate tests in order to catch any peaks of
toxicity and to account for daily variations in effluent quality. All tests shall be conducted on
a control (0%) and the following dilution concentrations at a minimum: 100.0%, 50.0%,
25.0%, 12.5%, and 6.25%.
b. If control mortality exceeds 10% for either species in any test, the test(s) for that species
(including the control) shall be repeated. A test will be considered valid only if control
mortality does not exceed 10% for either species.
2.	The toxicity tests specified above shall be conducted once every two months until six valid
bimonthly tests have been completed, and once every six months thereafter for the duration of the
permit, unless notified otherwise by the permit issuing authority. These tests are referred to as
"routine" tests.
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3. a. If unacceptable acute toxicity (an LC» of 100% or less occurs in either test species in any of
the four separate grab sample tests within the specified time) is found in a "routine" test, the
permittee shall conduct two additional acute toxicity tests in the same manner as the "routine"
test on the specie(s) indicating unacceptable acute toxicity. For each additional test, the
sample collection requirements and test acceptability criteria specified in Section 1 above must
be met for the test to be considered valid. The first test shall begin within two weeks of the
end of the "routine" tests, and shall be conducted weekly thereafter until two additional, valid
tests are completed. The additional tests will be used to determine if the toxicity found in the
"routine" test is still present.
b. Results from additional tests, required due to unacceptable acute toxicity in the "routine"
test(s), must be reported on the Discharge Monitoring Report (DMR) Form for the month in
which the test was begun. Such test results must be submitted within 45 days of completion
of the second additional, valid test.
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ACUTE SALTWATER LANGUAGE - 1/22/96
PART IV
Whole Effluent Toxicity Testing Program
As required by Part J_ of this permit, the permittee shall initiate the series of tests described below
beginning in month, year to evaluate whole effluent toxicity of the discharge from outfall	. All test
species, procedures and quality assurance criteria used shall be in accordance with Methods for Measuring
the Acute Toxicity of Effluents to Freshwater and Marine Organisms. EPA/600/4-90/027F, or the most
current edition. The dilution/control water and effluent used will be adjusted to a salinity of 20 parts per
thousand using artificial sea salts as described in EPA/600/4-90/027F, Section 7 (or the most current
edition). A standard reference toxicant quality assurance test shall be conducted concurrently with each
species used in the toxicity tests and the results submitted with the discharge monitoring report (DMR).
Alternatively, if monthly QA/QC reference toxicant tests are conducted, these results must be submitted
with the DMR.
1.	a. The permittee shall conduct 96-hour acute static-renewal multi-concentration toxicity tests
using the mysid shrimp (Mvsidopsis bahia-) and the inland silverside (Menidia beryllina). All
tests shall be conducted on four separate grab samples collected at evenly-spaced (6-hr)
intervals over a 24-hour period and used in four separate tests in order to catch any peaks of
toxicity and to account for daily variations in effluent quality. All tests shall be conducted on
a control (0%) and the following dilution concentrations at a minimum: 100.0%, 50.0%,
25.0%, 12.5%, and 6.25%.
b. If control mortality exceeds 10% for either species in any test, the test(s) for that species
(including the control) shall be repeated. A test will be considered valid only if control
mortality does not exceed 10% for either species.
2.	The toxicity tests specified above shall be conducted once every two months until six valid
bimonthly tests have been completed, and once every six months thereafter for the duration of the
permit, unless notified otherwise by the permit issuing authority. These tests are referred to as
"routine" tests.
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3. a. If unacceptable acute toxicity (an LCjo of 100% or less occurs in either test species in any of
the four separate grab sample tests within the specified time) is found in a "routine" test, the
permittee shall conduct two additional acute toxicity tests in the same manner as the "routine"
test on the specie(s) indicating unacceptable toxicity. For each additional test, the sample
collection requirements and test acceptability criteria specified in Section 1 above must be met
for the test to be considered valid. The first test shall begin within two weeks of the end of
the "routine" tests, and shall be conducted weekly thereafter until two additional, valid tests
are completed. The additional tests will be used to determine if the toxicity found in the
"routine" test is still present.
b. Results from additional tests, required due to unacceptable acute toxicity in the "routine"
test(s), must be reported on the Discharge Monitoring Report (DMR) Form for the month in
which the test was begun. Such test results must be submitted within 45 days of completion
of the second additional, valid test.
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ACUTE FRESHWATER LANGUAGE (monitoring only) - 1/22/96
PART IV
Whole Effluent Toxicity Testing Program
As required by Part J_ of this permit, the permittee shall initiate the series of tests described below
beginning in month, year to evaluate whole effluent toxicity of the discharge from outfall	. All test
species, procedures and quality assurance criteria used shall be in accordance with Methods for Measuring
the Acute Toxicity of Effluents to Freshwater and Marine Organisms. EPA/600/4-90/027F, or the most
current edition. The dilution/control water used will be a moderately hard water as described in
EPA/600/4-90/027F, Section 7 (or the most current edition). A standard reference toxicant quality
assurance test shall be conducted concurrently with each species used in the toxicity tests and the results
submitted with the discharge monitoring report (DMR). Alternatively, if monthly QA/QC reference
toxicant tests are conducted, these results must be submitted with the DMR.
1.	a. The permittee shall conduct 96-hour acute static-renewal multi-concentration toxicity bioassays
using the daphnid (Ceriodaphnia dubial and the fathead minnow (Pimephales promelasl.
Effluent samples shall be collected at four evenly-spaced (6-hr) intervals over a 24-hour period
and used in four separate tests in order to catch any peaks of toxicity and to account for daily
variations in effluent quality. All tests shall be conducted on a control (0%) and the following
dilution concentrations at a minimum: 100.0%, 50.0%, 25.0%, 12.5%, and 6.25%. Acute
toxicity will be demonstrated if an LC^ of 100% or less occurs in either test species in any
test within the specified time.
b. If control mortality exceeds 10% for either species in any test, the test(s) for that species
(including the control) shall be repeated. A test will be considered valid only if control
mortality does not exceed 10% for either species
2.	The toxicity tests specified above shall be conducted once every two months (bi-monthly) until six
valid tests are completed, and once every six months thereafter for the duration of the permit, unless
notified otherwise by the permit issuing authority. These tests are referred to as "routine" tests.
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ACUTE SALTWATER LANGUAGE (monitoring only) - 1/22/96
PART IV
Whole Effluent Toxicity Testing Program
As required by Part _L of this permit, the permittee shall initiate the series of tests described below
beginning in month, year to evaluate whole effluent toxicity of the discharge from outfall	. All test
species, procedures, and quality assurance criteria used shall be in accordance with Methods for
Measuring the Acute Toxicity of Effluents to Freshwater and Marine Organism*; EPA/600/4-90/027F,
or the most current edition. The dilution/control water and effluent used will be adjusted to a salinity
of 20 parts per thousand using artificial sea salts as described in EPA/600/4-90/027F, Section 7 (or the
most current edition). A standard reference toxicant quality assurance test shall be conducted
concurrently with each species used in the toxicity tests and the results submitted with the discharge
monitoring report (DMR). Alternatively, if monthly QA/QC reference toxicant tests are conducted, these
results must be submitted with the DMR.
1.	a. The permittee shall conduct 96-hour acute static-renewal multi-concentration toxicity tests
using the mysid shrimp fMvsidopsis bah i at and the inland silverside (Menidia bervllinaV All
tests shall be conducted on four separate grab samples collected at evenly-spaced (6-hr)
intervals over a 24-hour period and used in four separate tests in order to catch any peaks of
toxicity and to account for daily variations in effluent quality. All tests shall be conducted on
a control (0%) and the following dilution concentrations at a minimum: 100.0% 50 0%
25.0%, 12.5%, and 6.25%.
b. If control mortality exceeds 10% for either species in any test, the test(s) for that species
(including the control) shall be repeated. A test will be considered valid only if control
mortality does not exceed 10% for either species.
2.	The toxicity tests specified above shall be conducted once every two months until six valid
bimonthly tests have been completed, and once every six months thereafter for the duration of the
permit, unless notified otherwise by the permit issuing authority. These tests are referred to as
"routine" tests.
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CHRONIC FRESHWATER LANGUAGE - 1/22/96
PART IV
Whole Effluent Toxicity Testing Program
As required by Part J_ of this permit, the permittee shall initiate the series of tests described below
beginning in month, year to evaluate whole effluent toxicity of the discharge from outfall	. All test
species, procedures and quality assurance criteria used shall be in accordance with Short-term Methods
for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms
EPA/600/4-91/002, and Methods for Measuring the Acute Toxicity of Effluents to Freshwater and Marine
Organisms, EPA/600/4-90/027F, or the most current edition(s). The dilution/control water used will be
a moderately hard water as described in EPA/600/4-91/002, Section 7 (or the most current edition). A
chronic standard reference toxicant quality assurance test shall be conducted concurrently with each
species used in the toxicity tests and the results submitted with the discharge monitoring report (DMR).
Alternatively, if monthly QA/QC reference toxicant tests are conducted, these results must be submitted
with the DMR.
1. a. The permittee shall conduct multi-concentration daphnid (Ceriodanhnia duhial Survival and
Reproduction and Fathead Minnow (Pimephales promelas) Larval Survival and Growth Tests.
All tests shall be conducted on a control (0%) and the following dilution concentrations at a
minimum: 100.0%, fRWC + 1001/2%. RWC%. RWC/2%. and RWC/4%. Unacceptable
chronic toxicity will be demonstrated if either test results in a no observable effect
concentration (NOEC) less than	% effluent, which is the Receiving Water Concentration
(RWC) of the effluent at critical conditions.
b.	For each set of tests conducted, a minimum of three different 24-hour composite samples of
final effluent shall be collected and used per the sampling schedule of Section 8.3.2,
EPA/600/4-91/002 (or the most current edition). All test solutions shall be renewed daily.
If test results do not meet the acceptability criteria of Section 4.9.1, EPA/600/4-91/002 (or
the most current edition), that test shall be repeated. A chronic test will be considered valid
only if the acceptability criteria referenced above are met.
c.	If control mortality exceeds 20% for either species in any test, the test(s) for that species
(including the control) shall be repeated. A test will be considered valid only if control
mortality does not exceed 20% for either species.
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2. a. The permittee shall also conduct 96-hour acute static-renewal multi-concentration toxicity tests
on the daphnid (Ceriodaphnia dubia) and the fathead minnow fPimephales promelasl. The
composite samples collected under Section 1 (b) above shall be used in the acute tests. All tests
shall be conducted on a control (0%) and the following dilution concentrations at a minimum-
100.0%, 50.0%, 25.0%, 12.5%, and 6.25%. Unacceptable acute toxicity will be
demonstrated if an LCjo of 100% or less occurs in either test species within the specified time.
b. For each set of tests conducted, a fresh 24-hour composite sample of final effluent shall be
used at Day 1 and at
Day 3. If control mortality exceeds 10% for either species, the test for that species (including
the control) shall be repeated. An acute test will be considered valid only if control mortality
does not exceed 10% for either species.
3. The toxicity tests specified above shall be conducted once every two months until six valid
bimonthly tests have been completed, and once every six months thereafter for the duration of the
permit, unless notified otherwise by the permit issuing authority. These tests are referred to as
"routine" tests.
4. a. If unacceptable chronic toxicity (a NOEC less than	% in either test) and/or unacceptable
acute toxicity (an LC^ of 100% or less in either test) is found in a "routine" test, the permittee
shall conduct two additional toxicity tests based on the type of unacceptable toxicity found
(i.e., chronic and/or acute tests, as appropriate), on the specie(s) indicating unacceptable
toxicity. For each additional test, the sample collection requirements and test acceptability
criteria specified in Section 1(b), 1(c), and/or 2(b) above, as appropriate, must be met for the
test to be considered valid. The first test shall begin within two weeks of the end of the
"routine" test and the second test shall be conducted two weeks later. If either or both of
these tests are invalid, additional test(s) are to be conducted every two weeks until two valid
tests are completed (e.g., if the first test is valid and the second test is not, the permittee shall
continue to conduct tests until one more test is valid). The additional tests will be used to
determine if the toxicity found in the "routine" test is still present.
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1.	For "routine" tests with unacceptable chronic toxicity, additional daphnid (Ceriodaphnia
dubia) Survival and Reproduction and/or fathead minnow (Pimephales promelasl Survival
and Growth multi-concentration tests shall be conducted, as appropriate. All tests shall
be conducted on a control (0%) and the following dilution concentrations at a minimum-
1QM%, fRWC + 1001/2% - RWC%, RWC/2%. and RWC/4%. The sample collection
requirements specified in Section 1(b) above must be met.
2.	For "routine" tests with unacceptable acute toxicity, additional 96-hour acute static-
renewal multi-concentration toxicity tests using the daphnid (Ceriodaphnia duhial and/or
the fathead minnow (Pimephales promelasl shall be conducted, per EPA/600/4-90/027F.
Four separate grab samples shall be collected at evenly-spaced (6-hr) intervals over a
24-hour period and used in four separate tests. All tests shall be conducted on a control
(0%) and the following dilution concentrations at a minimum: 100.0%, 50.0%, 25 0%
12.5%, and 6.25%.
b. Results from additional tests, required due to unacceptable chronic and/or acute toxicity in the
"routine" test(s), must be reported on the Discharge Monitoring Report (DMR) Form for the
month in which the test was begun. Such test results must be submitted within 45 days of
completion of the second additional, valid test.
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CHRONIC SALTWATER LANGUAGE - 1/22/96
PART IV
Whole Effluent Toxicity Testing Program
As required by Part _I_ of this permit, the permittee shall initiate the series of tests described below
beginning in month, year to evaluate whole effluent toxicity of the discharge from outfall	. All test
species, procedures and quality assurance criteria used shall be in accordance with Short-term Methods
for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Marine and F.ctnarinp
Organisms, EPA/600/4-91/003, Short-term Methods for Estimating the Chronic Toxicity of Effluents and
Receiving Waters to Freshwater Organisms. EPA/fiOn/4-QI/ftn? and Methods for Measuring the Acuta
Toxicity of Effluents to Freshwater and Marine Organisms. EPA/600/4-90/027F, or the most current
edition(s), as appropriate. The dilution/control water and effluent used will be adjusted to a salinity of
20 parts per thousand using artificial sea salts (e.g., Forty Fathoms) as described in EPA/600/4-91/003,
Section 7 (or the most recent edition). A chronic standard reference toxicant quality assurance test shall
be conducted concurrently with each species used in the toxicity tests and the results submitted with the
monthly discharge monitoring report (DMR). Alternatively, if monthly QA/QC reference toxicant tests
are conducted, these results must be submitted with the monthly DMR.
1. a. The permittee shall conduct multi-concentration Mysid shrimp (Mvsidopsis bahial Survival,
Growth, and Fecundity and Inland Silverside fMenidia bervllina) Larval Survival and Growth
Tests. All tests shall be conducted on a control (0%) and the following dilution concentrations
at a minimum: 100.0%. fRWC + 1001/2%. RWC%, RWC/2%. and RWC/4%.
Unacceptable chronic toxicity will be demonstrated if either test results in a no observable
effect concentration (NOEC) less than 	% effluent, which is the Receiving Water
Concentration (RWC) of the effluent at critical conditions.
b.	For each set of tests conducted, a minimum of three different 24-hour composite samples of
final effluent shall be collected and used per the sampling schedule of Section 8.3.2,
EPA/600/4-91/003 (or the most current edition). All test solutions shall be renewed daily.
If test results do not meet the acceptability criteria of Section 4.9.1, EPA/600/4-91/003 (or
the most current edition), that test shall be repeated. A chronic test will be considered valid
only if the acceptability criteria referenced above are met.
c.	If control mortality exceeds 20% for either species in any test, the test(s) for that species
(including the control) shall be repeated. A test will be considered valid only if control
mortality does not exceed 20% for either species.
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a.	The permittee shall also conduct 96-hour acute static-renewal multi-concentration tests on the
Mysid shrimp (Mvsidopsis bahia) and the inland silverside (Menidia bervllinaV The composite
samples collected under Section 1(b) above shall be used in the acute tests. All tests shall be
conducted on a control (0%) and the following dilution concentrations at a minimum:
100.0%, 50.0%, 25.0%, 12.5%, and 6.25%. Unacceptable acute toxicity will be
demonstrated if an LCjq of 100% or less occurs in either test species within the specified time.
b.	For each set of tests conducted, a fresh 24-hour composite sample of final effluent shall be
used at Day 1 and at
Day 3. If control mortality exceeds 10% for either species, the test for that species (including
the control) shall be repeated. An acute test will be considered valid only if control mortality
does not exceed 10% for either species.
The toxicity tests specified above shall be conducted once every two months until six valid
bimonthly tests have been completed, and once every six months thereafter for the duration of the
permit, unless notified otherwise by the permit issuing authority. These tests are referred to as
"routine" tests.
a. If unacceptable chronic toxicity (a NOEC less than	% in either test) and/or unacceptable
acute toxicity (an LC^, of 100% or less in either test) is found in a "routine" test, the permittee
shall conduct two additional toxicity tests based on the type of unacceptable toxicity found
(i.e., chronic and/or acute tests, as appropriate), on the specie(s) indicating unacceptable
toxicity. For each additional test, the test acceptability criteria specified in Section 1(b), 1(c),
and/or 2(b) above, as appropriate, must be met for the test to be considered valid. The first
test shall begin within two weeks of the end of the "routine" test and the second test shall be
conducted two weeks later. If either or both of these tests are invalid, additional test(s) are
to be conducted every two weeks until two valid tests are completed (e.g., if the first test is
valid and the second test is not, the permittee shall continue to conduct tests until one more
test is valid). The additional tests will be used to determine if the toxicity found in the
"routine" test is still present.
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1.	For "routine" tests with unacceptable chronic toxicity, additional Mysid shrimp
(Mvsidopsis bahia) Survival, Growth, and Fecundity and/or inland silverside (Menidia
bervllina) Survival and Growth multi-concentration tests shall be conducted, as
appropriate. All tests shall be conducted on a control (0%) and the following dilution
concentrations at a minimum: 100.0%. fRWC + 1001/2%. RWC%. RWC/2%. and
RWC/4%. The sample collection requirements specified in Section 1(b) above must be
met.
2.	For "routine" tests with unacceptable acute toxicity, additional 96-hour acute static-
renewal toxicity tests using the Mysid shrimp fMvsidopsis bahia) and/or the inland
silverside (Menidia bervllinal shall be conducted, per EPA/600/4-90/027F. Four
separate grab samples shall be collected at evenly-spaced (6-hr) intervals over a 24-hour
period and used in four separate tests. All tests shall be conducted on a control (0%) and
the following dilution concentrations at a minimum: 100.0%, 50.0%, 25.0%, 12.5%,
and 6.25%.
b. Results from additional tests, required due to unacceptable chronic and/or acute toxicity in the
"routine" test(s), must be reported on the Discharge Monitoring Report (DMR) Form for the
month in which the test was begun. Such test results must be submitted within 45 days of
completion of the second additional, valid test.
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CHRONIC FRESHWATER LANGUAGE (when 7Q10 is zero) - 1/22/96
PART IV
Whole Effluent Toxicity Testing Program
As required by Part J_ of this permit, the permittee shall initiate the series of tests described below
beginning in month, year to evaluate whole effluent toxicity of the discharge from outfall	. All test
species, procedures and quality assurance criteria used shall be in accordance with Short-term Methods
for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms.
EPA/600/4-91/002, or the most current edition. The dilution/control water used will be a moderately
hard water as described in EPA/600/4-91/002, Section 7 (or the most current edition). A chronic
standard reference toxicant quality assurance test shall be conducted concurrendy with each species used
in the toxicity tests and the results submitted with the discharge monitoring report (DMR). Alternatively,
if monthly QA/QC reference toxicant tests are conducted, these results must be submitted with the DMR.
1. a. The permittee shall conduct multi-concentration daphnid (Ceriodaphnia dubial Survival and
Reproduction and Fathead Minnow (Pimenhales promelas') Larval Survival and Growth Tests.
All tests shall be conducted on a control (0%) and the following dilution concentrations at a
minimum: 100.0%, 50.0%, 25.0%, 12.5%, and 6.25%. Unacceptable chronic toxicity will
be demonstrated if either test results in a no observable effect concentration (NOEC) less than
100% effluent.
b For each set of tests conducted, a minimum of three different 24-hour composite samples of
final effluent shall be collected and used per the sampling schedule of Section 8.3.2,
EPA/600/4-91/002 (or the most current edition). All test solutions shall be renewed daily.
If test results do not meet the acceptability criteria of Section 4.9.1, EPA/600/4-91/002 (or
the most current edition), that test shall be repeated. A chronic test will be considered valid
only if the acceptability criteria referenced above are met.
c. If control mortality exceeds 20% for either species in any test, the test(s) for that species
(including the control) shall be repeated. A test will be considered valid only if control
mortality does not exceed 20% for either species.
13

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2. The toxicity tests specified above shall be conducted once every two months until six valid
bimonthly tests have been completed, and once every six months thereafter for the duration of the
permit, unless notified otherwise by the permit issuing authority. These tests are referred to as
"routine" tests.
3. a. If unacceptable chronic toxicity (a NOEC less than 100.0% in either test) is found in a
"routine" test, the permittee shall conduct two additional chronic toxicity tests on the specie(s)
indicating unacceptable chronic toxicity. For each additional test, the sample collection
requirements and test acceptability criteria specified in Section 1(b) and/or 1(c) above, as
appropriate, must be met for the test to be considered valid. The first test shall begin within
two weeks of the end of the "routine" test and the second test shall be conducted two weeks
later. If either or both of these tests are invalid, additional test(s) are to be conducted every
two weeks until two valid tests are completed (e.g., if the first test is valid and the second test
is not, the permittee shall continue to conduct tests until one more test is valid). The
additional tests will be used to determine if the toxicity found in the "routine" test is still
present.
1. For "routine" tests with unacceptable chronic toxicity, additional daphnid (Ceriodaphnia
dubia) Survival and Reproduction and/or fathead minnow (Pimephales promelas") Survival
and Growth multi-concentration tests shall be conducted, as appropriate All tests shall
be conducted on a control (0%) and the following dilution concentrations at a minimum:
100.0%, 50.0%, 25.0%, 12.5%, and 6.25% The sample collection requirements
specified in Section 1(b) above must be met.
b. Results from additional tests, required due to unacceptable chronic toxicity in the "routine"
test(s), must be reported on the Discharge Monitoring Report (DMR) Form for the month in
which the test was begun. Such test results must be submitted within 45 days of completion
of the second additional, valid test.
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CHRONIC SALTWATER LANGUAGE (if no dilution available) - 1/22/96
PART IV
Whole Effluent Toxicity Testing Program
As required by Part J_ of this permit, the permittee shall initiate the series of tests described below
beginning in month, year to evaluate whole effluent toxicity of the discharge from outfall	. All test
species, procedures and quality assurance criteria used shall be in accordance with Short-term Methods
for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Marine and Estuarine
Organisms, EPA/600/4-91/003 and Short-term Methods for Estimating the Chronic Toxicity of Effluents
and Receiving Waters to Freshwater Organisms. EPA/600/4-91/002, or the most current edition(s), as
appropriate. The dilution/control water and effluent used will be adjusted to a salinity of 20 parts per
thousand using artificial sea salts (e.g., Forty Fathoms) as described in EPA/600/4-91/003, Section 7 (or
the most recent edition). A chronic standard reference toxicant quality assurance test shall be conducted
concurrently with each species used in the toxicity tests and the results submitted with the monthly
discharge monitoring report (DMR). Alternatively, if monthly QA/QC reference toxicant tests are
conducted, these results must be submitted with the monthly DMR.
1. a. The permittee shall conduct multi-concentration Mysid shrimp (Mvsidopsis bahia) Survival,
Growth, and Fecundity and Inland Silverside (Menidia bervllinal Larval Survival and Growth
Tests. All tests shall be conducted on a control (0%) and the following dilution concentrations
at a minimum: 100.0%, 50.0%, 25.0%, 12.5%, and 6.25%. Unacceptable chronic toxicity
will be demonstrated if either test results in a no observable effect concentration (NOEC) less
than 100% effluent.
b.	For each set of tests conducted, a minimum of three different 24-hour composite samples of
final effluent shall be collected and used per the sampling schedule of Section 8.3.2,
EPA/600/4-91/002 (or the most current edition). All test solutions shall be renewed daily.
If test results do not meet the acceptability criteria of Section 4.9.1, EPA/600/4-91/003 (or
the most current edition), that test shall be repeated. A chronic test will be considered valid
only if the acceptability criteria referenced above are met.
c.	If control mortality exceeds 20% for either species in any test, the test(s) for that species
(including the control) shall be repeated. A test will be considered valid only if control
mortality does not exceed 20% for either species.
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2.	The toxicity tests specified above shall be conducted once every two months until six valid
bimonthly tests have been completed, and once every six months thereafter for the duration of the
permit, unless notified otherwise by the permit issuing authority. These tests are referred to as
"routine'' tests.
3.	a. If unacceptable chronic toxicity (a NOEC less than 100.0% in either test) is found in a
"routine" test, the permittee shall conduct two additional chronic toxicity tests on the specie(s)
indicating unacceptable chronic toxicity. For each additional test, the test acceptability criteria
specified in Section 1(b) and/or 1(c) above, as appropriate, must be met for the test to be
considered valid. The first test shall begin within two weeks of the end of the "routine" test
and the second test shall be conducted two weeks later. If either or both of these tests are
invalid, additional test(s) are to be conducted every two weeks until two valid tests are
completed (e.g., if the first test is valid and the second test is not, the permittee shall continue
to conduct tests until one more test is valid). The additional tests will be used to determine
if the toxicity found in the "routine" test is still present.
1. For "routine" tests with unacceptable chronic toxicity, additional Mysid shrimp
(Mysidopsis bahia) Survival, Growth, and Fecundity and/or inland silverside (Menidia
bervllina) Survival and Growth multi-concentration tests shall be conducted, as
appropriate. All tests shall be conducted on a control (0%) and the following dilution
concentrations at a minimum: 100.0%, 50.0%, 25.0%, 12.5%, and 6.25%. The sample
collection requirements specified in Section 1(b) above must be met.
b. Results from additional tests, required due to unacceptable chronic toxicity in the "routine"
test(s), must be reported on the Discharge Monitoring Report (DMR) Form for the month in
which the test was begun. Such test results must be submitted within 45 days of completion
of the second additional, valid test.
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CHRONIC FRESHWATER LANGUAGE (monitoring only) - 1/22/96
PART IV
Whole Effluent Toxicity Testing Program
As required by Part J_ of this permit, the permittee shall initiate the series of tests described below
beginning in month, year to evaluate whole effluent toxicity of the discharge from outfall	. All test
species, procedures and quality assurance criteria used shall be in accordance with Short-term Methods
for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms.
EPA/600/4-91/002, and Methods for Measuring the Acute Toxicity of Effluents to Freshwater and Marine
Organisms. EPA/600/4-90/027F, or the most current edition(s). The dilution/control water used will be
a moderately hard water as described in EPA/600/4-91/002, Section 7 (or the most current edition). A
chronic standard reference toxicant quality assurance test shall be conducted concurrently with each
species used in the toxicity tests and the results submitted with the discharge monitoring report (DMR).
Alternatively, if monthly QA/QC reference toxicant tests are conducted, these results must be submitted
with the DMR.
1. a. The permittee shall conduct multi-concentration daphnid (Ceriodaphnia dubial Survival and
Reproduction and Fathead Minnow (Pimephales promelas) Larval Survival and Growth Tests.
All tests shall be conducted on a control (0%) and the following dilution concentrations at a
minimum: 100 0%. FRWC + 1001/2%. RWC%. RWC/2%. and RWC/4% Chronic toxicity
will be demonstrated if either test results in a no observable effect concentration (NOEC) less
than	% effluent, which is the Receiving Water Concentration (RWC) of the effluent at
critical conditions.
b.	For each set of tests conducted, a minimum of three different 24-hour composite samples of
final effluent shall be collected and used per the sampling schedule of Section 8.3.2,
EPA/600/4-91/002 (or the most current edition). All test solutions shall be renewed daily.
If test results do not meet the acceptability criteria of Section 4.9.1, EPA/600/4-91/002 (or
the most current edition), that test shall be repeated. A chronic test will be considered valid
only if the acceptability criteria referenced above are met.
c.	If control mortality exceeds 20% for either species in any test, the test(s) for that species
(including the control) shall be repeated. A test will be considered valid only if control
mortality does not exceed 20% for either species.
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a.	The permittee shall also conduct 96-hour acute static-renewal multi-concentration toxicity tests
on the daphnid (Ceriodaphnia dubia") and the fathead minnow (Pimephales promelasV The
composite samples collected under Section 1(b) above shall be used in the acute tests. All tests
shall be conducted on a control (0%) and the following dilution concentrations at a minimum-
100.0%, 50.0%, 25.0%, 12.5%, and 6.25%. Unacceptable acute toxicity will be
demonstrated if an LC^, of 100% or less occurs in either test species within the specified time.
b.	For each set of tests conducted, a fresh 24-hour composite sample of final effluent shall be
used at Day 1 and at
Day 3. If control mortality exceeds 10% for either species, the test for that species (including
the control) shall be repeated. An acute test will be considered valid only if control mortality
does not exceed 10% for either species.
The toxicity tests specified above shall be conducted once every two months until six valid
bimonthly tests have been completed, and once every six months thereafter for the duration of the
permit, unless notified otherwise by the permit issuing authority. These tests are referred to as
"routine" tests.
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CHRONIC SALTWATER LANGUAGE (monitoring only) - 1/22/96
PART IV
Whole Effluent Toxicity Testing Program
As required by Part J_ of this permit, the permittee shall initiate the series of tests described below
beginning in month, year to evaluate whole effluent toxicity of the discharge from outfall	. All test
species, procedures and quality assurance criteria used shall be in accordance with Short-term Methods
for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Marine and Estuarine
Organisms. EPA/600/4-91/003. Short-term Methods for Estimating the Chronic Toxicity of Effluents and
Receiving Waters to Freshwater Organisms. EPA/600/4-91/002, and Methods for Measuring the Acute
Toxicity of Effluents to Freshwater and Marine Organisms. EPA/600/4-90/027F, or the most current
edition(s), as appropriate. The dilution/control water and effluent used will be adjusted to a salinity of
20 parts per thousand using artificial sea salts (e.g., Forty Fathoms) as described in EPA/600/4-91/003,
Section 7 (or the most recent edition). A chronic standard reference toxicant quality assurance test shall
be conducted concurrently with each species used in the toxicity tests and the results submitted with the
monthly discharge monitoring report (DMR). Alternatively, if monthly QA/QC reference toxicant tests
are conducted, these results must be submitted with the monthly DMR.
1. a. The permittee shall conduct multi-concentration Mysid shrimp (Mvsidopsis bahia) Survival,
Growth, and Fecundity and Inland Silverside ("Menidia bervllina) Larval Survival and Growth
Tests. All tests shall be conducted on a control (0%) and the following dilution concentrations
at a minimum: 100.0%. fRWC + 1001/2% RWC%. RWC/2%. and RWC/4%. Chronic
toxicity will be demonstrated if either test results in a no observable effect concentration
(NOEC) less than	% effluent, which is the Receiving Water Concentration (RWC) of the
effluent at critical conditions.
b.	For each set of tests conducted, a minimum of three different 24-hour composite samples of
final effluent shall be collected and used per the sampling schedule of Section 8.3.2,
EPA/600/4-91/002 (or the most current edition). All test solutions shall be renewed daily.
If test results do not meet the acceptability criteria of Section 4.9.1, EPA/600/4-91/003 (or
the most current edition), that test shall be repeated. A chronic test will be considered valid
only if the acceptability criteria referenced above are met.
c.	If control mortality exceeds 20% for either species in any test, the test(s) for that species
(including the control) shall be repeated. A test will be considered valid only if control
mortality does not exceed 20% for either species.
19

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2.	a. The permittee shall also conduct 96-hour acute static-renewal multi-concentration tests on the
Mysid shrimp ("Mvsidopsis bahiat and the inland silverside CMenidia bervllinal. The composite
samples collected under Section 1(b) above shall be used in the acute tests. All tests shall be
conducted on a control (0%) and the following dilution concentrations at a minimum:
100.0%, 50.0%, 25.0%, 12.5%, and 6.25%. Unacceptable acute toxicity will be
demonstrated if an LCjo of 100% or less occurs in either test species within the specified time.
b. For each set of tests conducted, a fresh 24-hour composite sample of final effluent shall be
used at Day 1 and at
Day 3. If control mortality exceeds 10% for either species, the test for that species (including
the control) shall be repeated. An acute test will be considered valid only if control mortality
does not exceed 10% for either species.
3.	The toxicity tests specified above shall be conducted once every two months until six valid
bimonthly tests have been completed, and once every six months thereafter for the duration of the
permit, unless notified otherwise by the permit issuing authority. These tests are referred to as
"routine" tests.
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PART I LANGUAGE
CHRONIC LANGUAGE
The effluent shall not be chronically toxic to, or produce adverse physiological or behavioral
responses in, aquatic animals. An effluent no observable effect concentration (NOEC) of less than
_% for any test species will constitute a violation of Florida Administrative Code (FAC) (April 25,
1993) Section 62-302.530(62) and the terms of this permit. The testing for this requirement shall
conform with Part IV of this permit.
ACUTE LANGUAGE
An LCjo of 100% or less in a test of 96 hours duration or less will constitute a violation of FAC
(February 2, 1994) Section 62-4.244(3)(a) and the terms of this permit. The testing for this
requirement must conform with Part IV of this permit.
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PART I LANGUAGE - MONITORING ONLY
CHRONIC LANGUAGE
The permittee shall conduct chronic whole effluent toxicity monitoring as specified in Part IV. The
effluent shall be monitored for chronic whole effluent toxicity once every two months until six valid
bimonthly tests have been completed and once every six months thereafter for the duration of the
permit. EPA will review these test results and the permit may be modified to establish a chronic
whole effluent toxicity limit, if needed, to ensure that the requirements of Florida Administrative
Code §17-302.530(62) (April 25, 1993) are maintained, as authorized and required by 40 CFR
§122.44(d)(l)(v).
ACUTE LANGUAGE
The permittee shall conduct acute whole effluent toxicity monitoring as specified in Part IV. The
effluent shall be monitored for acute whole effluent toxicity once every two months until six valid
bimonthly tests have been completed and once every six months thereafter for the duration of the
permit. EPA will review these test results and the permit may be modified to establish an acute
whole effluent toxicity limit, if needed, to ensure that the requirements of Florida Administrative
Code §17-302.500(l)(d) (April 25,1993) are maintained, as authorized and required by 40 CFR
§122.44(d)(l)(iv).
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SAMPLE FACT SHEET LANGUAGE
Chronic toxicity tests conducted by FDEP in April, 1989 on the City's effluent indicated a No Observable
Effect Concentration (NOEC) of 15% and an LC^ of 85%. Because the effluent has exhibited chronic
and acute toxicity, toxicity testing requirements and limits as contained in Item 8 on Page 1-2 and in Part
IV have been included to ensure that the effluent from Outfall 001 conforms with FAC Sections
17-302.530(62) (April 25, 1993) and 17-4.244(3)(a) (February 2, 1994) and are based on the rationale
contained in the Regional policy document, "Whole Effluent Toxicity Testing Policy for Florida", dated
May 5, 1986. The inclusion of a chronic whole effluent toxicity limit in the permit is authorized and
required by 40 CFR §122.44(d)(l)(v). The inclusion of an acute whole effluent toxicity limit in the
permit is authorized and required by 40 CFR §122.44(d)(l)(iv)
[OPTION 1]:
The Receiving Water Concentration (RWC) is used to determine the type of whole effluent toxicity testing
required in a permit. When the source of the facility's water supply is not the receiving water, the RWC
is calculated using the following equation:
RWC (%) = 100 x	Qw
(Qr + Qw)
where, Q„ = 1.1 MGD (design or appl. max. 30-day avg. flow)
Qr = Receiving water flow at appropriate low flow conditions (the 7Q10 flow in this
case)
= 8.1 cfs (5.2 MGD)
RWC (%) = 100 x 1.1/(1.1 + 5.2) = 17%
Based on a 7Q10 of 8.1 cfs for Holmes Creek, a receiving water concentration (RWC) of 17% will exist
at a design discharge flow of 1.1 MGD after complete mixing at low flow conditions.
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[OPTION 2]:
The Receiving Water Concentration (RWC) is used to determine the type of whole effluent toxicity testing
required in a permit. When the source of the facility's water supply is the receiving water, the RWC is
calculated using the following equation:
RWC (%) = 100 x	Qw
Qr
where, Q„ = 1.1 MGD (design or appl. max. 30-day avg. flow)
Qr = Receiving water flow at appropriate low flow conditions (the 7Q10 flow in this
case)
= 8.1 cfs (5.2 MGD)
RWC (%) = 100 x 1.1/5.2 = 21%
Based on a 7Q10 of 8.1 cfs for Holmes Creek, a receiving water concentration (RWC) of 21 % will exist
at a design discharge flow of 1.1 MGD after complete mixing at low flow conditions.
The effluent NOEC (%) is determined using the following equation:
effluent NOEC (%) >_ RWC
[OPTION A]:
For this facility, the RWC is greater than 1% of the receiving water flow at appropriate low flow
conditions; therefore, 7-day chronic toxicity requirements (expressed as NOEC) as well as acute toxicity
requirements were selected.
[OPTION B]:
For this facility, the RWC is less than 1 % of the receiving water flow at appropriate low flow conditions;
therefore, acute toxicity requirements were selected.
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ESTABLISHING WET PERMIT LIMITS OR MONITORING REQUIREMENTS
No Limitations
No Monitoring
Does the discharge have
the reasonable potential
to cause or contribute to WET
toxicity?
INSUFFICIENT
DATA
Monitor Until
Sufficient Data
Available
y
NO
No Limitation
WET Effluent Limit, WET
Monitoring Requirement, or
chemical limits if causative
agent known
Compliance Monitoring
Figure 1
Flowchart for establishing WET permit limits or conditions

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BASIS FOR ESTABLISHING REASONABLE POTENTIAL
NPDES regulations at 40 CFR 122.44(d) require the permit writer to establish effluent limitations for
pollutants, including whole effluent toxicity (WET), which are discharged in amounts that cause, have
the reasonable potential to cause, or contribute to an excursion above State water quality standards,
including State narrative objectives for water quality.
As required under 40 CFR 122.44(d)(l)(ii), the permit writer must consider a number of factors in
establishing reasonable potential including existing controls on point and nonpoint sources of
pollution, pollutant variability in the effluent, sensitivity of toxicity test species, and dilution in the
receiving water. The following discussions outline the tiered methodology followed when conducting
a reasonable potential evaluation. Regulations supporting reasonable potential determinations are
discussed in the TSD (see Chapter 3).
Justification for imposing water quality-based effluent limitations based on reasonable potential is
required in the statement of basis, or fact sheet [see 40 CFR 122.44(d)(vi)(C)].
ESTABLISHING REASONABLE POTENTIAL WITH FACILITY-SPECIFIC DATA
Where facility-specific effluent data are available, reasonable potential is evaluated in a sequential
(i.e., tiered) process. The first-tier analysis may be performed by using a simple steady-state mass
balance equation. The mass balance equation relates the mass of pollutants upstream of a point source
discharge, to the mass of pollutants downstream after mixing of the discharge in the receiving water is
complete. The general mass balance equation for the recommended steady-state model (see Training
Manual for NPDES Permit Writers EPA 833-B-93-003, March 1993, pp. 6-10) is:
QdCd + QsCs = QrCr, where
Qd — waste discharge flow in million gallons per day (MGD), or cubic feet per second (cfs)
Cd = waste discharge pollutant concentration in toxic units for WET (TUa or TUc)
Qs = background in-stream flow in MGD or cfs above point of discharge during critical
flow conditions
%Qs= percent of upstream flow allowed by mixing zone standard
Cs =	background in-stream pollutant concentration in toxic units for WET (TUa or
TUc)
Qr =	resultant in-stream flow after discharge in MGD or cfs: %Qs + Qd
Cr =	resultant in-stream pollutant concentration in toxic units for WET (TUa or
TUc) in the stream reach (after complete mixing)
For reasonable potential determinations, this equation is rearranged to solve for the resultant in-stream
concentration (Cr) at the edge of the mixing zone:
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Cr = (OdKCcD + tosVCsl
Qr
Using the mass balance equation, Cr should be calculated using conservative (i.e., critical)
assumptions for background in-stream receiving water flow (Qs), background in-stream receiving
water pollutant concentration (Cs), waste discharge flow (Qd) and waste discharge pollutant
concentration (Cd). Critical waste discharge conditions should be represented by the highest observed
pollutant concentration and waste discharge flow. Critical background in-stream receiving water
flows are: 1) the 1Q10 flow (1-day low flow over a 10-year recurrence interval) for calculating acute
effects and 2) the 7Q10 flow (consecutive 7-day low flow over a 10-year recurrence interval) for
calculating chronic effects. The State of Alaska uses 30Q2 (consecutive 30-day low flow over a 2-
year recurrence interval). Where possible, background in-stream pollutant concentrations should
correlate with critical background in-stream flows, as critical pollutant concentrations occur during
low flows, or are associated with stormwater. For WET, Regions 9 and 10 recommend that
background be assumed to be zero, unless data are available. Ambient low flow data, developed by
the U.S. Geological Survey, are available through STORET.
Once the projected maximum in-stream pollutant concentration (Cr) is calculated, this value can be
compared to the appropriate water quality criterion (WQC). Where Cr is greater than the WQC,
reasonable potential is established for that pollutant at the specified effect level (i.e., acute or
chronic). When reasonable potential is demonstrated, water quality-based effluent limitations must
then be developed for WET.
If the projected maximum resultant in-stream pollutant concentration (Cr) is less than the WQC, the
permit writer must then exercise judgement to determine whether reasonable potential exists. This
judgement depends on how large the difference is between Cr and the applicable WQC, the
uncertainty of maximum effluent concentrations, type of discharger, and the sensitivity of the
receiving water. To assist in making this judgement, a second-tier assessment may be performed that
statistically addresses the uncertainty of maximum effluent concentrations for individual pollutants.
The second-tier analysis is a six step process (see TSD, Box 3-2, p. 53) and is conducted for an
effluent pollutant data set as follows:
1.	Calculate the coefficient of variation (CV), where the CV is the standard deviation
over the mean (alfi) (see TSD, Appendix E). For sample sizes less than 10 (k < 10)
a default CV of 0.6 can be used (see TSD, Box 3-2, p. 53).
2.	Choose uncertainty multiplier from Table 3-1 or 3-2 (see TSD, p. 54) using k and the
CV. The 99% confidence level and 99% probability basis (Table 3-1) is
recommended.
3.	Calculate the adjusted maximum effluent concentration by multiplying the uncertainty
multiplier times the highest observed effluent concentration (Cd).
4.	Re-calculate the maximum resultant in-stream pollutant concentration (Cr) using the
adjusted maximum effluent concentration (Cd) and the mass balance equation.
5.	Compare Cr with the applicable criterion Reasonable potential is established when
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Cr exceeds the criterion.
When reasonable potential is established by either first- and/or second-tier analyses, a water
quality-based effluent limitation must be included in the permit for WET. A case example is
presented at the end of this appendix.
ESTABLISHING REASONABLE POTENTIAL WITHOUT FACILITY-SPECIFIC EFFLUENT
DATA
Where facility-specific effluent data are lacking, the permit writer may still conduct a reasonable
potential evaluation. Establishing reasonable potential under such circumstances requires a systematic
consideration of all applicable factors in 40 CFR 122.44(d)(l)(ii) (see TSD, pp. 50-51 and Box 3-1,
p. 49) including:
•	Existing ambient water quality data;
•	Available dilution in the receiving water;
•	Type of receiving water and designated uses;
•	Industry/POTW type and nature of the discharges;
•	Compliance history and historical toxic impacts; and
•	Information from permit application or DMRs.
If a review of ambient monitoring data shows in-stream exceedances or near exceedances of a
criterion for toxicity and WET is present in the discharge, reasonable potential is clearly established
and effluent limitations for WET should be included in the permit. The in-stream exceedance of the
toxicity criterion indicates that the receiving water body cannot assimilate any additional load of
toxicity. Consequently, compliance with the criterion for toxicity must be met at the end-of-pipe
(i.e., no dilution).
FINDING NO REASONABLE POTENTIAL
Where existing effluent monitoring data show no reasonable potential for excursions above ambient
applicable criteria, the permit need not contain water quality-based effluent limitations. However, the
permit writer may include monitoring requirements in the permit to continue to re-affirm initial
reasonable potential determinations and to monitor for effluent changes (see TSD, pp. 59, 64).
CASE EXAMPLE
Facility Description
A regional wastewater treatment plant (Regional Plant) discharges to a river. The Regional Plant
treatment train consists of coarse screening, aerated grit chambers, primary sedimentation, pure
oxygen activated sludge, secondary clarification, and disinfection using chlorination/dechlorination
systems. The river in the vicinity of the discharge is influenced by tides and slack flows and flow
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reversals may occur. In order to insure rapid mixing in the receiving water and prevent a breakdown
in jet diffusion, the secondary effluent is diverted to an on-site emergency storage basin. Once the
river flow is sufficient for adequate mixing of the effluent, the discharge is resumed Design effluent
flow is 180 MGD.
Data
Based on information provided, the 7Q10 is estimated to be 7500 cfc. Using the design flow of 180
MGD, this would correspond to an instream dilution of 26:1 Based on the analysis provided for the
diversion of the effluent during low flow periods, a minimum dilution of 14:1 would occur
infrequently, as a result of extreme high tides and low flow conditions, is a short-duration event (less
than 1-hour in duration), and is used to assess for the exceedance of the CMC (i.e., acute effects).
The following table is a summary of the results of 20 chronic tests conducted by the facility. Based
on those results, the value for k is 20, the highest effluent concentration of WET observed was 16
TUc, and the CV is 0.9. The uncertainty multiplier from Table 3-1 is 3.2.
Toxicity test results, TUc
2,2,2,4,4,4,2,2,2,2,2,4,4,4,8,2, > 16,16,2,2,8
ACR
10
Chronic dilution
26:1
Acute dilution
14:1
CV
0.9
k
20
Uncertainty multiplier (RPF)
3.2
In order to evaluate reasonable potential for the acute criterion, the chronic results need to be
converted to TUa, i.e., 16/ACR = 1.6 TUa.
Acute: (1.6 TUa)(3.2)/14 = 0.4 TUa
0.4 TUa > 0.3 TUa (acute criterion)
Chronic: (16 TUc)(3.2)/26 = 1.9 TUc
1.9 TUc > 1.0 TUc (chronic criterion)
Based on these results, both acute and chronic criteria for toxicity have demonstrated a reasonable
potential to be exceeded. Permit limits for toxicity must be developed this discharge.
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TYPICAL COMMENTS ON WET AND SUGGESTED RESPONSES
CHRONIC WET
COMMENT: The imposition of a chronic WET limit is premature until
EPA completes its study through the Pellston workshop of
the accuracy of the test.
RESPONSE: Existing state and federal regulations require the
imposition of a chronic WET limit if it is determined
that the discharge causes or has the reasonable potential
to cause an excursion of applicable water quality
criteria. The existing chronic WET data support such a
determination. Preliminary conclusions from the Pellston
workshop indicate that the current WET program is
technically sound, that current statistical methods are
in wide use and are accepted in the scientific community,
and that additional laboratory to field validation is not
essential. EPA also notes that the freshwater chronic
WET test was promulgated as an approved Part 136 method
on October 16, 1995.
COMMENT: Currently, no published TIE procedure is available for
low-level chronic toxicity. A chronic WET limit should
not be required until such methods are available.
RESPONSE: A Phase I chronic TIE manual was published in 5/92.
Phase II and III chronic TIE manuals were published in
9/93. Current state and federal regulations and
SS 301(b)(1)(c) and 402(a) of the Act mandate the
imposition of chronic WET limits where it is determined
that a discharge causes an excursion of applicable state
water quality criteria.
COMMENT: Species sensitivity variability, test methodology
variability, and variability in correlation to receiving
water impacts raise concerns about the appropriateness of
the chronic WET test.
RESPONSE: The freshwater chronic WET test was promulgated as an
approved Part 136 method on October 16, 1995. EPA
believes the facility's concerns are also adequately
addressed through the Technical Support Dnnnnpnt (TSD)
and the conclusions from the Pellston workshop.
COMMENT: Insufficient inter laboratory validation data exist for
use of the chronic WET test as a Part 136 method.
RESPONSE: Interlaboratory data are available which validate the
Ceriodaphnia chronic WET test. In a round robin inter-
laboratory study supervised by EPA Region 4 in 1989

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2
(EPA/505/2—90—001), 36 (80%) of 45 tests were
successfully completed. The endpoints (No-Observed-
Effect Concentrations, or NOECs) of 35 of the 36 tests,
fell on two adjacent concentrations. Also, an
interlaboratory study of the Ceriodaphnia dubia 7-day
chronic test conducted by the San Francisco Bay Regional
Water Quality Control Board (Environ. Toxicol. Chem.
10:143-145, 1991), resulted in a coefficient of variation
of 29%, demonstrating good precision.
EPA agrees that methods approved under Part 136 must be
scientifically validated. Further, EPA recognizes that
an interlaboratory study (round robin) is a useful and
desirable means of validating an analytical method.
However, EPA does not consider such a study to be a
requirement for approval under Part 136 because each
interlaboratory study conducted with aquatic toxicity
tests methods was preceded by intralaboratory studies
that showed satisfactory precision, and where the Agency
does not have interlaboratory data for a species,
adequate data on intralaboratory precision are available.
EPA does consider the precision of candidate methods in
approving such methods under Part 136. The essential
criterion is that the precision of the methods are
approximately within the range of other Part 136 methods,
and that the methods provide valid results. The
variability of some chemical methods, e.g., Mn, exceeds
that of the toxicity test methods (EPA/505/2-90-001). A
large amount of intra- and inter-laboratory precision
data are available on these toxicity tests, and
representative data sets are included in the methods
manuals. On the basis of these data, EPA is comfortable
with the conclusion that whole effluent toxicity tests
are no more variable than chemical analytical methods in
Part 136 and, therefore, stands behind the recommendation
that toxicity tests be used in NPDES permits.
COMMENT: The QA/QC requirements of chronic WET tests do not
adequately account for variability. A practical
quantitation limit (PQL) is needed.
RESPONSE: PQLs are not recognized in the NPDES program and are not
recommended for use by the TSD. EPA's recent
promulgation of the chronic WET test as a Part 136 method
is an indication that variability from use of this method
is within acceptable limits.

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3
COMMENT: Ceriodaphnia is a very sensitive nonindigenous species -
its use may result in indications of chronic toxicity
that do not actually exist. Applicable state rules for
toxicity do not specify this organism as a statewide
indicator of chronic toxicity.
RESPONSE: The State's regulations do not designate any specific
species that must be used in acute or chronic WET tests.
As discussed in the TSD, WET test species do not have to
be indigenous to the receiving stream of interest. EPA
believes that Ceriodaphnia's sensitivity is
representative of other species that may exist in the
receiving stream. Also, data for nonindigenous species
were included in the data bases for all numeric toxic
criteria adopted by the State for protection of aquatic
life.
COMMENT: No data have shown that low-level chronic WET limits
correlate to instream impacts. Chronic WET tests should
not be used to trigger enforcement actions until such
results have been correlated with instream assessments or
biological surveys. The proposed permit improperly
classifies any exceedance of the limit as a violation.
RESPONSE: Numerous EPA policies and Environmental Appeals Board and
judicial decisions have supported the position that
exceedance of any permit limit, including a WET limit, is
a permit violation.. The facility's comment that
enforcement should wait until instream impact has been
correlated contradicts EPA's policy of independent
application and is contrary to the intent of the Clean
Water Act and state and federal regulations. A draft
summary of the Pellston conference also indicates that
correlation of effluent results to instream impacts is
not needed.
COMMENT: The State's regulations do not define chronic toxicity as
being equal to an NOEC i IWC.
RESPONSE: The State's reasonable potential procedures do define
chronic toxicity as NOEC * IWC. The State's
interpretation is consistent with and based on the TSD.

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4
COMMENTi Federal and state regulations state that a WET limit need
not ^ be applied if chemical-specific limits are
sufficient. Because the historical source of toxicity
has been identified, a WET limit is not appropriate.
RESPONSE: Under 40 CFR 5122.44(d) (1) (v), the permit fact sheet must
document that chemical-specific limits are sufficient to
control chronic toxicity if no WET limit is included in
the permit. The fact sheet for this permit does not
contain such a demonstration. EPA also notes that the
permit fact sheet contains no assurance that the
chemical-specific limits in this permit are sufficient to
control chronic toxicity and maintain water quality
standards.
COMMENT: The multiple dilution concentrations are inappropriate in
light of the rapid dilution achieved by the discharge.
RESPONSE: The permittee has misinterpreted why multiple dilutions
are being required; the number of dilutions has nothing
to do with the available dilution of the receiving
waterbody. Per the July 1994 chronic WET protocol
manual, multiple dilutions are recommended to assess
NPDES compliance for all WET tests (pg. 42). They
provide more information about the dose-response of the
test, increase the statistical power of the test, and
decrease the inherent variability found in conducting a
single test concentration with a control. EPA's TSD
(pg.58) also discourages the use of single concentration
toxicity tests.
COMMENT: The chronic toxicity test species are not representative
of species that inhabit the waters affected by the
discharge from this facility. Further, because of
problems inherent in laboratory tests (such as the
inability to replicate field conditions) the use of
bioassay data should only be a part of a broader approach
to perceived water quality problems. Such em approach
should include one or more other evaluations that could
include in-stream tests, biological monitoring, and other
forms of field assessments.
RESPONSE: The use of standard laboratory species in toxicity tests
is consistent with EPA's TSD, national policies regarding
WET, and the various EPA toxicity test protocols, which
were promulgated as Part 136 NPDES methods on October 16,
1995. The use of indigenous species is not practical due
to:

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5
1.	sensitive organisms may not be present in the
receiving water due to previous exposure to the
effluent or other pollutants;
2.	the difficulty in collecting and handling organisms
of the desired age and condition (free from
disease) from the receiving water;
3.	the lack of extensive quality control and
range-of-sensitivity data for such species; and
4.	the lack of information on the diet of such
indigenous organisms.
EPA disagrees with the comment's contention that
laboratory tests cannot replicate field conditions.
EPA's complex effluent toxicity testing program
correlated receiving water chronic toxicity measured by
EPA (laboratory) toxicity tests to in-stream observed
impacts (Reference: TSD, pg. 7-11).
Regarding the comment about a "broader approach", EPA
cites the following section of the TSD to support its
position: "To more fully protect aquatic habitats and
provide more comprehensive assessments of aquatic life
use nonattainment, EPA recommends that States fully
integrate chemical-specific, whole effluent, and
bio-assessment approaches into their water quality-based
toxics control programs. It is EPA's position that the
concept of "independent application" be applied to water
quality-based situations. Since each method has unique
as well as overlapping attributes, sensitivities, and
program applications, no single approach for detecting
impact should be considered uniformly superior to any
other approach. For example, the inability to detect
receiving water impacts using a bio-survey alone is
insufficient evidence to waive or relax a permit limit
established using either of the other methods. The most
protective results from each assessment conducted should
be used in the effluent characterization process. The
results of one assessment technique should not be used to
contradict or overrule the results of the other(s)."
(pg.22)
COMMENT: Renewal of short-term chronic toxicity test solutions
with fresh effluent sample, instead of with the initial
(same) sample, introduces a degree of variability that
obscures the meaning of the test results and makes the
identification of responsible toxicants almost
impossible.
RESPONSE: The commenter incorrectly suggests that the variability
in the tests results caused by variability in the
toxicity of the effluent is undesirable. However, the
objective of short-term chronic toxicity tests is to

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6
determine the toxicity of the effluent (or receiving
water), including its day-to-day variability. EPA
recognizes that results of tests performed with the same
sample may differ from the results of tests performed
with multiple, fresh samples. However, the use of fresh
samples for daily test solution renewal more accurately
reflects the temporal variability of the toxicity of the
effluent or receiving water. Because of the possible
loss of volatile toxics such as ammonia, chlorine, and
toxic organics, and adsorption of toxics on the surfaces
of the sample container, holding and using a single
sample for the entire length of the test could result in
a significant underestimation of toxicity, and would
fail to incorporate any of the possible effects of spikes
or lows in effluent or receiving water toxicity. Use of
fresh sample in renewal of test solutions is preferred,
but is not always feasible due to logistical constraints.
However, a ™-inimnm of three fresh samples must be
employed to achieve the objectives of the short-term
chronic survival, growth, and reproduction tests. The
samples must be collected and used as prescribed in the
freshwater and marine short-term chronic toxicity test
methods. Regardless of the number of fresh samples
collected for the tests, daily renewal of test solutions
is required.
COMMENT: Only survivors should be used for Ceriodaphnia
reproduction analysis.
RESPONSE: EPA disagrees. The exclusion of reproduction data from
females that do not survive to the end of the test would
bias the results in favor of the organisms that are more
tolerant to pollution. Therefore, EPA believes that it is
best to use the reproduction data from all the test
organisms in the analysis, except for those from
concentrations that have significantly greater mortality
than the controls. Data from the latter are not included
in the determination of the reproductive endpoint.

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ACUTE WET
COMMENT: The multiple.dilution concentrations are inappropriate in
light of the rapid dilution achieved by the discharge.
RESPONSE: The permittee has misinterpreted why multiple dilutions
are being required; the number of dilutions has nothing
to do with the available dilution of the receiving
waterbody. Per the August 1993 acute WET protocol
manual, multiple dilutions are recommended to assess
NPDES compliance for all WET tests (pg. 47-48). They
provide more information about the dose-response of the
test, increase the statistical power of the test, and
decrease the inherent variability found in conducting a
single test concentration with a control. EPA's
Tec.inical Supppt-i- nnnimpnt (TSD) (pg.58) also discourages
the use of single concentration toxicity tests.
COMMENT: Given the lack of correlation between the prescribed test
methodology and actual conditions in the receiving
saltwater, an exceedance could not support a finding of
acute toxicity nor could it justify the imposition of
effluent limits or additional system improvements.
RESPONSE: EPA strongly disagrees with the permittee's premise that
there is no correlation between the prescribed test
methodology and actual conditions in the receiving
saltwater. The permittee has provided no documentation
for its assertion. EPA's TSD clearly demonstrates such
a correlation (EPA/505/2-90-001, March, 1991, pg.7-11).
Based on ambient and effluent toxicity data collected at
4 different saltwater sites, a 94% accuracy rate was
found when using marine and estuarine toxicity tests to
predict receiving water impacts. A testing frequency of
twice/year does not impose an unreasonable cost burden on
the permittee.
COMMENT: By failing to adjust test data with control mortality,
EPA's acute WET protocol introduces an uncompensated
variable and results in false indications of potential
toxicity.
RESPONSE: This comment is unfounded and shows a lack of familiarity
with EPA's test protocols. EPA's August 1993 acute WET
protocol recommends (pg. 47) that acute WET tests be
conducted on a control and 5 concentrations of diluted
effluent. The use of multiple concentrations provides
more information about the dose-response of the test.
The endpoint of a multi-concentration acute test is a
point estimate of the effluent concentration which is
lethal to 50% of the test organisms during the test
duration. Section 11 of EPA's August 1993 acute WET
protocol contains 4 different statistical methods that

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8
may be used .to calculate the point estimate (LCS0) from
a given multi-concentration test. Each method requires
that responses in each concentration be adjusted by
mortality in the control using Abbott's formula (See
Section 11.2.2.3.1 and 2, pg. 78, Graphical Method;
Section 11.2.3.3.1 and 2, pg. 81, Spearman-Karber Method;
Section 11.2.4.3.1 and 2, pg. 84, Trimmed Spearman-Karber
Method; Section 11.2.5.4.3, pg. 88, Probit Method). As
stated in the protocol (pg. 56), for the test results to
be acceptable, survival in controls must be at least 90%
(i.e., no more than 10% mortality is allowed in a 96 hr
test). Tests in which the control survival is less than
90% (i.e., mortality exceeds 10%) are invalid and must be
repeated.
The example provided in the comment is based on a control
and a single test concentration. In such an acute WET
test which is allowed by EPA's August 1993 acute WET
protocol, the objective is to determine if the survival
in that single treatment (i.e., effluent) is
significantly different from the control survival (pg.
91, EPA August 1993 protocol). This determination must
be accomplished by hypothesis testing - whether there is
a significant difference between the treatment and the
control. Because the treatment and the control are being
compared directly against each other in this case, it is
entirely inappropriate to allow the treatment survival to
be adjusted by the control survival. The procedure for
this statistical analysis is found on pg. 101-105 of the
EPA protocol and does not allow for such an adjustment.
COMMENT: Variations in salinity alone affect the survival of Mysid
shrimp based on the EPA Environmental Research Brief
"Optimization of Environmental Factors During the Life
Cycle of Mvsidopsis bahia" (EPA/600/M-87/004, June 1987).
RESPONSE: An examination of this article finds several significant
differences in the way that this test was conducted from
that which is required by EPA's August 1993 acute WET
protocol (EPA/600/4-90/027f). Such differences do not
allow for a valid scientific extrapolation of the
article's findings to tests conducted under EPA's acute
WET protocol. First, the article indicates that the test
organisms were fed daily with an abundance of Art em i a.
Such feeding is not allowed by EPA's protocol - the
inclusion of an abundance of live food may result in
oxygen depletion and a build-up of toxic levels of
ammonia and nitrite, possibly contributing to observed
Mysid mortality. Thus, the mortality observed is not
necessarily representative of that which may occur in an
acute test using the EPA protocol. Second, mortality
data are based on a one week exposure. The acute tests

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9
required in the facility's permit are a maximum of 4 days
(96 hr). Thus, it is expected that any observed
mortality due to stress from salinity and/or temperature
will be significantly lower than that cited in this
article. Third, the article indicates that test
solutions were renewed every third day. EPA's protocol
requires that test solutions be renewed at 48 hr in -a 96
hr test. Accumulation of waste products from Mysids and
ArtRTnia may have contributed to the Mysid mortality
observed in the article. Fourth, the article indicates
that 15 mysids were placed in each salinity/temperature
exposure. The EPA protocol requires that a Tn-i-n-iTmun of 20
organisms be used in each dilution. Fifth, the article
suggests that only 1 dilution of each
salinity/temperature exposure was prepared. The EPA
protocol recommends that multiple dilutions be used. Use
of a single test concentration yields higher variability.
The article does state that 95% survival occurred at the
approximate range of 15-28*i and 23-32 «> C. after one week
of exposure. Mysid tests are typically conducted at a
salinity of 20fe and a temperature of either 20® or 25°
C., well within or near these optimal conditions.

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Judicial and Administrative Decisions
Regarding Whole Effluent Toxicity
1.	Hercules, Inc. v. EPA. 598 F.2d 91 (D. C. Cir. 1978).
Hercules, Inc. and velsicol Chemical Corporation petitioned to
review EPA regulations limiting discharges of toxaphene and
endrin to U.S. waters. Petitions were combined. Hercules and
Velsicol objected to EPA's categorical approach using tests of
several aquatic species as opposed to standards based on local
species and local receiving waters. After an analysis of EPA's
methodology and of the legislative history of the Clean Water Act
in this regard, the court upheld the regulations setting
standards for discharges of toxaphene and endrin.
2.	American Petroleum Institute v. E.P.A., 787 F.2d 965
(5th Cir. 1986) . Four conpanies and a trade association appealed
issuance of two NPDES permits for offshore drilling rigs.
Specifically, they contended that EPA overreached its authority
in imposing five of the discharge limitations and requiring two
test methods in the permits. On appeal the court remanded one
discharge limitation but left intact the remaining features of
the permit, including the use of a standard bioassay test for
drilling mud. The court noted that EPA had not selected an
irrational methodology and that even the petitioner conceded the
test to be the most widely accepted benchmark for toxicity
evaluations by EPA.
3.	Natural Resources Defense Council v. U.S.E.P.A.. 859
F.2d 156 (D.C. Cir. 1988). This is a consolidation of challenges
to NPDES regulations by industry and environmental groups. The
following are pertinent issues which the court ruled on.
Permit writers may express technology-based or water quality-
based limits in terms of toxicity. The Court reasoned that the
CWA authorizes the use of toxicity to regulate effluents that are
pollutants through the Act's broad definition of "pollutant,"
although toxicity appears to be an attribute of pollutants rather
Mian a pollutant itself. Also, industry challenged EPA's
decision making process for 40 C.F.R. § 125.3(c)(4)r however, the
Court found that EPA is not barred from an information-gathering
process while an informal rulemaking is pending.
4.	Natural Resources Defense Council v. U.S. E.P.A.. 863
F.2d 1420 (9th Cir.- 1988) . Industry, environmental groups and
the State of Florida filed petitions for review of a general
permit authorizing discharge of pollutants from oil and gas
operations to the Gulf of Mexico. Industry challenged the
validity of the bioassay test EPA selected to establish the
toxicity level. The Court ruled that the choice of the bioassay
test was a matter of agency discretion, that it was an
appropriate effluent limitation gauge and that because many of
the pollutants covered by the permit do not have approved test

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2
methods, EPA could use a test procedure specified in the permit.
On this issue, the permit was upheld, but it was remanded on
other issues.
5.	American Paper Institute v. U.S.E.P.A.. 996 F.2d 346
(D.C. Cir. 1993). Consolidated challenges by industry to CWA
regulations, primarily regarding the rule requiring permit
writers to use one of three methods to interpret state water
quality standards containing "narrative criteria" so as to create
precise chemical-specific effluent limitations. The regulations
were found to be reasonable and petitions were denied.
In dictum the court discussed the problem of arriving at effluent
limitations using narrative criteria and noted the inconsistency
in translating criteria into permit limitations when standards
containing narrative criteria were enforced through the use of
whole effluent discharge limitations based on biological
monitoring techniques, which is one method of testing compliance
with narrative criteria.
6.	In re City of Hollywood. Florida. NPDES Appeal No.
92-21 (March 21, 1994). The City challenged aspects of the
permit's whole effluent toxicity limitation and biological
testing provisions based on inconsistency with EPA policy and
practice; these challenges were rejected. The City also objected
to the test species used on the ground that those species are not
indigenous to the aquatic community affected by the City's
discharge. The Agency requested that this issue be remanded for
further consideration as to whether the City's objection raised a
material issue of fact warranting an evidentiary hearing. The
EAB requested briefs regarding testing in 100% effluent as
opposed to the City's proposal of testing at 30% of full strength
in view of the seemingly contradictory federal anti-backsliding
requirement and Florida Administrative Code requirement. The
City argued that testing organisms in a constant concentration as
opposed to a declining concentration of toxicants would not
simulate actual conditions. This argument was rejected. The
City's challenge to proposed grab sampling requirements was
rejected also.
7.	In the Matter of American Cvanamid Company, NPDES
Appeal No. 92-18; In the Matter of Jefferson Smurfit Corporation.
NPDES Appeal No. 92-8 (September 27, 1993). Two consolidated
petitions for review. Both companies objected to effluent
toxicity limitations and biomonitoring requirements, specifically
the permit language stating that any single failed toxicity test
constitutes an enforceable violation of the permit. The EAB held
that such testing methods are necessary and appropriate and that
the characterization of a single exceedance as a violation is
consistent with the plain meaning interpretation of the pertinent
Florida rule. The EAB also found that Jefferson's objection to

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3
permit language requiring follow-up testing was not stated with
sufficient specificity. Petitions.were denied.
8.	In the Matter of Broward County, Florida. NPDES Appeal
No. 92-11 (June 7, 1993). In its petition for review, the County
raised the following issue relating to whole effluent toxicity:
whether the test species are significant to the indigenous
aquatic environment; if not, will they accurately predict how
indigenous species would fare when exposed to the county's
effluent. Based on Florida regulations, the County also objected
to the requirement for testing in 100 percent effluent
concentrations. The EAB ordered the Region to modify the permit
to allow the County to conduct testing in effluent diluted to
thirty percent based on Florida regulations. Because the record
did not contain the necessary information, on remand the Region
must provide a detailed explanation of the basis for selecting
the test species and whether the County's effluent is causing or
contributing to a violation of Florida's water quality standards.
9.	In the Matter of City of Denison. Texas. NPDES Appeal
No. 91-6 (December 8, 1992). The City based its appeal on issues
relating to toxicity testing and biomonitoring requirements. The
City objected to the requirement for a Toxicity Reduction
Evaluation (TRE), contending that only biomonitoring was required
for state certification. The Board responded that the TRE
requirement is an element of biomonitoring. The City also
contended that EPA erroneously denied its hearing request as to
toxicity test protocols and test organisms. The Board agreed
with EPA that Denison's failure to raise these concerns during
public comment did not preserve the issue for review, nor did its
objection to the appropriateness of biomonitoring.
10.	In the Matter of General Electric Company. Hooksett,
New Hampshire, NPDES Appeal No. 91-13 (January 5, 1993) . GE
objected to the inclusion of WET testing requirements in its
permit. This appeal was denied on the grounds that the State of
New Hampshire certified the permit and adopted the federal permit
as the state permit. According to the Board, this approval was
evidence that the State believed the permit requirements were
necessary and could not be made less stringent.
11.	In re Florida Pulp and Paper Association & Buckeye
Florida. L.P.. NPDES Appeal Nos. 94-4 and 94-5 (May 17, 1995).
Among other things, Florida Pulp and Paper Association appealed
on the basis of whether the species proposed for use in chronic
toxicity tests were representative of species found in affected
waters. The EAB remanded this issue so that an evidentiary
hearing could be held because it was a genuine issue of material
fact which had been raised with sufficient specificity.
12.	In the Matter of City of Jacksonville. District II
Wastewater Treatment Plant. NPDES Appeal No. 91-19 (August 4,

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4
1992). The City appealed the question of the "single excursion"
provision in its permit. The Board ruled that this requirement
was necessary to insure compliance with Florida's toxicity
standards and denied the appeal since this is a legal issue, not
a factual issue.
13.	In the Matter of J & L Specialty Products Corp.,
Louisville, Ohio, NPDES Appeal No. 92-22 (February 2, 1994).
J & L appealed on a number of issues, including the effluent
limitations for whole effluent toxicity and biomonitoring
requirements. J & L contended that toxicity testing is not
sufficiently developed to be used for enforcing WET limitations
and these test procedures would be invalid until published in
40 C.F.R. The Board disagreed, saying the Agency had been using
such testing since 1984 and that publication was not necessary
for the procedures to be valid. J & L argued successfully as to
whether its effluent will cause, contribute to or have a
reasonable potential to cause or contribute to a violation of the
state's water quality standard for WET and therefore whether such
limitations and requirements can be imposed. The Board concluded
that J & L raised material issues of fact in this regard and
remanded for an evidentiary hearing. Since EPA indicated a
willingness to accommodate J & L's concerns about biomonitoring
requirements, the Board felt review of that requirement was not
warranted.
14.	In the Matter of Miami-Dade Water and Sewer Authority
Department, NPDES Appeal No. 91-14 (July 27, 1992). The Board
ruled that the following pertinent issue was a factual one which
should be heard at hearing: whether the test species would
predict how indigenous species would fare when exposed to the
effluent as required by the permit.
15.	In the Matter of Louisiana-Pacific Corporation and
Simpson Paper Company. Docket No. NPDES 09-87-0005, July 27,
1992. In this Initial Decision, the ALJ held that a suite of
tests should be used for chronic toxicity testing. In addition,
he ruled that EPA had the authority to include acute toxicity
testing in the permit but that the species originally recommended
for use was insufficiently sensitive and that mysid shrimp should
be used in a continuous flow-through methodology.

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CRITERIA FOR USE OF HPItiBWUB (AWBRKMK) 6SBCXK8
SFFLDSBT TOXICITY TESTS
WILLIAM H. PELTIER
U.S. FnuI iiiiimnnfnl Protection Agency
REGION IV
The species 111 i >¦¦< inilinl fay BPA for a££lwat toxicity tests
in the HPDKS prograa wze selected to represent a 'pacfonaaeft
standard* or indicator o£ appropriate sensitivity to toxicity for
a given phylogenetic category. Therefore, to obtain
authorisation to nee o species other than tho recoaenrnded
species, the permittee aust provlcSe supporting data on species
criteria to the permitting imtborlty to dreomtrnf that tha
proposed indigenous test species is at least as sensitive as the
recouended test species for that phylogenetic category. The
required criteria for indigenous species are as follows*
•	Sufficiently sensitive species - The speoiee
recoanended by BPA as standard test species for use in
the HPDES prograa were selected based on their
sensitivity to the types of toxic substances i r—nnly
occurring in effluents. The proposed indigenous
species oust be at least as sensitive to toxic
substances as the recoanended BPA species representing
that phylogenetic category.
•	Related phytogenetically and having ecological and
functional requirements similar to SPA i nr ni— imlart
species - Indigenous species used in toxicity testing
must be similar phytogenetically and have similar
ecological and functional requirements similar to the
recomaended species (i.e. game fish for a game fish,
shrimp for a shrimp, alga for an alga, etc.).
•	Important ecologically, cuiirr inlly, and/or
recreationally - Host be critical in the food chain and
trophic scheme; ecologically important, (i.e., forage
fish)| ccsmercially important, (i.e., shrimp) and/or
recreationally important, (i.e., game fiah).
•	Early-life stage and readily available year round -
Since the most sensitive stage of species used in
aquatic toxicity testing has generally been documented
in peer reviewed journals as the early life stage of
the species, the early life stage is to be used in
assessing the impact of an effluent. The testing
frequencies in HPS8S permits vary weekly, monthly, bi-
monthly or quarterly and it is a requirement that an
early life stage of the organism, be readily available
year-round for use in testing. This requirement may
best be met by culturing the organisms.

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•	Tolerate handling and laboratory conditions - The
indigenous specie# to be need in testing must tolerate
handling without excessive disruption of its life
processes, and be amenable to col taring, rearing,
maintenance and testing under controlled laboratory
conditions.
•	Give consistent, reproducible response to toxicants —
The permittee must establish that acute and/or short-
term chronic teats with the proposed indigenous species
provide consistent and reproducible results vhen using
KPA-npproved standard reference toxicants, by
conducting a alniaaa of five I ntrnl nhnrntory acute
and\or short-term chronic toxicity tests. Hie precis ion
of t-Jhf* results is evaluated on the basis of data in the
published literature and EPA guidance documents.
•	Standardised, validated, test protocols must be
available - A protocol for test organism cultaring and
toxicity testing must be developed and available for
the indigenous species* The protocol must: hove
undergone intra-laboratory testing that evaluates
sensitivity to test methods variables (ruggedness) and
single laboratory/single operator precision from at
least three laboratories. Che interim protocol
nuboitted for approval nut include the full methods
description, information on ruggedness, mandatory and
optional test conditions, guidance on data analysis,
intralaboratory precision, etc* If approved by the
permitting authority, the protocol would now be
considered a -standard" protocol, interlaboratory
validation of the protocol requires data from a minimum
of six laboratories.
The completed protocol and all supporting data relating to
the criteria for indigenous species characteristics for effluent
toxicity testing, must be submitted to the permitting authority
for approval prior to the use of the indigenous species in the
HPDBS permit.
If, after public commit and review by the permitting
authority, the supporting data on species character! sties
submitted by the permittee are found to meet the above criteria,
the permitting authority may approve the use of the protocol for
the indigenous species in the HPDBS pezsit.

-------
KENTUCKY WHOLE EFFLUENT TOXICITY SURVEY
Dear Colleague:
We have been implementing both acute and chronic WET limits in our
NPDES permits for several years. A number of municipalities in our
state have filed a petition with EPA Administrator Browner
requesting fundamental changes in the NPDES regulations regarding
chronic WET. To better respond to that petition and assist us in
understanding how our program (particularly for POTWs) compares
with others in the rest of the country, we request your help in
completing the following survey. We have structured the survey so
that it will take at most 1 hour to compile and complete. We will
send a summary of the results to all those that complete it.
Please submit your responses by Wednesday, August 10, to:
Charlie Roth
Division of Water
KY Dept. for Environmental Protection
14 Reilly Road
Frankfort, KY 40601
(502) 564-3410 - phone
(502) 564-4245 - fax
Do not hesitate to call if you have any questions. Thank you in
advance for your assistance and time.
PERMIT ISSUING AUTHORITY: 	
The following are the basic permitting situations/assumptions that
the survey is based on:
-	Major Municipal POTW w/ approved pretreatment program
-	freahwater discharge to a fish & wildlife-classified
receiving stream, Tier 1 - no endangered species; no
outstanding resource waters
-	no diffuser
-	this facility's expiring NPDES permit contains no WET
monitoring or limits. The permit application contains
historical WET data and the permit writer has concluded that
reasonable potential to exceed State WQS now exists.
General Question: Please designate what, "receiving stream
critical low-flow" in general means for your
state (i.e. 7Q10, 1Q10, etc)	
PLEASE PROVIDE THE ACTUAL PERMIT LIMIT/MONITORING PAGE(S) AND
REQUIREMENTS THAT YOU WOULD USE IN EACH OF THE FOLLOWING SITUATIONS
A-l

-------
Situation ilz POTW design flow is 2.2 MGD
Receiving stream critical low-flow to protect
aquatic life is 0 MGD.
1. would the permit contain: (circle all that apply)
an acute WET limit	(go to a. below)
a chronic WET limit	(go to a. below)
acute WET monitoring only (go to c. below)
chronic WET monitoring only (go to c- below)
no WET monitoring/limit (please explain)
a.	would a compliance schedule for the WET limit be given?
YES How long? 	 (go to b. below)
NO	(go to b. below)
b.	is each WET test failure a permit violation?
YES	(go to 2)
NO	(go to 2)
c.	if acute or chronic WET monitoring only is required,
when (if ever) would a limit be imposed?
(go to 2)
2.	Are multiple dilutions required?
YES/NO
3.	What test species are required? (circle all that apply)
Ceriodaphnia
Daphnia magna/pulex
fathead minnow
Other:
a. Does the permit allow for testing to be conducted on only
the more sensitive test species at some point during the
permit term?
YES/NO/NA
4.	Does the permit require additional WET tests after a WET test
failure?
YES/NO
5.	Does the permit require that a TIE/TRE be conducted? (provide
language if not already done above)
YES/NO
6.	In KY, the sequence is: permit contains chronic WET limit-if
WET test is failed, that's a violation & permit requires additional
chronic WET tests-if additional failures occur, permit requires
TIE/TRE-formal enforcement action occurs if TIE/TRE doesn't resolve
problem. Please describe on the back your sequence fir where formal
enforcement action (if any) would occur.
A-2

-------
Situation #2: POTW design flow is 2.2 MGD
Receiving stream critical low-flow to protect
aquatic life is 8.1 MGD.
1. would the permit contain: (circle all that apply)
an acute WET limit	(go to a. below)
a chronic WET limit	(go to a. below)
acute WET monitoring only (go to c. below)
chronic WET monitoring only (go to c. below)
no WET monitoring/limit (please explain)
a.	would a compliance schedule for the WET limit be given?
YES How long? 	 (go to b. below)
NO	(go to b. below)
b.	is each WET test failure a permit violation?
YES	(go to 2)
NO	(go to 2)
c.	if acute or chronic WET monitoring only is required,
when (if ever) would a limit be imposed?
(go to 2)
2.	Are multiple dilutions required?
YES/NO
3.	What test species are required? (circle all that apply)
Ceriodaphnia
Daphnia magna/pulex
fathead minnow
Other:
a. Does the permit allow for testing to be conducted on only
the more sensitive test species at some point during the
permit term?
YES/NO/NA
4.	Does the permit require additional WET tests after a WET test
failure?
YES/NO
5.	Does the permit require that a TIE/TRE be conducted? (provide
language if not already done above)
YES/NO
6.	In KY, the sequence is: permit contains chronic WET limit-if
WET test is failed, that's a violation & permit requires additional
chronic WET tests-if additional failures occur, permit requires
TIE/TRE-formal enforcement action occurs if TIE/TRE doesn't resolve
problem. Please describe on the back your sequence & where formal
enforcement action (if any) would occur.
A-3

-------
Situation #3	POTW design flow is 2.2 MGD
Receiving stream critical low-flow to protect
aquatic life is 3000 MGD.
1. would the permit contain: (circle all that apply)
an acute WET limit	(go to a. below)
a chronic WET limit	(go to a. below)
acute WET monitoring only (go to c. below)
chronic WET monitoring only (go to c. below)
no WET monitoring/limit (please explain)
a.	would a compliance schedule for the WET limit be given?
YES How long? 	 (go to b. below)
NO	(go to b. below)
b.	is each WET test failure a permit violation?
YES	(go to 2)
NO	(go to 2)
c.	if acute or chronic WET monitoring only is required,
when (if ever) would a limit be imposed?
(go to 2)
2.	Are multiple dilutions required?
YES/NO
3.	What test species are required? (circle all that apply)
Ceriodaphnia
Daphnia magna/pulex
fathead minnow
Other:
a. Does the permit allow for testing to be conducted on only
the more sensitive test species at some point during the
permit term?
YES/NO/NA
4.	Does the permit require additional WET tests after a WET test
failure?
YES/NO
5.	Does the permit require that a TIE/TRE be conducted? (provide
language if not already done above)
YES/NO
6.	In KY, the sequence is: permit contains chronic WET limit-af
WET test is failed, that's a violation & permit requires additional
chronic WET tests-if additional failures occur, permit requires
TIE/TRE-formal enforcement action occurs if TIE/TRE doesn't resolve
problem. Please describe on the back your sequence & where formal
enforcement action (if any) would occur.
A-4

-------
SUMMARY OF KY WET SURVEY
46 states require a minimum of 2 species (fish and invertebrate)
in doing WET testing
27 states allow for a compliance schedule
34 states require chronic WET limits; 14 of these 34 states
require both acute and chronic WET limits (where appropriate)
41 states consider each WET failure as a permit violation
25 states allow for testing of the most sensitive species after
some period of time
39 states require multiple dilution tests
38 states require additional tests after an initial failure
34 states - permit contains a TIE/TRE requirement if initial WET
failure occurs

-------
RESULTS OF A NATIONAL SURVEY ADDRESSING
WHOLE EFFLUENT TOXICTY (WET) REQUIREMENTS:
KENTUCKY DEPARTMENT FOR ENVIRONMENTAL PROTECTION
DIVISION OF WATER
BIOASSAY SECTION
14 REILLY ROAD
FRANKFORT, KENTUCKY 40601
This report has been appr
Date

-------
Results of a National Survey Addressing Whole Effluent
Toxicity (WET) Requirements
by
Charles A. Roth
Division of Water
Bioassay Section
March 13, 1995

-------
ACKNOWLEDGEMENT
I would like to thank Marshall Hyatt of EPA Region IV for his.
assistance in designing and distributing this survey. This effort
would not have been possible without his input.

-------
TABLE OF CONTENTS
Page
Acknowledgement		 iii
Introduction 		1
Results of the Survey	2
Tables: Summary of Survey Results
Table 1. States of EPA Region I	4
Table 2. States of EPA Region II	6
Table 3. States of EPA Region III	8
Table 4. States of EPA Region IV	11
Table 5. States of EPA Region V	15
Table 6. States of EPA Region VI	19
Table 7. States of EPA Region VII	21
Table 8. States of EPA Region VIII	23
Table 9. States of EPA Region IX	25
Table 10. States of EPA Region X	27
Figure 1: States Which Allow For WET
Compliance Schedules 		29
Figure 2: States Where Each Toxicity
Test Fail is a Permit Violation	30
Figure 3: States with WET Limits for a 2.2 MGD POTW
and a 0.0 MGD 7Q10 Receiving Stream	31
Figure 4: States with WET Limits for a 2.2 MGD POTW
and an 8.1 MGD Receiving Stream	32
Figure 5: States with WET Limits for a 2.2 MGD POTW
and a 3 000 MGD Receiving Stream	33
Appendix A: Kentucky Whole Effluent Toxicity Survey . . . A-l
iv

-------
INTRODUCTION
In August 1994, Kentucky with the assistance of EPA Region IV,
distributed nationwide a whole effluent toxicity (WET) survey. The
intent of this survey was to gain some knowledge of the way EPA
Regions and states were implementing their WET programs.
The survey was designed as a series of questions regarding any
WET requirements for a facility meeting the following basic
permitting situations/conditions:
- major municipal POTW with approved pretreatment
program.
freshwater discharge to a Fish & Wildlife
classified receiving stream, Tier 1 - no endangered
species; no outstanding resource waters,
no diffuser
this facility's expiring permit contains no WET
monitoring or limits. Their permit application
contains historical WET data and the permit writer
has concluded that reasonable potential to exceed
state water quality standards now exists.
The survey then identified three different scenarios involving
a POTW with a design flow of 2.2 mgd. Namely receiving stream
critical low-flows of:
1)	0.0 mgd
2)	8.1 mgd
3)	3000 mgd
Participants were then asked to respond to questions
describing their WET requirements for each of these situations.
These questions addressed issues such as permit limits, compliance
schedules, violations, test species and enforcement procedures.
This report is a summary of completed surveys received from
all 50 states plus the District of Columbia. Again the intent of
this survey was to provide an overall view of how WET programs are
being implemented nationally and how consistent these programs are
between states and EPA regions.
1

-------
RESULTS OF THE SURVEY
Programs relating to WET testing are present in every state as
well as the District of Columbia. The implementation of these
programs varies, ranging from recently promulgating regulations for
WET to well-defined WET strategies.
The manner in which effluent toxicity testing is incorporated
into individual permits does vary from state to state and EPA
region to region. WET requirements may be only a monitoring
requirement in some states, while a permit limit in other states.
Enforcement strategies also vary among states and regions.
Furthermore, toxicity reduction evaluations (TREs) are a permit
required response to effluent toxicity in some areas but are
addressed outside the permit in others.
There is some consistency in the types of tests conducted and
the test species used. Most states use EPA's acute and chronic
toxicity testing manuals. The water flea fCeriodaphnia dubia) and
the fathead minnow Pimephales promelas are the most commonly
utilized test species.
Several generalizations can be made from this study:
( Numbers include the District of Columbia)
~ 27 states allow for some type of compliance schedule
WET.
-	41 states consider each toxicity test failure a
permit violation.
-	36 states use 7Q10 as the receiving stream low-flow
measurement for determining the instream waste
concentration (IWC).
All states use Ceriodaphnia dubia and the fathead
minnow as the required freshwater test species.
~ 25 states allow for the testing of a most sensitive
species.
39 states require multiple concentration tests.
38 states require additional tests after an initial
failure.
~ 34 states utilize the TRE as a permit required
respdnse to a toxic effluent.
When given a specific permitting situation for WET, the
responses are summarized as follows:
1) Situation: POTW average design flow =2.2 mgd
receiving stream 7Q10 =0.0 mgd
7 states with acute limits
-	24 states with chronic limits
11 states with both acute & chronic limits
-	9 states with no WET limits or monitoring only
2

-------
2)
Situation: POTW average design flow = 2.2 mgd
Receiving stream 7Q10 = 8.1 mgd
6 states with acute limits
21 states with chronic limits
15 states with both acute & chronic limits
9 states with no WET limits or monitoring only
3) Situation: POTW average design flow = 2.2 mgd
Receiving stream 7Q10 = 3000 mgd
33 states with acute limits
3	states with chronic limits
4	states with both acute and chronic limits
11 states with no WET limits or monitoring only
A summary of WET requirements and conditions required by
individual states are presented in the following tables and
figures.
3

-------
TABLE 1: States of EPA Region I
SUIA/feYI XLil
STATE
Low
Compliance
Each Fall A
If Monitoring Only
Multiple Cone
Test
Sensitive
Addl Tests
Permit Requite
'EPA Issued
Flow
Schedule ?
Violation ?
When Limit ?
Reaulred ?
Species
Species
After Fall ?
TRE/TIE ?








		
		
REGION 1















CONNECTICUT
7Q10
Yes
Yes
If toxicity and TRE. then
No
OP
Yes
Yes
Yes


Up to 3 years

limit
Pass/Fall
FM

Relest wllhin
2 consecutive or





Definitive tests


30 days
3 annual violations





may be reaulred








to verify






•


compliance.






*







•MAINE
7QI0
No
Yes
NA
Yes
CD
Yes
No
No
•MASSACHUSETTS

Only ME WQSfds



FM


TRE required through
•NEW HAMPSHIRE

provide for schedule





308 Order or A O




















VERMONT
7QI0
Yes
Yes
If *reasanable potential*
Yes
CD
Yes
NO
Yes


If not In compliance

determined then limit.

FM
In certain
May be required



then schedule Is set.




situations
In TRE





















RHODE ISLAND
7Q10
No
No
NA
Yes
CD
Yes
No
Yes



Presently no WET


FM






enforcement









program.

















-------
TABLE 1: Continued.
SUKVtYtB KUi
U1
S1ATE
«l POTW 2 2 MGD—7QI0=00MGD
02 POTW 2 2 MGD— 7Q10-8 1 MGD
03 POTW 2 2 MGD—7Q10=3000MGD
"EPA Issued







REGION 1




Acute Umlt—NOAEl> 100*
Acuta Umlt-NOAEl>100%
Acute Umlt
CONNECTICUT
Chronic protection based on 5% of the LC50 the
Same as for situation 41
Actual allocation would determine llmll Unlikely

Noael Is assumed to be 1 /3 LC50 (ACR»6 66) At
Most POTWS have llmll of NOAEL* 100%,
loaet >1001--lf dlffuser then likely to get 100 I

IWC >5%-llmlt Is NOAEL> 100% Toxicity Is <90%
>67%. or monitoring.
dilution

survival where dilution li limited "No Klir al 100% off



Complianceaoo sla mortality at cone >or- IWCX20/3
•


up to 100%



"


• MAINE
Acute/Chronic limit
Acute/Chronic Umlt
Acute Umlt
'MASSACHUSETTS
1060-100%: C-NOEC>-fWC(l00%)
LC50-100%: C-NOEC>-IWC(21%)
LC50>-50%
•NEW HAMPSHIRE
quarterly testing.

2/yr testing.

SNC-Revlew last 6 test* by type: SNC If (1) >-3vlolattortt
and (2) result* are <- £0%. A.O. for TRE





VERMONF
Acuta Limit
Acute Umlt
Acute Umlt





If fall then: (1) priority pollutant metals analysis; (2) If cause b known, men report and ftc If cause unknown then TRE plan within 60 days No specific

language for formal enforcement action.






RHODE ISLAND
Acute/Chronic Umlt
Acute/Chronic Umlt
Acute Umlt





















-------
TABLE 2: States of Region II.
suuvtyz xls

STATE
low
Compliance
Each Fall A
If Monitoring Only
Multiple Cone.
Test
Sensitive
Addl Tests
Permit Require
'EPA Issued
Flow
Schedule 7
Violation 7
When Umll 1
Reaulred ?
Species
Species
After Fall 7
TRE/TIE 7










REGION II



















NEW JERSEY
7Q10
Yes
Yes
NA
Yes
CD
Yes
No
Yes


Minimum of 3 years
After 3 years


FM

part of TRE
2 falls/18 months


Proposing S years





Proposing more
Proooslng TRE








frequent testing
based on % of falls
			







prior to TRE
during addl testing











-







NEW YORK




Yes
CO
Yes
Yes
Yes






DM/DP





'



FM














WET monitoring applied when
1) substance Is present v/tth no criteria: (2) hlah natural background cones, preclude development of TMDl/WiA (3) for

substances where criteria are available. WQ8EU are below analytical detection: (4) effluent b chemically complex or synergistic; (5) aquatic

life Impairments observed Instream. ] I III


» one or more of these then: 1
fA acute testing: If 2 fallsfvear then chronic also: If continued falls then TRE; If still toxic then limit








































































-------
TABLE 2: Continued.
iUHV/fcY2b XLS
~-J
STATE
HI POIW2.2MGO—7Q\0-O.OMGD
#2 POtW 2.2 MGD—7Q10-8.1 MGD
S3 POTW 2.2 MGD—7QI0-3000MGD
•EPA Issued







REGION II







NEW JERSEY
Chronic Umlf-NOEC/IC25-IOO*
Chronic Umll-NOEC/IC25»22%
Acute Umll-I.C50> «S0%

Most sensitive endpolnl Is used
Most sensitive endpolnt Is used.
Minimum stofe standard




















NEW YORK
Acute/Chronic Monltortna
Acute Monltortna
No WET Monltortna or limit

Would consider limit In thli extreme ease.

Given pollution specific toxics control and dllutloi

NY would have itronaV suaaested oltemale site.

monitoring would only be required II one of the



6 previous conditions are met













































-------
TABLE 3: States of Region III.
iuuvtya xib
00
StATE
Low
Compliance
Each Fall A
It Monltortna Only
Multiple Cone
Test
Sensitive
Addi Tests
Pornil Require
'EPA Issued
Flow
Schedule ?
Violation 7
When Umll ?
Reaulred ?
Species
Species
After Fall ?
TRE/TIE ?









Yes
REGION III







	 - - -








DISTRICT OF
7QI0-ch
YES
Yes
Umit It monltortna
Yes
CD
Yes
Yes
COLUMBIA
IQIO-oc
3 Years

Indlcotes "reasonable

DM/DP

		
It toll twice In any on<




potential* as defined by

FM

monlh




talilna 2/4 tests.



































DELAWARE
7Q10-ch
Yes
Yes
Based on "reasonable
Yes
CD
Yes
Yes
Yes

IQlO-ac


potential'
If determined
DM/DP


If fall lest and one (1)





necessary by
FM


confirmation test





screenlna tests.
























MARYLAND
30Q5-ch
No
Yes
Bawd on "reasonable
Yes
CD
Yes
Yes
Yes

Acute not


potential."

DM/DP


If fall 2 tests In 12

based on




FM


months, then retest

stream







If fall retest. then TRE

flow.




























PENNSYLVANIA
7QI0
Yes
Yes
Based on "reasonable
Yes
CD
Yei
Yes
Yes


3 Years

potential.'

DM/OP









FM























WEST VIRGINIA
7Q10
No
No
NA
Yes
CD
Yes
Yes
Yes






DM/DP


2 falls for TRE






FM




-------
TABLE 3: Continued.
MJHVtYJ XLi


















No
Yes
VIRGINIA
7Q10-ch
Yes
Yes
Based on "reasonable
Yes
CD
DM/DP
Yes

IQlO-ac
4 yean
potential'






FM
















VO

-------
TABLE 3: Continued.
bUHVfcYUB *U>
STAIE
#1 POTW2 2MGO—-7Q10-OOMGO
#2 POTW2.2MGD—7Q10-B.I MGO
#3 POTW 2 2 MGD-—7Q10=3000MGD
"EPA Issued'







REGION til







DISTRICT OF
Umlf- based on NOEC-II "reasonable potential" Is demonstrated. Defined as falling 2 out of 4 tests Umlt expressed as TUa or TUc
COLUMBIA
Testing freauencv Is quartertv for the life of the permit.










DELAWARE
Chronic monitoring—NOEC
Acute/Chronic monitoring
Acute monltorlnQ—LC50


LC50/NOEC


If Initial test falls then 2 confirmation tests. If 1 confirmation test falls then TOE and possible limit.









MARYLAND
Acute/Chronic monitoring
Acute/Chronic monitoring
Acute/Chronic momltorlng

LC50 IWC then no more testing If It Is leu than IWC then retest. If NOEC or LC50 < IWC for 2 consecutive tests then TRE








WEST VIRGINIA
Acute/Chronic limit—LC60/NOEC > 100%
Acute/Chronic llmlt-LC50/NOEC >IWC{22*
Acute limit—LC50>°40%








VIRGINIA
Chionlc limit—TUc
Acute or Chronic limit-based on
Acute llmlt-TUa


'reasonable potential" analysis TUo oi TUc






Quarterly testlng-lf 6 of 8 tests (75%) exceed WLA then
TRE and WET limit


-------
TABLE 4: States of Region IV.
SUUVfcY4 xu>
SfATE
Low
Compliance
Each Fall A
It Monitoring Or\ty
Multiple Cone
Test
Sensitive
Addl Tests
Permit Require
TRE/TIE ? "
"EPA lisued
Flow
Schedule ?
Violation ?
When limit 1
Reaulred 1
Species
Species
After Fall ?










REGION IV







	








ALABAMA
76110-ch
No
Yes
NA
Yes-wlth
CD
No
No-Depl carweq
NoDept canreq

IQlO-ac



application
FM

addl tests If
TRE If problems





Now/OMR


problems exist.









Acute-1 /wk-4wks









Chronic-1 /wk-2wks




















'FLORIDA
7Q10
No
Yes
NA
Yes
CD
No
Yes-2 additional
No-may be required






FM

tests
under A O




















GEORGIA
7Q10
Yes
Yes
NA

CD
Yes








FM























KENTUCKY
7QI0
Yes-I year
Yes
NA
Yes
CD
Yes
Yes
Yes






FM

Accelerated
Depends on * and








testing-total of 6
degree of test falls








tests





















MISSISSIPPI
7Q10
Yes
Yes
Monitoring 1st year If no
Yes
CD
No
Yes
Yes

7Q2-slorm

data. Results compared
FM

Relest after tall
45 days after 2nd

water

to matrix to determine H




lest fall




limits applied.
















-------
TABLE 4: Continued
SUKVfcY4.XLS









NO
NORTH CAROLINA
7Q10
No
Yes
NA
No
CD
NA
Yes





But available
FM for
May
Quarterly testing
IRE Is considered






acute
request alt
goes to monthly o
loalcol response to







species If
single fall-back to
noncompliance







more
quarterly next pass







sensitive.






















SOUTH CAROLINA
7QI0
Yes
Yes
After 2 failures, may
No
CD
NA
Yes
Yes


Up to 3 years.

modify permit to Include




11 tesl falls then IRE




limit or Issue Consent









Order.

























TENNESSEE
3Q20
No
Yes-Acute
NA
Yes
CD
No
Yes
Yos

1020

No-Chronic permit


FM


SIq. fatl-4/5NOELor



Umll Is averaged.





LC60 2 consecutive









slfl. falls or 3 In 12









months-. IRE.









2years tof completlor











-------
TABLE 4: Continued.
bUHVfcY/Iti XLb
STAIE
fll POIW 2 2 MGO—7QI0=00MGD
#2 POTW 2 2 MGO—7QI0-6 1 MGD
03 POTW2 2MGD--7Q10-3000MGD
•EPA issued







REGION IV




Chronic limit-no tig. difference of 100%.
Chronic llmlt-no slo, difference at 22%.
Acute llmlf-LC50> 100% If dlffuser then IWC base
ALABAMA


on model. Umll <»I0% mortality at IWC ot ZIO












•flORlOA
Chronic llmlt-NOEC>"100%
Acute/Chronic limits
Acute limit


LC50>)00%: NOEC>-2)%
LC50>IC0%












GEORGIA
Chronic Umll-NOEC>-100%
Chronic llmlt-NO£C>-2l%
No llmlt/monlforln(j?












KENTUCKY
Chronic Um!(—TUc» 1.0
Chronic llmlt-TUc«4 76
Acute llmll-TUai 1 0

IC25>»l009f>
IC25>-21%
LC50>-!00%








MISSISSIPPI
Chronic limit
Chronic limit
Acute limit

IC25>-100%
1C25>»2I%
IC50>-IWC X 3 not lo exceed 100%








NORHT CAROUNA
Chronic limit
Chronic limit
Acute limit

(IWC) Max llmlt-90%

No dlftuseMlmll=90% Dlffuser-allowance for



diffused cone on cose-by-case basis

For IWC>- 1%-Chronlc limit (NC chloric tests or NC phase II chronic procedure) No observed Inhibition In reproduction or sla mortality at IWC

For lWC0.25%-48- hour Oaphnld acute test Umlt-LC60>»l00 X IWC.


For lWC<«0.25%-24 hour minnow acute test. No sla. mortality pass/fall test


-------
TABLE 4: Continued.
MJIWfcY4U.XLS








SOUTH CAROLINA
Chronic limit
Acute/Chronic limit
Acute limit

Sfo. difference at IWC
Slo. difference ot IWC.
Pau/Fall





Wllh no tilffuser: If IWC 0-10% then Acute



If IWC 10-60% then Acute and Chronic


l( IWC <30-10096 then Chronic










TENNESSEE
Acute/Chronic limit
Acute/Chronic limit
No limit

ocute—500 1

chionlc-
-------
TABLE 5: States of Region V.
SiUUVtYi XLb
STATE
Low
Compliance
Each Foil A
l( Monitoring Only
Multiple Cone.
Tes1
Sensitive
Addl Tesls
Permit Require
*EPA Issued'
Flow
Schedule ?
Violation ?
When Limit 7
Reaulred ?
Species
Species
After Fall ?
TIJEAIE ?








	
Yes
REGION V
















ILLINOIS
7Q10
NA
NA
No limit
Yes
CD
Yes
Yes




only monltorlnn

FM

If 2 months fail
If 2 months fall then






SC-aiaae


possible TRE




















OHIO
7Q10-ch
Yes
Ye*
Once demonstrated
Yei
CD
Yes
No
No

JOQlO-oc
3 yeais

WET Is severe and

FM

Maybe required
Maybe required




consistent then TRE and



under A O
under A O




limit.















INDIANA
7Q10
NA
NA
If toxicity Is demonstrate)
No
CD
Yes
Yes
Yes




then WET limit and/or
pass/fall
FM

Follow-up test
If fall any 2 tests




limits on additional
Definitive as


after 1st (oil





toxicants.
follow-up to









screen fall.
























MINNESOTA
7Q10
yes
Yes
NA
Yes
CD
No
Yes
Yes


1 to 3 years



FM

3 additional tests
It 2 or more tolls.








after I si fall





















WISCONSIN
76210/4
Yes
Yes
If terrible toxicity problen
Yes
CD
No
Yes
Yes


2 to 3 years

then permit mod for

0M/0P

2 retests at each
If persistent toxicity



•
WET limit. If persistent

FM

foil
(fall permit test and




toxicity then WET at




2 retests) then TRE




reissuance.




before limit Is









effective

-------
TABLE 5: Continued.
bUUVfcYb xu>




















MICHIGAN
Lowest
Yes
Ye»
If monltorino and TRE,
Yet
CO or
Yes
No
No

of 12 mo.
iveois
For acute
then limit at tame time as
DM/DP

Howevei. It no llml
Bui con be leaulred

flows
case-by-case.
For ctvortc If month!
TRE of after TRE

FM

and test falls then
on case-by-case

exceeded
average exceeded.
comDletton (3 veara).



Additional monll
basis If limit Is

95% of






required.
exceeded

the time.



















-------
TABLE 5: Continued.
bUNVfcYSb XUs
STATE
#1 POIW 2.2 MGD—7Q10-0 OMGO
12 POIW 2 2 MGD—7QIO-8.1 MGD
»3 POTW2 2 MGD—7QI0-3000MGD
"EPA Issued







REGION V




Acute monltortna
Acute monltortna
Acute monltoilno
ILLINOIS




Dilution*-100,1-ocutefl-C50); Dilution* 100:1 -Chronlc(NOEC). Chronic monltortna mav be reaulred at < 100:1 dilution on case-by-cose boils

Frequency Is 1 /month for 6 months. If toxicity then additional requirements or limits (numerical for specific toxicants) may be Imposed

Can uis blosurveyi.










OHIO
Monltor1no-acute(.3TUa), chronlcO TUc).
Monltorlna-ocutetf 1 %), chronlc(21 %).
Monltorlna-acule(409TUa).chronlc( 1364 6!Uc)





Acute criteria It ,3TUo, chronic crlterlo Is l.OTUc. (acute-LCSO; chrontc-NOEC). WET rnontlorlno lor T veorfmonthly-ocule. quarlerly-chronlc). II WET Is

severe and consistent then TRE by Director's Flndlnas & Orders (F&O). 3 yeors compliance. Additional WET monltortna In F&O or permit modification

with WET limits.










INDIANA
Chronic monltortno-NOEC
Chronic monltorlna-NOEC
Acute monltorlna-LCSO





Frequency and duration depend upon WET tests submitted with application and/or facility history. Acute or chronic toxicity after permit Issued then

follow-up tests. If any 2 tests ton then TRE. Reopener allows for WET limits and/or Dmlts for additional toxicants. Formal enforcement If TRE falls

to solve problem.










MINNESOTA
Chronic limit
Chronic limit
Acute limit

TUc-1 IC25 or NOAEL
TUc-1.05 IC25 or NOAEL.
TUo-1 LC50





Frequency Is 4 tests In tsl 6 months. If no toxicity then ao to I per year. TRE considered formal enforcement action

-------
TABLE 5: Continued.
SUKVfcYbtl.XUS








WISCONSIN
Would probably not allow a facility of this ilze to locate
Acute/Chronic monitoring,
Acute/Chronic monltoilna

on Ihli itfoom.







IfdltuHonc- 100:1 then acute ond chronic monllorlna
Currently very few permlti with limit*.









MICHIGAN
Acute/Chronic limit
Acute/Chronic limit
Acute limit

TUo»IO:TUc-t.O
TUa« 1,0: IVIc« 1.9(53%)
TUa-1.0


Standards allow mbdna wtth no more than



25% of low flow.






-------
TABLE 6: States of EPA Region VI.
bUUVtYft xu>
 above
Same as obove
Same
Some
Same as obove
Some as above
•NEW MEXICO
4Q3
•
•
•
•
•
•
•
•
'OKLAHOMA
7 Q2
•
•
•
•
•
•
¦
•
•TEXAS
7Q2
S
»
¦
•
¦
¦
•
•

























































































































-------
TABLE 6: Continued.
SUHVtVOH xu>
STATE
#1 POTW2 2MGD—7QI0-0.0MGD
#2 POTW2.2 MGD—7QIO-0 1 MGD
#3 POTW 2 2 MGD-~7QI0=3000MGD
"EPA Issued







REGION VI







ARKANSAS
Chionlc llmU
Chronic limit
Chronic limit








¦LOUISIANA
Chronic limit
Chronic limit
Chronic limit
•NEW MEXICO
Chronic limit
Chronic limit
Chronic limit
•OKLAHOMA
Chronic limit
Chronic limit
Acute limit
•TEXAS
Acute limit
Chronic limit
Acute limit









All mojors and minors with known toxic potential are first requited to perform monitoring test* only. If permittee falls 'survivor portion of either acute

or chronic test, additional testing Is requited. If toxicity persists (1 of 2 or 3 additional tests In the following 4S-60 days) a TRE Is triggered (26 months

allowed for completion). At the conclusion of the TRE the permit Is modified to Include WET limit at critical dilution for lethality only * May be given

comDllance schedule of up to 3 years. Eoch failure is a violation and ft acted upon by the enforcement branch.









.































-------
TABLE 7: States of EPA Region VII.	suuvly/xib
STATE
Low
Compliance
Each foil A
If Monllorlna Only
Multiple Cone
Test
SertsHlve
Addi Tests
Peimll Requite
"EPA Issued
Flow
Schedule 7
Violation ?
When Umll 7
fteaulred ?
Soecles
Species
After Foil ?
THE/TIE 7










REGION VII



















IOWA
7QI0
No
Yei
no limits at this lime
NO
CD
No
Yei
Yes






FM

Mulllconcenlratlor
If fan screen ana








test after screen
follow-up test








loll.





















KANSAS
Soma
Same as above
Same as above
Same as above
Same as above
Same
Same
Same oj above
Same os above
Missouri
•
¦
•
•
•
KS allows
•
¦
•
NEBRASKA
•
»
•
•
¦
DP
¦
¦
¦



































































































































-------
TABLE 7: Continued.
SUHVfcY/UXLS
to
N>
STATE
M POTW2 1 MGD—7QIO=OOMGD
#2 POTW 2.2 MSD—7Q10-8.1 MGD
03 POTW 2.2 MGD—-7QI0o3000MGD
"EPA Issued.







REGION VII







IOWA
Acute limit
Acute limit
Acute limit
KANSAS
Acute limit
Acute limit
Acute limit
MISSOURI
Acute limit
Acute limit
Acute limit
NEBRASKA
Acute limit
Acute limit
Acute limit









Tvolcolly streams with 7G10 <1 .Ocfs are unclassified and numeric standards are not applied. EPA Is working to ensure at least acute standards are

applied In these streams. Most stales are applying WET as acute monitoring only. Chronic limits are not being required as most state standards

do not require Imposition of chronic limits. Some states are proposing downgrading itreams where chronic toxicity Is becoming an Issue

[ t

For classified .streams, mixing zone is 25% of the 7Q10. Z1D Is 1/10 of the mWng zone(2.S% of 7Q10). Effluent must show no staHstlticalty significant acut

toxicity (or 0.3 TUa) at edae of ZlD. Dilution for screenln
3 test Is based on design flow mMng with allowed flow In the 2JD. this approach does not

work for effluent dominated itreams.



























-------
TABLE 8:
States of EPA Region VIII.
SUNVtYB XL!)
STATE
Low
Compliance
Each Fall A
If Monitor! na Only
Multiple Cone.
Tett
Seruilhve
Addl Tests
Permit Reautre
•EPA issued.
Flow
Schedule 7
Violation ?
When Umit ?
Reaulred ?
SdocIos
SDeclet
After Fall ?
TRErtlE ?










REGION VIII



















COLORADO
3003
Yes
Yet
Monitoring may be
Yet
CD
No
Yes
Yes


up to 3 yean

ImDOsed with orovlilon

FM
After 1






of limit IF toxicity ti shown.

Quarters of









no toxicity.









may ao to









alternating









ipeclet.






















MONTANA
7QI0
Some oi above
Same at above
Some at above
Yet-oc,No-ch
Same
Same
Same
Yes
NORTH DAKOTA
7QI0
Same at above
Same at above
Same as above
Ye»-ac,No-ch
Same
Same
Same
Yes
SOUTH DAKOTA
7Q5
Same oi above
Same as above
Same as above
Yei-oc,No-ch
Same
Some
Some
Yes
UTAH
7€310
Same oi above
Same as above
Same as above
Yet-ac.No-ch
Same
Some
Some
Yet
WYOMING
7Q10
Same o» above
Same as above
Same at above
Yet-oc,No-ch
Same
Same
Some
NO







































































-------
TABLE 8: Continued.
suuvtYtm XLb
NJ
STATE
#1 POTW 2 2 MGD—7Q10»O.OMGD
#2 POTW 2.2 MGD—7QIO=>8.1 MGD
#3 POTW 2 2 MGD— 7QI0-3000MGD
"EPA Issued:







REGION VIII







COLORADO
Chronic limit
Chronic limit
Acute limit

No statistically sla. difference.
No statistical! sla difference.
LC50

(acute limit If lower classified stream)
(acute Omit If lower classified stream)




'" 	 — - - 	

.


MONTANA
Same as above
Same as above
Same as above "
NORTH DAKOTA
•
¦
• ~ .
SOUTH DAKOTA
•
•
•
UTAH
•
¦
•
WVOMING
¦
•
•



1







• •



'

























-------
TABLE 9: States of EPA Region IX.
SUNVbVV XIS
NJ
L/l
STATE
Low
Compliance
Each Fall A
If Monltorlna Only
Multiple Cone
Test
Sensitive
Addi Tests
Permit Reauire
'EPA Issued.
Flow
Schedule ?
Violation ?
When Umlt ?
Reaulred ?
Species
Species
After Foil ?
TRE/TIE 7










REGION IX



















CALIFORNIA

No
No
NA
Yes
CD
Yes-after
Yes
Yes



Monthly median or


FM
screen wltr
With monlhly
Based on number



2 tests In X time


SC-oiaae
3 chronic
median can do 3
of falls



pe (tod or 2



tests for 3
tests within o month



consecutive falls



months,
or Increased









testing offer first









fall





















ARIZONA

Some ot above
Some os above
Same as above
Same as above
Same
Some
Some as above
Same os above
HAWAII

•
•
•
¦
•
•
¦
•
NEVADA

•
•
t
•
•
•
¦
¦







































































-------
TABLE 9: Continued.
SUUVfcYVB Xlh
STATE
»1 POTW 2.2 MGD—7QIO-O.OMGD
#2 POTW 2.2 MGD—7Q10-8. t MGO
03 POTW 2.2 MGD—7QIO-3OO0MGD
'EPA luued'







REGION IX







CALIFORNIA
Acuta and/or Chronic limit
Acute ond/o< Chronic limit
Acute and/or Chronic limit

Chronlc-NOEC baled on 1) ony monthly median of
Bated on 20% of streom now


2) ony one test result areolar frion £0% effect.










ARIZONA
Soma as above
Same oi obove
Same ot above
HAWAII
II
•
»
NEVADA
•
•
¦

















































-------
TABLE 10: States of EPA Region X.
SUUVtY IU XLS
M
STATE
Compliance
Each Foil A
If Monitoring Only
Multiple Cone
Sensitive
Addl Tests
Permit Require
Issued
Schedule 7
violation 7
When Umlt 7
Required 7
Species
Species
After Fall 7
TRE/TIE 7
REGION X
ALASKA
7QI0-Ch
Not In permit
Cose-by-Cose
IQlO-ac
Compliance order
o biweekly tests
If oil 6 chronics foil
or 1 shows acute
necessary.
toxicity
•IDAHO
2SXW.
Not In permit
Case-by-Cose
25%vol
Compliance order
6 biweekly tests
If all 6
chronics
at 7Q10-C
If necessary.
or I shows acule
iQiO-a
Oregon
SEE NEXT PAGE
WASHINGTON
SEE NEXT PAGE

-------
TABLE 10: Continued.
!>UUVtYI I.XLb
STATE
#1 POTW 2 2 MGD—7Q10=0 OMGD
#2 POTW 2 2 MGD—7Q10-8 1 MGD
M POTW 2 2 MGD—7Q10°3000MGD
*EPA Issued







REGION X







•ALASKA
Chronic limit
Chronic Omit
Acute limit

NOEC-100%
NOEC-21%
IC50.



Alaska has no acute criteria (or toxicity

Frequency Is quarterly. No enforcement policy for Wei.



Aj of 7/2S/94 Region X has not luued any WWTP



permit? with WET limit!



-


•IDAHO
Chronic limit
Chronic limit
Acute Limit

NOEC-100%
NOEC-21%
LCSO








OREGON
No permits with WET limits. All malors do Include provisions for monitoring. If fall 2 consecutive tests then submit plan to meet water quality standards

(may or may not be THE). Reaulre dye studies to determine dilution ratios to determine "reasonable potential1 It toxicity then permit may be

reopened to Include Omit. Chronic and acute tests-no statistically slanlflcant difference at IWC.









WASHINGTON
Recently promulflated new reaulations to Implement WET














-------
Figure 1: States Which Allow for WET Compliance
Schedules

-------
Figure 2: States Where Each Toxicity Test
Fail is a Permit Violation

-------
Figure 3:
States With WET Limits for a 2.2 MGD POTW
and a 0.0 MGD Receiving Stream
Limits
Acute (7)
Chronic (24)
HUB Both (11)
I None (9)

-------
u>
ro
Figure 4: States With WET Limits for a 2.2 MGD POTW
and an 8.1 MGD Receiving Stream

Llmts
Acute (6)
Chronic (21)
Both (15)
None (9)

-------
Figure 5: States With WET Limits for a 2.2 MGD POTW
and a 3000 MGD Receiving Stream
Limits
Acute (33)
Chronic (3)
Both (4)
None (11)

-------
BIOCIDE/CHEMICAL TREATMENT
WORKSHEET-FORM 101
The following calculations are to be performed on any biocidal products ultimately discharged to the surface waters of
North Carolina. This worksheet must be completed separately for each biocidal product in use. This worksheet is to be
returned with all appropriate data entered into the designated areas with calculations performed as indicated.
Facility Name.
NPDES # NC.
County	
Receiving Stream 				__7Q10	(cfs)
(All above information supplied by the Division of Environmental Management)
What is the Average Daily Discharge (A.D.D.) volume of the water handling systems to the receiving water body?
A.D.D. =	(in M.G.D.)
Please calculate the Instream Waste Concentration (IWC in percent) of this discharge using the data entered
above.
nA/r	(A.D.D.) x 100	( ) X 100
(7Q10 x 0.646) +(A.D.D.) ~( )(0.646) +	(	)	
=	%
This value (IWC) represents the waste concentration to the receiving stream during low flow conditions.
What is the name of the whole product chemical treatment proposed for use in the discharge identified in Part I?
Please list the active ingredients and percent composition:
			—				%
			—		%
			—			%
			—				%
					 	%
Note: If ingredients contain the metals copper, chromium, or zinc, also complete page 4. Supplemental Metals Analysis
What feed or dosage rate (D.R.) is used in this application? The units must be converted to grams of whole
product used per day.
D.R = 	grams/day
D.E.M. Form 101 (7/92)
1

-------
Facility Name:
NPDES #: NC.
Estimate total volume of the water handling system between entry of biocidal product and NPDES discharge
point On an attached sheet please provide justification for this estimate (system volume, average cycles pei
blowdown, holding lagoon size, etc.)
Volume= 	million gallons
What is the pH of the handling system prior to biocide addition? If unknown, enter N/A. 	
What is the decay rate (D.K.) of the product? If unknown, assume no decay (D.K-O) and proceed to asterisk.
The degradation must be stated at pH level within 1/2 pH standard unit within handling system. Enter the half
life (Half Life is the time required for the initial product to degrade to half of its original concentration). Please
provide copies of the sources of this data.
H.L. =	Days
The decay rate is equal to X 0.69 =	=Decay Rate (D.K)
Calculate degradation factor (D.F.). This is the first order loss coefficient.
DF- - + (D-K-' = j—1:	) + ( )=	
Calculate Steady State Discharge Concentration:
DischgCone. = p p j(Vdume)(3785) = (	Si	^ )(37B5) = 	mg"
Calculate concentration of biocide instream during low flow conditions.
(Receiving Stream Concentration)
(Dischg. Cone) x (IWC%)	(	) x f	1 _	n
100	=	100	" 	
Receiving Stream Concentration
III. Calculate regulated limitation.
List all LC50 data available for the whole product according to the following columns. (Note that units should be
in mg/l). Please provide copies of the sources of this data.
Organism	Test Duration	LC50(mg1)
D.E.M. Form 101 (7/92)	2

-------
Facility Name:
NPDES #: NC
Choose the lowest LC50 listed above:
Enter the LC50: 	
If the half life (H.L.) is less than 4 days, perform the following calculation.
Regulated Limitation = 0.05 x LC50 = 	mg/l
If the half life (H.L.) is greater than 4 days or unknown, perform the following calculation.
Regulated Limitation = 0.01 x LC50 = 	mg/l
Choose the appropriate regulated limitation from the calculations immediately above and place in this blank:
	mg/liter
From Part II enter the receiving stream concentration:
	mg/liter
IV. Analysis.
If the receiving stream concentration is greater than the calculated regulated limitation, then this biocide is
unacceptable for use.
Name (Print)
Signature	Date
Person in Responsible Charge
D.E.M. Form 101 (7/92)
3

-------
Facility Name:
NPDE& #: NC.
Supplemental Metals Analysis
If copper, zinc, or chromium are present in the proposed biocidal compound, complete this worksheet A
separate form must be used for each metal and/or metal compound present in the biocide. List the metal,
its chemical formula, molecular weight (MW), formula weight (FW), and the concentration of the metal
compound in the biocide (MCC). Complete a separate form for every metal present in the biocide.
Metal	Cherniwl Fprmul? Molecular Weight of Metal Form^a Weight
EXAMPLE
Copper	CuSOj5H£)	63.546 g/mole	249.680 gemote
Concentration in Biocide
0.2%
Dosage rate of Biocide (DR) (from page 1):
DR =	grams/day
Average Daily Discharge (ADD) (from page 1):
ADD = 	million gallons/day
Discharge Concentration (DC) of Biocide:
DR	(	orams/dav)
DC =
ADD " (_
.million gallons/day)
Convert DC to micrograms/liter (ppb):
DC (ng/l) =	DC (grams/million gal) x
1 x 106 ud/q
.grams/million gallons
3.785 x 106 liters/million gal.
jig/l
Calculate the fraction of metal in the metal-containing compound (MF):
FW (_
.grams/mole)
_grams/moIe)
Calculate the fraction of metal in the biocidal compound (BF):
%
BF = MF x-MSSp.
(100)
Calculate the concentration of metal in the discharge (M):
M = DCx BF = 	ng/l x 	 = 	
-H9/1
Calculate the instream metal concentration (IMC) at low-flow conditions:
IMC = M x IWC (%) =
100
jig/i x
%
100

Regulated limitation of metal (from below):
Jig/I
NC General Statutes 15A NCAC 2B.0211 deline:
Copper- 7 jig/1 water quahty action level* Zinc- SO (ig/I water quality action level*
Chromium- 50 p.g/1 water quality standard
('Values which exceed action levels must be addressed directly by aquatic toxicity testing.)
D.E.M. Form 101 (7/92)	4

-------
NATIONAL WET
COMPLIANCE/ENFORCEMENT
PERSPECTIVE

-------
WET Compliance and
Enforcement: EPA's
Perspective
¦	Overview of EPA National policy and
guidance
¦	Common issues raised by regulated
community
¦	Examples of EPA Regional approaches
to WET violations
Talking Points:
¦	This portion of the workshop will provide an overview of
EPA's National policy and guidance relating to the
enforcement of WET permit requirements.
¦	This module will focus on the two most common enforcement
issues raised regarding WET and EPA's response.
¦	Examples of EPA Regional and State approaches to WET
enforcement will be provided.
¦	A State representative will then discuss how their State
addresses WET compliance and enforcement.
2

-------
Overview of National Policy
and Guidance
1989 "Whole Effluent Toxicity Basic Permitting
Principles and Enforcement Strategy"
-	Permits must be protective of water quality
and written to ensure enforceability.
-	Any violation of a WET limit should be
addressed promptly, and it may or may not
result in a formal action.
-	A toxicity reduction evaluation (TRE) can be
required through permit condition or
enforcement action.
Talking Points:
1989 "Whole Effluent Toxicity Basic Permitting Principles and Enforcement Strategy"
•	Strategy was the result of a Regional/State workgroup and the goal was to promote
consistency in WET permit requirements and enforcement
•	2 key permitting principles:
1)	Permits must be protective of WQ
-	all majors, and minors of concern should be considered for WET requirements
-	WET limits should be included where necessary to meet WQS
2)	Permits must be written to ensure enforceability
-	limits should be included in Part I of permit
-	specifics {protocols, species, frequency of tests) should be spelled out
•	Enforcement principles:
1)	Permittees are responsible for attaining, monitoring, and maintaining compliance
2)	Regulators will evaluate self-monitoring data quality
3)	Regulators will assess compliance through inspections, audits, and data reviews
4)	Regulators will enforce effluent limits and compliance schedules to eliminate
toxicity
•	Requiring a TRE may be an appropriate response - if included in an enforcement
action and where corrective action and a final compliance date are required (see
Region IV examples provided in handouts).
3

-------
1989 "Enforcement
Management System"
¦	States that any violation of an NPDES permit is
a violation of the CWA for which EPA
encourages some type of enforcement response
¦	Recommends an escalating response to
continuing violations of any parameter
¦	Includes recommended enforcement responses
for various types of violations -- WET examples:
-	infrequent violation of a WET limit, causing no known
harm: letter of violation or AO
-	known harm or continuing violations: AO, APO, or
judicial action
Talking Points:
•	The Enforcement Management System is EPA's comprehensive NPDES
compliance and enforcement guidance which encourages timely and
appropriate enforcement action for any violation. It includes a response
guide for various types of violations. The EMS recommends that an
appropriate response be determined within 30 days of the identification of
any violation. The EMS was updated in 1989, and WET violations were
added to the response guide.
•	EPA treats WET like any other parameter and recommends timely and
appropriate action for WET violations. All relevant facts—including
magnitude, frequency, and duration of violation, environmental harm, and
compliance history of the facility—are considered in determining an
appropriate enforcement response. The response should reflect the nature
and severity of the violation. For example, although EPA generally
recommends an escalating response to continuing violations, a first-time
violation causing significant environmental harm may result in a formal
action with a penalty. EPA's recommended response to an infrequent
violation of a WET limit causing no known harm is a "letter of violation"
(informal action) or an "Administrative Order" (formal action but does not
include a penalty). A stronger response—administrative order,
administrative penalty order, or judicial action with penalty—is
recommended for infrequent violations causing harm or for continuing
violations.
4

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Enforcement Issues
Commonly Raised by
Regulated Community
¦	Single exceedance of WET limit should
not be considered a violation
¦	Facilities with inconclusive TRE results
should get relief
Talking Points:
•	Single exceedance of WET limit should not be a violation
-	Section 309 of CWA says any violation of a permit requirement is a
violation of the Act and is subject to enforcement. EPA recommends
timely and appropriate action for any permit violation, taking into
consideration all the relevant facts-magnitude, frequency, and
duration of the violation, impact on public health or the environment,
and compliance history of the facility.
•	Some facilities with inconclusive TREs should get relief
-the 1989 WET permitting and enforcement strategy does say that
technical assistance and civil penalty relief may be warranted in a
few highly unusual cases where a facility is otherwise in compliance,
has made a good faith effort to conduct a TRE, but fails to attain
compliance with its WET limits
-	the Agency considers all relevant facts—including a facility's good
faith effort to conduct a TRE—in determining an appropriate
enforcement response
•	The Office of Water and Office of Regulatory Enforcement jointly issued a
guidance memo last August to specifically address these issues.
5

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August 14, 1995 EPA National
Policy Memorandum
¦	Issued jointly by OWM and ORE to clarify
existing policy re: single exceedances and
inconclusive TREs
¦	Section 309 of CWA says any violation of permit
limit or condition is subject to enforcement;
EPA's guidance recommends an escalating
response to continuing violations
¦	EPA's guidance does not recommend that the
initial response to a single exceedance of a WET
limit, causing no known harm, be a formal
enforcement action with a civil penalty

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August 14, 1995 EPA National
Policy Memorandum (cont.)
¦	Section 505 of the CWA allows citizens
to sue anyone alleged to be In violation
- case law
¦	OW has provided funds to SETAC to
establish an independent expert panel
Talking Points:
•	This memo reiterated EPA's existing guidance:
-	EPA generally recommends an escalating response to continuing
violations: our guidance does NOT recommend that the initial
response to a single exceedance of a WET limit causing no known
harm - be a formal enforcement action with a civil penalty.
-	Case law suggests that citizens cannot sue a permittee on the
basis of a single violation of a permit limit. In Gwaltney of
Smithfield, Ltd. v. Chesapeake Bay Foundation, Inc., the Supreme
Court held a requirement that citizen-plaintiffs allege a state of
either continuous or intermittent violation.
•	Memo committed EPA to providing technical support to facilities with
inconclusive TREs. Office of Water is supporting a number of activities
to address technical WET issues:
-1995 SETAC Pellston WET conference
-	implementation meetings
-	training workshops
-funding to SETAC for independent expert panel to provide
technical assistance to permittees on issues such as inconclusive
TREs
-	AMSA/WEF compilation of TIE/TRE case histories
7

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Examples of Regional
Approaches to WET Violations
¦ Regions IX and X use an escalating
enforcement response to continuing
violations.
Talking Points:
• Regions IX and X
-	If toxicity as defined in the permit is detected, then the
permittee shall conduct six more tests, bi-weekly (every two
weeks), over a twelve-week period. If implementation of the
TRE workplan indicates the source of toxicity (for instance, a
temporary plant upset), then only one additional test is
necessary. If any of the test during the accelerated period
demonstrates toxicity, the permittee should initiate a TRE/TIE.
The TRE could be as simple as investigating housekeeping or
chemical usage or something more complicated.
-	Chapter 5 on Enforcement Guidelines for WET violations in the
Interim Final Regions 9 and 10 Guidance for Implementing
Whole Effluent Toxicity Testing Program, dated May 31,
1996, highlights the Regions' enforcement responses.
8

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WET COMPLIANCE
AND ENFORCEMENT:
A STATE PERSPECTIVE

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¦
Consistency
¦
Permittee/Regulator Interaction
¦
Permittee's Responsibility
¦
Enforcement/Enforcement Relief
¦
Complete the Process
¦
Preliminary Activities
¦
In-Depth Activities
Talking Points:
•	Establish communications between regulator and regulated.
Develop an iterative approach to toxicity reduction. This will
require knowledgeable staff.
•	Compliance must be established as the permittee's
responsibility. The regulator should refrain from defining the
path to compliance.
•	A fair system of regulatory relief (e.g. AO) should credit
responsible movement toward compliance.
•	How to start the process of toxicity reduction.
•	In-depth follow-up activities in a TRE.

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Handling WET Non-compliance
¦ Treat same as other parameters
- treating compliance with WET limits the
same as other parameters to the greatest
extent possible will add the strength of
consistency and precedent to the activity
Talking Points:
•Treating WET consistently with other parameters will lessen
the need for specific support of approaches.
•	The farther you move away from existing compliance and
enforcement strategies, the more you move into new
problems. Permittees are familiar with the existing systems
and are uncomfortable with unknown thresholds for
enforcement.
•	Rigidly defined enforcement activities such as automatic civil
penalties create a political climate which disfavors the
programs leading to those actions.
•	Response to WET violation should be certain and rapid yet
equitable and realistic.
3

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Regulatory Relief
¦	How to handle extended periods of
noncompliance?
¦	AOs/Consent Orders/Decrees
¦	Incentives to solve problem
Talking Points:
•	Intermittent violations of WET limits (which do not cause
measurable instream effects) should receive some regulatory
response and likewise, response from the permittee. These
events may be difficult to control outside of preventative
measures.
•	Extended non-compliance may reasonably require an extended
period of evaluation and problem solving. The regulatory
process must acknowledge and accommodate this.
•	Formal regulatory relief should be a reasonably available option
to allow a facility to achieve WET compliance. This should be
an interactive process with defined responsibilities for both
parties.
•	Incentives to solve non-compliance should include a
reasonable opportunity to extend relief but the regulator must
retain the option for stipulated penalty for failure to move
decisively toward compliance.
4

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Permittee Education
¦	Face to face outline of problems/
solutions
¦	Written guidance
¦	Contractor selection
Talking Points:
•	The regulator should consider permittee education about WET
and the toxicity reduction process not only an important
responsibility but also a valuable opportunity to proactively
reduce future questions and problems.
•	Several examples exist of educational resources about WET
(EPA methods manuals, NC toxicity information packet) as
well as toxicity reduction (EPA TIE & TRE manuals, NC toxicity
reduction guidance document).
•	The information provided should contain significant guidance
on quality assurance and methods by which to judge data
quality (EPA toxicity laboratory evaluation manual).
•	Laboratory certification/validation programs will frequently not
address TRE experience. Guidance provided should address
suggestions/criteria for selection of experienced toxicity
reduction consultants.
5

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Compliance is Permittee
Responsibility
¦	Establish that the permittee is in non-
compliance and that compliance is expected
¦	Emphasize it is the permittee's responsibility
to achieve compliance
¦	Need to let permittee define their own path
¦	The permittee should develop and present
the means by which compliance will be
achieved
Talking Points:
•	At the occurrence of a non-compliant event, the situation and
liability should be brought immediately to the attention of the
permittee. A notice of violation (NOV) can be used to establish
the situation and expectations.
•	The permittee should be made aware that liability for
compliance rests on them.
•	Consistent with other parameters, the solution for compliance
should be left up to the permittee. The regulator should remain
involved by providing guidance and evaluation of the propriety
of steps proposed.
6

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Regulatory Activities
¦	Initiate enforcement
¦	Require immediate response to non-
compliance, e.g. TRE plan, more frequent
testing
¦	Regulatory follow-up on TRE logic
¦	Periodic update and evaluation of progress
reports
¦	Allow plans to work
¦	Requiring complete follow-through
¦	Feedback loop
Talking Points:
•	The regulator should establish responsibilities, expectations,
and provide continued feedback on whether actions being
taken are appropriate.
•	If not already a permit requirement, the regulator can suggest
increased monitoring frequency to minimize the potential
duration of non-compliance.
•	If non-compliance continues, the regulator should initiate
discussion of regulatory relief (if appropriate) and establish
that this as an available option.
•	The regulator should remain active in the feedback loop during
evaluation and toxicity reduction, allow reasonable time for
activities to occur, and insure completion of needed/proposed
activities.
7

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Preliminary TRE Activities
¦	What is a TRE and when is it used?
¦	Important elements of a TRE
¦	Logical starting place is a thorough
information gathering phase.
¦	Preliminary investigation and
information evaluation of potential
causes/sources of toxicity, effluent
variability, treatment system
efficiency, etc.
Talking Points:
•	The definition of a TRE varies widely. NC interprets a TRE as
any actions taken to resolve WET non-compliance. Thus, a
TRE is expected following a single event.
•	A TRE can take many paths but all should include a logical
pathway that ends only with compliance. Several guidance
documents are available which list important elements.
•	A significant amount of information can be gathered by a
permittee about possible causes of toxicity before introduction
of consultants to the toxicity reduction process. This work is
frequently viewed as an acceptable first response to non-
compliant events. Not surprisingly, this increased scrutiny of
wastes entering the treatment system and the treatment
process itself frequently results in a reduction of toxicity.
•	It will also be problematic and expensive for a consultant to
begin toxicity identification work prior to treatment process
being stabilized and optimized.
8

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For POTWs This Could Include:
¦	The NPDES permit
- what are the potential toxicants identified in
the permitting process?
¦	Treatment system design criteria, flow
diagrams, descriptions of treatment
elements
¦	Is the system operating up to design
specifications, e.g. removal efficiencies?
Talking Points:
•	The NPDES permit and history of compliance with established
limits, particularly toxic parameters, can give useful clues
about potential toxicants.
•	What are the possible physical sources of wastewater? What
are the major contributions in volume? What are the most
likely toxicant sources?
•	Is the treatment facility operating at designed efficiency?
9

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For POTWs This Could
Include (cont.)
¦	Sources and relative contributions of
wastewater?
¦	Influent and effluent flow data
¦	How do flows relate to observed toxicity?
¦	Effluent toxicity data and trends and
correlation with other effluent data
¦	Short or long term variation in toxicity
related to flow, rainfall, industrial user
operating schedules, seasons.
Talking Points:
•	How do measured parameters (flow, loads, concentrations)
vary with observed toxicity. How is this related to various
contributions?
•	Were SlUs open/closed during significant toxicity? Have operating
schedules or processes of SlUs changed? Are climatic events
coinciding with toxicity/lack of toxicity?
10

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For POTWs This Could
Include (cont.)
¦	Process control and operational data and
histories
¦	In-plant chemical usage (polymers,
coagulants, chlorine)
¦	Treatment upset histories and reports
¦	Pretreatment information
Talking Points:
•	Is sufficient information available about chemical inventory/use
by SlUs?
•	Is toxicity being added in-plant or by in-plant processes?
11

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For POTWs This Could
Include (cont.)
¦	Industrial waste surveys
¦	Industrial user self-monitoring reports
¦	Industrial user operational schedules and
flow patterns
¦	Waste hauler monitoring and manifests
¦	Hazardous waste inventories
Talking Points:
• Is toxicity related to non-SIU contributions (waste haulers,
commercial operations, domestic sources)?
12

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Initial Review for Industrials
¦	Housekeeping
¦	Process and wastewater generating
process diagrams and descriptions
¦	Diagrams and descriptions of non-
process wastewater sources( cooling
towers, boilers, floor drains)
¦	In-plant flow records and water usage
Talking Points:
•	industrial facilities usually will have better knowledge and
control of wastewater sources and composition.
•	Housekeeping is often a frequent first step to insure that
wastewater is what is believed. Often not the case!
•	Evaluation of sources
•	Evaluation of composition
•	Evaluation of treatability of chemicals
•	Evaluation of monitoring data
13

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Initial Review for Industrials
(cont.)
Chemical labels, MSDS, and toxicity
information
WWTP operational data and histories
Wastewater monitoring records
(chemical and toxicity)
Chemical inventories and usage
records

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Initial Review for Industrials
(cont.)
¦	Chemical treatability information (e.g.
BRI Aquatox® Approach)
¦	Production records
¦	Operating schedules with emphasis
on how these schedules affect
wastewater flow/composition
¦	Effluent toxicity data and trends and
correlation with other effluent data

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Continued TRE Activities
¦	Waste reduction/reuse opportunities
-	many states now have programs designed to
provide free technical assistance to facilities to
assist with reduction, re-use, recycling.
¦	Date logs
-	maintain log of activities, treatment changes,
chemical use, etc. to correlate with changes in
toxicity
¦	Other innovative approaches-e.g. toxicity
treatability of process chemicals
Talking Points:
•	NC has non-regulatory, free-of-charge pollution prevention,
waste reduction, reuse audits.
•	Careful record keeping to correlate changes in process,
operations, chemicals, toxicity reduction activities, with
observed toxicity.
•	Generation of databases on chemical treatability-risk
assessment/risk management approaches to inventory.
•	IMPORTANT- Solutions to toxicity should become SOP. TRE
activities should not be once in time, but rather, continuing
activities. It's expensive to do it all over!
•	Above activities Show up over and over in difficult success
stories.

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Handling TIEs
¦	Regulatory requirements
-	advantages disadvantages from
permittees perspective
¦	Where to end TIE?
-	is there a need to extend TIE and/or TRE
beyond the point of compliance?
Talking Points:
•	From the perspective of the permittee, a required TIE
represents a shift of responsibility. If established as an only
means to the end of compliance, failure of a strictly defined
TIE to solve the problem shifts liability to the regulator. TIEs
are tools and should only be part of the toolbox.
•	TIEs/TREs are being required that extend past the point of
regained compliance.
•	The TIE/TRE should not be established as an end in and of
itself. It should only be a part of the enforcement process as
long as non-compliance exists. Compliance is the goal. Once
compliant, the regulator has no real need for the TIE/TRE to
continue. Again, it is the liability of the discharger to solve
toxicity. It is most likely beneficial to the permittee to identify
causative toxicants but frequently this may not occur even
though toxicity diminishes.
17

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TIE - Toxicity Identification
Evaluation
¦	Acute/chronic
¦	Characterization
¦	Identification
¦	Confirmation
Talking Points:
•	The TIE is a process of characterization, identification, and
confirmation of causative toxicants in a complex waste
through a process of chemical/physical manipulations of
samples.
•	While causative toxicants may be identified through a trial-
and-error approach, the cost of doing such may quickly
surpass the cost of switching to a more organized approach
like the EPA TIE.
•	Permittees will frequently rely on existing consultants
(frequently wastewater engineering firms) to solve toxicity
problems. Caution should be given about the different kinds of
considerations necessary in toxicity reduction and the benefits
of association with firms with TRE experience.
18

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Chemicals identified as
causing the toxicity are:
Zinc, nickel, copper
Ammonia
Surfactants
Chlorine
Salinity (TDS)
Diazinon, chlorofenvinphos, dichlorvos,
carbofuran, malathion

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Cationic Metals as Suspect
Toxicant(s) When:
Toxicity reduced by EDTA additions
Toxicity reduced by sodium thiosulfate
additions
Toxicity reduced by C18 SPE column
and toxicity not recovered in methanol
eluate
Erratic dose response

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Cationic Metals as Suspect
Toxicant(s) When (continued)
¦	Graduated pH test shows toxicity
differences
¦	Toxicity removed by filtration and
recovered in pH 3 dilution water
extract
¦	Toxicity removed by cation exchange
resin (e.g., zeolite)

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Ammonia is Suspect Toxicant
When:
¦	Toxicity reduced with low pH's
¦	Toxicity is greater to fathead minnows
than Ceriodaphnia
¦	Post-zeolite column effluent is non-
toxic and ammonia levels are reduced
¦	Large surface/volume air-stripping
reduces toxicity
¦	Total ammonia measured is >5 mg/L

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Oxidants are Suspect
Toxicant(s) When:
¦	Toxicity reduced by sodium thiosulfate
additions
¦	Toxicity reduced by aeration
¦	Toxicity of the sample less over time
¦	C. Dubia are more sensitive than fathead
minnows
¦	0.1 mg/L at the effluent LC50
¦	Toxicity reduced by addition of S02-
saturated water

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Phase I TIE - Characterization
¦ Chemical/physical manipulations of
samples to characterize classes of
toxicants, behavior of toxicants
-e.g., pH adjustment, aeration, C18 solid
phase extraction, filtration
Talking Points:
•	Chemical and/or physical characteristics or behaviors of
toxicants or classes of toxicants are characterized in Phase I
TIEs.
•	The results of these tests are often indications of what
toxicants are not likely. This can be as informative as the
inverse.
24

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Phase II TIE - Identification
¦	Further analyses to narrow in on specific
toxicant(s), help ID multiple toxicants
¦	Filtration
¦	SPE Fractions
¦	Ammonia-grad. pH, zeolite, air stripping
¦	Metals-EDTA, sodium thiosulfate, grad.
PH
Talking Points:
•	Either by the lack of results in Phase I or through identification
of suspect toxicants, more closely refined procedures are used
at Phase II to provide more well defined identification.
•	Innovative or ad hoc approaches become more frequently used
at this stage.
25

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Phase III TIE - Confirmation
¦ Confirm that identified toxicant(s) are
correct
-	sample spiking
-	correlation
-	symptoms
-	mass balance
Talking Points:
•	Once suspect toxicants have been identified, Phase III
procedures are used in an attempt to confirm. A variety of
approaches may be attempted to replicate effects through
surrogate sample spiking, to mathematically approximate
similar results from calculated toxicity of the compound(s), or
to account for observed toxicity through mass balance
approaches.
•	This stage is helpful in identifying the presence of masked
toxicants that may still cause problems when primary
toxicants are removed/reduced. Multiple toxicants are
frequently identified or implicated in non-compliant situations.
26

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Chronic TIEs
¦	EPA has basic methods published
¦	Performed regularly
¦	More subtle effects than acute
¦	Tiered to make efficient use of time
¦	Innovative Approaches
- experiments designed ad hoc to answer
specific questions
Talking Points:
•	Past argument has been made by permittees that chronic TIE
methods were not published and thus TREs for chronic toxicity
was not possible. This again emphasizes the importance of
establishing permittee liability for compliance. Through that
same period, experienced consultants were routinely assessing
and solving chronic toxicity problems by developing innovative
tools.
•	Currently, EPA has published chronic TIE methods: very similar
to acute approaches. However, these are only one tool
among many to achieve compliance.
27

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Treatability
¦	Cost/benefit decisions between source
reduction and treatment
¦	Modifications to existing
works/processes
¦	Bench/Pilot modifications
¦	Innovative Approaches
- current vs. future availability of
processes/products/chemicals and
regulatory requirements
Talking Points:
•	While source reduction of toxicants should be the preferred
method of toxicity reduction, a concurrent or alternative
approach is the assessment of the treatment of toxicity. Past
complaints have been raised by the discharging community
that WWTPs were not designed to remove toxicity. While this
may be true, many plants, particularly biological facilities do a
tremendously good job of accomplishing just that.
•	Cost/benefit decisions often drive decisions between source
reduction or treatment.
•	Paths taken to address treatability most often take the form of
either trials of modified treatment procedures in existing
works, or the evaluation of different procedures or works
through bench or pilot scale simulation.
•	Some innovative approaches have looked at pushing existing
technology or chemicals and existing cost/benefit paradigms.
28

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POTWs-Different Approaches
¦	RTAs
-	bench simulations of treatment process (Act.
Sludge) to determine toxicity pass-through
¦	Pretreatment Toxicity Limits
-	treatability
-	effects on flow moratoria
¦	Public Education
-	environmental and cost considerations of
household waste disposal practices
-	consideration of hazardous waste collection
activities
Talking Points:
•	Though useful in some industrial settings, refractory toxicity
assessments (RTAs) are most commonly applied in POTW
TREs which generally have less control over source reduction.
RTA approaches are necessary to evaluate whether toxicity
entering a plant is treated by existing processes and/or to
simulate treatability of proposed waste sources.
•	NC has required RTA procedures to evaluate the effect of
additional proposed industrial waste in POTW situations that
otherwise would remain under flow addition moratorium.
•	Public education is becoming a more frequently utilized tool in
POTW toxicity reduction evaluations. Commonly used
household pesticides like diazinon have been implicated as
toxicity sources in some POTWs. Educating the public about
environmental and even monetary costs of improper disposal
of toxic substances is a logical response to reduce this diffuse
source. Current public sentiment about the environment makes
it that much more likely such an approach can work.
29

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Take Home Points
Handle compliance consistent with other
limits
-	don't set up a system that will need its own
defense
Compliance is the liability of the
permittee
-	don't assume the responsibility of designing
and supporting the TRE
-	success or failure is the responsibility of the
permittee and not suggested methods.
-	support innovative approaches

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Take Home Points (cont.)
Emphasize a process with periodic
feedback and allow it to work
Support a logical, step-wise process of
toxicity reduction that will follow through
to successful completion

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GENERIC TOXICITY REDUCTION EVALUATION WORKPLAN (TRE)
INDUSTRIAL
Information and Data Acquisition
a.	Regulatory information
i.	NPDES permit limits
ii.	Trigger
b.	Facility monitoring data
i.	NPDES monitoring data
ii.	In-house monitoring data
iii.	State agency monitoring data
c.	Plant and Process Description
i.	Process and treatment plant description
(1)	numbers and types of streams
(2)	their size
(3)	scheduled changes or events in process stream operation
(4)	types and configurations of equipment
(5)	flow equalization facilities
(6)	records of treatment plant upsets
ii.	Physical/chemical monitoring data
(1)	chemical analyses of process streams
(2)	physical/chemical analyses of treatment streams
Housekeeping
a.	Initiation of housekeeping study
i.	Identify areas which may contribute to toxicity
ii.	Reduce these contributions through best management practices (BMPs),
administrative, and procedural controls
b.	Evaluation of housekeeping practices
i.	Review of plant policies
ii.	"Walk-through" inspection
c.	Identification of potential problem areas
i.	Probability of release of toxic material
ii.	Type and frequency of release which may occur
iii.	Quantity of toxic substances involved
iv Toxicity of substances released
v.	Potential downstream impact of the substances released
vi.	Effect of release on final effluent
d.	Identification of corrective measures
i.	Area cleanup
ii.	Process or operational changes
iii.	Material loss collection and recovery
iv.	Chemical and biological testing of contained waters prior to release from
diked storage areas
v.	Increased storage capacity for contained waters
vi.	Equipment modifications or changes
e.	Selection of corrective measures
f.	Implementation of corrective measures
Treatment Plant Optimization
a. Evaluation of influent wastestreams
1

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i.	Raw chemicals or materials used in the process
ii.	Byproducts or reaction products produced during the process
iii.	Reaction vessels, valves, piping systems, overflow points, and other
mechanical aspects of the system
iv.	Wastestreams produced, volumes, and routing paths
v.	Non-point sources
Description and evaluation of the treatment system
i.	Design basis for each constituent, including variability in flow conditions and
concentrations
ii.	Treatment sequence
iii.	Performance projections by constituents
iv.	Operational flexibility of each process
v.	Treatment objectives and projected effluent standards
Analysis of treatment system operation
i.	Flow loading
ii.	Mass loading
iii.	Frequency and impact of shock loadings
(1)	normal cleaning and maintenance
(2)	spills and upsets
iv.	Changes in operating procedures
4. Chemical optimization
a.	Information gathering
i.	Examination of wastestreams produced by specific production processes
ii.	Chemicals and raw materials and their contaminants and by-products used in
the process
iii.	Chemicals used in treatment
iv.	Chemicals and material use rates
v.	Percentage of chemical in final product
vi.	Chemical reuse and waste recycling activities
b.	Process chemical review
i.	List all chemicals used
ii.	List all quantities
iii.	Determine pounds per product
iv.	Determine pounds per gallon of wastewater discharged
c.	MSDS information review
i.	Obtain MSDS for all process chemicals discharged
ii.	Highlight MSDS sections on aquatic toxicity
iii.	Examine Hazardous Ingredient section and note "hazardous substances" listed
iv.	Categorize all chemicals by hazard and irritation potential and use standard
references to obtain aquatic toxicity information, if possible
d.	Chemical composition screen of incoming raw materials
e.	Outcome of chemical optimization phase
i.	List of all chemicals used in processing and manufacturing the product
ii.	MSDS and literature reviews will be on file when needed
iii.	List of all chemicals and raw material purchased on a monthly basis and a
record of production volumes during the same time period
b.
c.
2

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GENERIC TOXICITY REDUCTION EVALUATION (TRE) WORKPLAN
POTW
1. Information and Data Acquisition
a. Operations and performance review
i.	NPDES permit requirements
(1)	Effluent limitations
(2)	Special Conditions
(3)	Monitoring data and compliance history
ii.	POTW design criteria
(1)	Hydraulic loading capacities
(2)	Pollutant loading capacities
(3)	Biodegradation kinetics calculations/assumptions
iii.	Influent and effluent conventional pollutant data
(1)	Biochemical oxygen demand (BODs)
(2)	Chemical oxygen demand (COD)
(3)	Suspended solids (SS)
(4)	Ammonia
(5)	Residual chlorine
(6)	pH
iv.	Process control data
(1)	Primary sedimentation-hydraulic loading capacity and BOD ans SS
removal
(2)	Activated sludge-Food -to-raicroorganism (F/M) ratio, mean cell
residence time (MCRT), mixed liquor suspended solids (MLSS),
sludge yield, and BOD and COD removal
(3)	Secondary clarification-hydraulic and solids loading capacity, sludge
volume index and sludge blanket depth
v.	Operations information
(1)	Operating logs
(2)	Standard operating procedures
(3)	Operations and maintenance practices
vi.	Process sidestream characterization data
(1)	Sludge processing sidestreams
(2)	Tertiary filter backwash
(3)	Cooling water
vii.	Combined sewer overflow (CSO) bypass data
(1)	Frequency
(2)	Volume
viii.	Chemical coagulant usage for wastewater treatment and sludge processing
(1)	Polymer
(2)	Ferric chloride
(3)	Alum
b. POTW influent and effluent characterization data
i.	Toxicity
ii.	Priority pollutants
iii.	Hazardous pollutants
iv.	SARA 313 pollutants
1

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v. Other chemical-specific monitoring results
c.	Sewage residuals (raw, digested, thickened and dewatered sludge and incinerator ash)
characterization data
i.	EP toxicity
ii.	Toxicity Characteristic Leaching Procedure (TCLP)
iii.	Chemical analysis
d.	Industrial waste survey (IWS)
i. Information on IUs with categorical standards or local limits and other
significant non-categorical IUs
ii Number of IUs
iii.	Discharge flow
iv.	Standard Industrial Classification (SIC) code
v.	Wastewater flow
(1)	Types and concentrations of pollutants in the discharge
(2)	Products manufactured
vi.	Description of pretreatment facilities and operating practices
vii.	Annual pretreatment report
viii.	Schematic of sewer collection system
ix.	POTW monitoring data
(1)	Discharge characterization data
(2)	Spill prevention and control procedures
(3)	Hazardous waste generation
x.	IU self-monitoring data
(1)	Description of operations
(2)	Flow measurements
(3)	Discharge characterization data
(4)	Notice of slug loading
(5)	Compliance schedule (if out of compliance)
xi.	Technically based local limits compliance reports
xii.	Waste hauler monitoring data and manifests
xiii.	Evidence of POTW treatment interferences (i.e., biological process inhibition)
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INDUSTRIAL CASE STUDIES
These are a few industrial TRE case studies prepared by the Texas Natural Resource Conservation
Commission (TNRCC). The TNRCC does not mandate that permittees utilize any particular TRE
protocol. They found that most permittees began the TRE process using EPA protocols and later
modify these protocols as necessary to accommodate the TRE findings. Overall, the TNRCC's
experience monitoring TREs has been educational and positive They observed several complicating
events or planning problems in many of these TREs. The following list of TRE
shortcomings/complications will be useful to environmental managers and consultants involved in
future TREs.
•	Failure to collect adequate sample volume necessary to perform chemical analysis and
characterization tests in the event that a biomonitoring sample is toxic.
•	Failure to follow-up with characterization tests when an effluent sample is acutely or
chronically toxic.
•	Failure to correlate the presence or absence of toxicity with operational changes
•	Inability to interpret multiple characterization test results.
•	Devoting unnecessary time and effort to studies of potential surrogate test species.
•	Complications due to infrequent toxicity.
•	Limiting the TRE effort to routine biomonitoring tests.
•	Failure to utilize abbreviated screening tests to track effluent toxicity when routine
biomonitoring tests are not required.
•	Failure to recognize patterns of toxicity.
•	Failure to scrutinize artificial sea salts for toxic contaminants.
Phillips Petroleum Company
This refinery and petrochemical complex is located near Sweeny in Brazoria County, Texas. The
permit issued on September 27, 1990, required the permittee to conduct the chronic 7-day survival
and reproduction test with the water flea, Ceriodaphnia dubia and the chronic 7-day larval survival
and growth test with the fathead minnow, Pimephales promelas using samples from outfall 001. A
September 15, 1991, permit amendment retained this requirement. Treated process wastewater is
discharged at this outfall. The discharge enters Linnville Bayou which flows to Caney Creek. The
defined critical dilution is 98% effluent. The dilution series defined in the permit is 6,12, 25, 49 and
98% effluent.
This facility began a TRE effort in December 1989 as a requirement of its NPDES permit Both test
organisms demonstrated sensitivity to the effluent. From October 1990 through May 1993, 13 of 29
Ceriodaphnia dubia tests demonstrated statistically significant mortality at the critical dilution. Of the
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failed tests, survival NOEC values ranged between 12 and 50% effluent. Where survival was not
affected at the critical dilution, 15 out of 16 C. dubia tests demonstrated statistically significant
reproduction effects at the critical dilution.
From October 1990 through May 1993, 19 of 28 fathead minnow tests demonstrated statistically
significant mortality at the critical dilution. Of the failed tests, survival NOEC values ranged between
6 and 50% effluent. Where survival was not affected at the critical dilution, 4 out of 9 tests
demonstrated statistically significant growth effects at the critical dilution.
Characterization tests conducted between November 1991 and March 1992 indicated that effluent
toxicity was attributable to three sources: (1) chloride, (2) ammonia, (3) one or more organic
chemicals. Continued Ceriodaphnia reproduction effects were attributed to effluent chloride levels
(approximately 700 - 800 mg/L). For this reason, Phillips is now beginning an effort to evaluate the
ionic makeup of the effluent. In recent characterization studies, effluent toxicity to fathead minnow
was removed by solid phase extraction with a C18 resin. Phillips has considered napthenic acids as a
possible cause of toxicity although information thus far has not been conclusive A powdered
activated carbon treatment pilot plant test and powdered activated carbon tests effectively controlled
the toxicity due to the unknown organic constituent(s).
Effluent toxicity and ammonia levels have decreased over the past year. Phillips attributes this
success to a number of waste improvement projects throughout the refinery. Additionally, Phillips
began operating a new waste water treatment system in April 1993 (2-staged activated sludge system
with a ZIMPRO powdered activated carbon process).
Bell Helicopter Textron. Inc.
This facility manufactures components for the aircraft industry and assembles complete helicopters in
Fort Worth, Texas. The permit issued on November 14, 1991, required the permittee to conduct the
chronic 7-day survival and reproduction test with the water flea, Ceriodaphnia dubia and the chronic
7-day larval survival and growth test with the fathead minnow, Pimephales promelas using samples
from outfall 001. Waste streams permitted at this outfall include air conditioning condensate and
stormwater runoff. The discharge enters a railroad ditch which enters Valley View Branch, which
flows to Walker Branch, which enters the West Fork of the Trinity River. The defined critical
dilution is 76% effluent. The dilution series defined in the permit is 59, 67, 76, 86 and 98%
effluent.
The November 1991 permit recognized that Bell Helicopter had already initiated the TRE process
since the NPDES permit effective in September 1991, specified a WET limit. The Ceriodaphnia was
the most sensitive species tested. NOEC values for Pimephales survival ranged between < 6 and
98% for 22 tests between October 1991, and June 1993. The TNRCC database reflects only one
statistically significant survival failure at the critical dilution However, Bell Helicopter's historical
biomonitoring data collected as a result of earlier federal requirements was not reflected in the
TNRCC database. Six fathead minnow tests demonstrated statistically significant growth effects at the
critical dilution. For tests that were conducted from October 1991 through June 1993, only 1 of 21
Ceriodaphnia dubia survival tests revealed statistically significant effects at the critical dilution.
Under the TRE effort, Bell Helicopter implemented rigorous outside housekeeping improvements.
Bell Helicopter began washing fleet vehicles off-site, plugged storm drains near potential
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contamination sources such as chemical and hazardous waste storage areas, improved housekeeping
and containment for raw material drum storage areas, and covered and installed containment sumps.
Bell Helicopter has recently implemented a stormwater pollution prevention plan. Statistically
significant effluent toxicity has not been demonstrated for a year and a half. A single EDTA
characterization chelation test performed in January 1992 failed to yield significant information as
about the effluent toxicity. A permit amendment issued on July 30, 1993, specifies a WET limit that
goes into effect m July 1994.
Intercontinental Terminals Company (TTO
This bulk liquids storage terminal and commercial waste water treatment facility is located in Deer
Park. The permit issued on March 21, 1990, required the permittee to conduct the acute static
renewal 48-hour test with the Atlantic mysid, Mysidopsis bahia and the acute static renewal 48-hour
with the sheepshead minnow, Cyprinodon variegatus using samples from outfall 002. Treated
industrial wastewater is discharged at this outfall. The discharge enters drainage ditches that flow to
Tucker Bayou which enters the Houston Ship Channel The defined critical dilution is 30% effluent.
The dilution series defined in the permit is 11, 18, 30, 50 and 83% effluent.
This facility began the TRE effort in January 1991. The mysid has been the most sensitive species
tested. From June 1990 through June 1993, 31 of 43 Mysidopsis bahia tests demonstrated statistically
significant mortality at the critical dilution. The majority of the NOEC values were less than 11%
effluent. Since, October 1992, the Mysidopsis bahia test for survival passed at the 30% critical
dilution (NOECs of 50 and 83% effluent). From June 1990 through June 1993, only 3 of 41
Cyprinodon variegatus tests demonstrated statistically significant mortality at the critical dilution.
Five initial characterization efforts in 1991 were inconclusive No particular class of chemicals was
implicated as a probable cause of effluent toxicity. ITC then launched a program of source
segregation where various waste streams were routed away from the treatment system to determine if
elimination of the segregated stream resulted in a reduction of effluent toxicity. ITC isolated various
third party streams and in-plant wastewaters. The program revealed that a particular third party
stream treated at ITC's facility was highly toxic. ITC ceased accepting this third party stream m June
1992. Since then, test results have demonstrated a continuous reduction in effluent toxicity. ITC
reports that there have been no other operational changes since removal of the suspected third party
stream.
Central Power and Light - J.L. Bates Station
This steam electric station is located near the City of Mission in Hidalgo County, Texas. The permit
issued on March 22, 1988, required the permittee to conduct the chronic 7-day survival and
reproduction test with the water flea, Ceriodaphnia dubia and the chronic 7-day larval survival and
growth test using the fathead minnow, Pimephcdes promelas, using samples from outfall 001. Waste
streams permitted at this outfall include cooling tower blowdown, low volume wastewater, metal
cleaning wastes, and storm water runoff. Hie discharge enters a drainage ditch which flows to the
Arroyo Colorado. The defined critical dilution is 100% effluent. The dilution series defined in the
permit is 6.25, 12.5, 25, 50 and 100% effluent.
This facility began the TRE effort in June 1989. Effluent toxicity based on survival was intermittent
throughout this TRE effort. The water flea was the most sensitive species tested. Ceriodaphnia
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dubia survival NOEC values ranged between 6 and 100% effluent for 34 tests between June 1988,
and December 1992. Eleven Ceriodaphnia tests demonstrated statistically significant mortality at the
critical dilution. Thirty Ceriodaphnia tests demonstrated statistically significant reproduction effects
at the critical dilution. Test results have revealed statistically significant mortality for only 2 of 26
fathead minnow tests conducted between June 1988 and August 1990. Growth effects at the critical
dilution were indicated in 3 of these tests.
This TRE has been complicated by intermittent lethal toxicity sometimes associated with turn-around
events. Recent TRE findings have indicated several probable effluent toxicants. Probable sources of
toxicity include: 1) tributyltin (TBT) used in periodic cooling tower treatment, 2) water treatment
process polymers, and 3) copper originating within the steam cycle system.
A January 1993, effluent sample revealed significant lethality to Ceriodaphnia. Subsequent
investigation revealed that the cooling tower was treated with TBT in December 1992, and that the
Unit 1 cooling tower was drained while the January 1993, biomonitoring sample was collected.
Chemical analyses of the effluent indicated a whole effluent TBT concentration of 1.696 ppb.
Interestingly, the TBT concentration determined in a filtered effluent sample was 0.541 ppb.
Characterization tests revealed that toxicity was removed by filtration at every pH. Toxicity was also
reduced in samples passed through the Ci8 Solid Phase Extraction (SPE) column Past TBT
treatments appear to correlate well with past toxicity events.
Methanol elution of the SPE column failed to recover a toxic fraction. CP&L believes that this
characteristic is indicative of surfactant or polymer behavior. CP&L reports that it is possible that the
January 1993 effluent sample contained one of two polymers used for water treatment. These are
Chemlink IPC 6115 (which contains formaldehyde as a component) and Betz Polymer 1192. CP&L
indicated that backwash from the water treatment filtration unit may accumulate in the cooling tower
Unit 1 basin when the unit is not operating.
Based on this information, CP&L performed 48-hour acute range-finding tests on non-toxic effluent
dosed with the suspect polymers. No acute toxicity was demonstrated with IPC6115. At
concentrations of 10 and 100 mg/L, Betz polymer 1192 was acutely toxic to C. dubia. Reproduction
effects were apparent at concentrations above 0.63 mg/L. CP&L suspects that the maximum expected
effluent concentration for this polymer should be somewhere between 1 and 10 mg/L.
CP&L recently conducted waste stream surveys to investigate sources of copper within the plant.
Primary copper sources are indicated within the boiler and boiler cooling circuits. Unit 1 copper
levels are consistently higher that those associated with Unit 2 Because the condenser for the Unit 1
boiler contains brass tubes (copper and nickel), CP&L representatives speculate that copper may
readily go into solution at the low pH (6.7) of the cooling water. Smce nickel and zinc are
consistently present in the final effluent, CP&L continues to evaluate their potential contribution to
the overall effluent toxicity.
MUNICIPAL CASE STUDIES
The California San Francisco Regional Water Quality Control Board (Regional Water Board) in
Oakland, California supplied information for various POTWs in the San Francisco Bay Area. The
Regional Water Board has revised many of the NPDES permits for POTWs and some industries to
include self-implementing TEE language. Permittees are required to call the Regional Water Board if
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they have any violations and then they are to follow up the call by letter or by including the notice
with their discharge monitoring reports (DMRs). The Regional Water Board has found generally had
good cooperation from the facilities.. About eight POTWs and more than four refineries have
performed at least a Phase I TIE. One POTW has completed a Phase HI, confirmation study. The
various studies conducted at facilities in Regional Board indicated probable causes of toxicity as the
pesticide diazinon, ammonia, possible poor lab quality assurance, hardness, and methods used for
culturing test organisms.
Central Contra Costa Sanitary District CCCCSD1
CCCSD began a lib investigation in early 1992, and completed the Phase HI confirmation study in
early 1994. The primary cause of toxicity was found to be diazinon. As an effort to reduce the
toxicity from diazinon, the district recently began a public information campaign describing how
homeowners and others should use and dispose of diazinon to lessen the environmental impacts
In performing the studies, the CCCSD found that if high conductivity (or TDS) is a suspected
toxicant, then it is useful to compare the toxicity of nitrified samples to de-nitrified samples. If the
toxicities of nitrified and denitrified samples were not different, then TDS would not account for the
difference in toxicity. In addition, as a control for conductivity effects, CCCSD increased the
conductivity of the lowest concentration of the combined effluent to the level found in the 100 percent
concentration of the combined solution. Then CCCSD compared the conductivity of the concurrent
reference toxicant tests to the 100 percent effluent concentration. If the values in the reference
toxicant tests were well above the 100 percent effluent concentration, conductivity was eliminated as a
suspect toxicant.
Also a metabolic inhibitor, piperonyl butoxide was used to prevent the activation of indirect acting
organophosphates (OPs) such as diazinon to their toxic form. This is one test to help identify the
presence of OP toxicity in effluents. CCCSD also concluded that analytical methods with detection
limits under 0.1 ug/L are needed to detect OPs in effluent matrix.
City of South San Francisco
The City of South San Francisco initiated a Phase I TIE m September 1992. Their contractor
modified the EPA TIE methods by using a Q, instead of C18 column for the SPE tests. The
contractor had previously found that some of the nonpolar organics do not elute from C18 columns
even with 100 percent methanol. After performing the initial Phase I tests, the contractor identified
that toxicity may have been related to the sodium meta-bisulfite used to dechlorinate the effluent. The
facility adjusted their dosing of the bisulfite and came back into compliance with their toxicity limit
East Bay Dischargers Authority (EBPA")
The results of an initial Phase I TIE study for EBDA indicated nonpolar organics as possible causes
of toxicity. Because of the high level of toxicity to Ceriodaphnia dubia, EBDA concluded that any
further chemical analyses should also target nitrogen, OPs, and sulfur-based pesticides.
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City of Palo Alto
After submitting and following a detailed TIE study plan, the facility identified that toxicity was
caused by hardness effects on the green alga Selenasrrum capri cornutum. The study consisted of
toxicity characterization, POTW performance evaluation, TIE, toxicity source evaluation, in-plant
control evaluation, toxicity control selection, and control implementation with follow up monitoring.
In exploring the toxicity to Selenasrrum, the facility found that metals, anions and elevated hardness
play major roles. Other tests performed suggested that zinc was the prime suspect in metal toxicity.
In performing the aeration tests, the facility found reduced toxicity at pH 11. Toxicity was eliminated
at pH 3. This could mean that toxicity was caused by compounds volatile under acidic conditions
(e.g., hydrogen sulfide) or by short chain acidic organics. Alternatively, the results could mean that
under acidic conditions, insoluble precipitates are formed and that this reaction is catalyzed by the
mixing associated with aeration.
In addition, a loss of toxicity via aeration may also be caused by surfactants. In order to evaluate that
possibility, the facility redissolved residual materials in the aeration vessels in clean water and then
tested for toxicity. Upon finding no toxicity, the facility concluded that surfactants were not a cause
of toxicity.
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TIE/TRE BIBLIOGRAPHY
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State of North Carolina
Department of Environment,
Health and Natural Resources
Jomes B. Hunt, Jr., Governor
Jonathan B. Howes, Secretary
Steven J. Levitas, Deputy Secretary
WHOLE EFFLUENT TOXICITY REDUCTION GUIDANCE r». 9/*
This document has been prepared by the N.C. Water Quality Section's Aquatic Survey and Toxicology
Unit to aid NPDES permittees, their consultants, and state regulatory staff with the development of whole
effluent toxicity identification and reduction plans. As has been the operating strategy of the Water Quality
Section in the past, this document will not attempt to present a strictly defined procedure to accomplish
toxicity reduction, as such defined processes limit the ability to design programs custom fitted to the
situation of each discharge. Rather, this document will attempt to present a series of logical steps that can
be initiated to help track effluent toxicity sources, trends, and investigate possible avenues of toxicity
reduction. Nearly all steps presented herein have originally been described by documents published by the
U.S. Environmental Protection Agency including:
Mount, D.I. and L. Anderson-Carnahan. 1988. Methods for Aquatic Toxicity Identification
Evaluations: Phase I. Toxicity Characterization Procedures. EPA/600/3-88/034. Sept 1988
Environmental Research Laboratory, Duluth, Minn
United States Environmental Protection Agency. Toxicity Identification Evaluation: Characterization
of Chronically Toxic Effluents, Phase I. EPA/600/6-91/005F. May 1992. EPA Office of Research
and Development Washington, D.C.
United States Environmental Protection Agency. 1989. Methods for Aquatic Toxicity Identification
Evaluations- Phase IE Toxicity Confirmation Procedures. EPA/600/3-88/036. Environmental
Research Laboratory, Duluth, Minn.
United States Environmental Protection Agency. 1993. Methods for Aquatic Toxicity Identification
Evaluations- Phase III Toxicity Confirmation Procedures for Samples Exhibiting Acute and
Chronic Toxicity. EPA/600/R-92/081
United States Environmental Protection Agency. 1993. Methods for Aquatic Toxicity Identification
Evaluations- Phase III Toxicity Confirmation Procedures for Samples Exhibiting Acute and
Chronic Toxicity. EPA/600/R-92/081. Office of Research and Development, Washington
D.C. September 1993.
United States Environmental Protection Agency. 1989. Methods for Aquatic Toxicity Identification
Evaluations- Phase II Toxicity Identification Procedures. EPA/600/3-88/035. Environmental
Research Laboratory, Duluth, Minn.
United States Environmental Protection Agency. 1993. Methods for Aquatic Toxicity Identification
Evaluations- Phase II Toxicity Identification Procedures for Samples Exhibiting Acute and
Chronic Toxicity. EPA/600/R-92/080. Office of Research and Development, Washington.
D.C. September 1993.
United States Environmental Protection Agency. Toxicity Reduction Evaluation Protocol For
Municipal Wastewater Treatment Plants. EPA/600/2-88/062. April 1989. EPA Risk Reduction
Engineering Laboratory. Cincinnati, Ohio.
United States Environmental Protection Agency. 1989. Generalized Methodology for Conducting
Industrial Toxicity Reduction Evaluations (TREs). EPA/600/2-88/070. EPA Risk Reduction
Engineering Laboratory. Cincinnati, Ohio.
These documents should be available from the: National Technical Information Service
5285 Port Royal Rd.
Springfield Va. 22161
(703) 487-4650

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Anyone developing a toxicity reduction plan should obtain copies of one or more or these documents
as appropriate in order to better understand the strengths and weaknesses of each step and be better able to
plan further actions. Included in this guidance are outlines of general toxicity reduction evaluations for
both industrial and municipal wastewater treatment facilities. These outlines demonstrate decision paths
that may be encountered in performing a TRE, as described in detail by the EPA documents.
A logical starting place for investigations of toxicity sources and reduction is a thorough information
gathering phase. At this stage, several preliminary issues should be investigated and information evaluated
for potential causes/sources of toxicity, effluent variability, treatment system efficiency, etc.
For municipal waste water treatment plants(WWTP) this information may include:
•The NPDES permit
•Treatment system design criteria, flow diagrams, descriptions of treatment elements
•Influent and effluent flow data
•Influent and effluent conventional pollutant data and removal efficiencies
•Effluent toxicity data and trends
•Process control and operational data and histories
•In-plant chemical usage (polymers, coagulants, chlorine)
•Treatment upset histories and reports
•Pretreatment information
•Industrial waste surveys
•Industrial user self-monitoring reports
•Industrial user operational schedules and flow patterns
•Waste hauler monitoring and manifests
•Hazardous waste inventories
For industrial waste water treatment plants(WWTP) this information may include:
•Process and wastewater generating process diagrams and descriptions
•Production records
•Diagrams and descriptions of non-process wastewater sources( cooling towers, boilers, floor
drams)
•In-plant flow records and water usage
•Chemical inventories and usage records
•Chemical labels, MSDS, and toxicity information
•Operating schedules with emphasis on how these schedules affect wastewater flow/composition
•WWTP operational data and histories
•Wastewater monitoring records(chemical and toxicity)
The overall importance of this initial information gathering phase to the success of a toxicity reduction
evaluation cannot be overestimated both in terms of eventual outcome as well as the efficiency with which
that outcome is achieved. In many instances, it is likely that sources or toxicity can be escalated or de-
escalated as targets for investigation by simple calculation rather than further testing, greatly reducing the
cost of the investigation. This information gathering phase will often be accomplished by the permittee
prior to contact with any paid consultants and before any actual testing takes place. By carefully reviewing
the information gathered and comparing trends in flow patterns, treatment efficiency, wastewater loading
and effluent constituents with toxicity patterns observed over time, the permittee may be able to narrow the
scope of further investigations and possibly even identify problem constituents.
Following information gathering, emphasis should next be placed on maximizing in-house treatment
efficiency and assuring that housekeeping practices are not contributing unnecessarily to final effluent
toxicity practices. Waste treatment efficiency must be maximized during this process in order that it does
not present a moving target in itself. Industrial facilities should identify and regulate all possible
contributions to the wastewater system, including even floor drain discharges where unwanted materials
may be disposed without the knowledge of wastewater treatment staff. In addition to the obvious
industrial process waste streams, side streams such as cooling tower discharge, boiler blowdown, or
airwash discharges should be reviewed for the presence of sometimes very toxic chemicals. Toxicity
information is now readily available for many of the more commonly used biocidal compounds used in

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these treatments. Municipal systems should investigate the toxicity of any added treatment chemicals and
review effluent toxicity vs. use records of such chemicals. Effluent chlorination and ammonia frequently
prove to be two of the most commonly encountered causes of effluent toxicity. Over-chlorination and
excess variation in chlorination should be high on the list as potential toxicity problems.
At this point a good informational base should have been developed and wastewater treatment
efficiency should be at peak sustainable levels. If still necessary, a Toxicity Identification Evaluation (TIE)
could now be initiated to help characterize the chemical/physical nature of the problem constituents. This
series of tests, described by EPA/600/3-88/034, EPA/600/3-88/035, and EPA/600/3-88/036, referenced
on the first page consists of a series of chemical/physical manipulations of the wastewater followed by
subsequent toxicity tests which can identify or rule out possible toxicants. A TIE will most likely require
the expertise of a group of biologists, chemists, and possibly wastewater engineers experienced with these
protocols due to the case by case decisions that must be made in development and interpretation of results.
Some of the complex analytical procedures required by lib protocols may not be available in most
wastewater laboratories. As such, consultants specializing in toxicity reduction may need to be sought A
TIE series should be carefully planned and monitored through its duration with interim findings translated
back to the collection and treatment system managers for immediate investigation. Where these interim
findings point to a possible source of toxicity, modifications in chemicals being used or disposal/use
patterns modified so as to efficiently solve toxicity problems.
Once problem constituents have been characterized and/or identified, in either an individual industrial
discharge or in a municipal system, the sources of these constituents can be tracked to the source and
addressed through product substitution, waste reduction, or recycle/reuse systems. With few exceptions,
removal of these problem constituents will be more cost effective by these means than by treatment If
treatment is necessary, the TRE can shift to bench or pilot scale treatability studies.
In municipal treatment systems where the sources of problem constituents are not known, a procedure
referred to as Refractory Toxicity Assessment (RTA) can be accomplished, as described by EPA/600/2-
88/062. This approach utilizes bench scale treatment of various iifluent sources to determine which
wastestreams pass through the treatment system without effective detoxification.
For either industrial or municipal discharges, custom designed toxicity tests to answer specific
questions can be beneficial. If well organized, these tests can be significantly reduced in both complexity
and cost from those required for NPDES compliance testing. Such tests could address the toxicity of
individual streams contributing to the whole effluent or tests of process or treatment chemicals to address
relative toxicity. Consultants experienced with performing toxicity reduction work should be able to
design such tests and significantly reduce costs by doing so. As stated earlier, the position of the N.C.
Water Quality Section is that the goal of this process is to reduce observed toxicity in the effluent Finding
the most efficient means of reaching this goal is encouraged so that unnecessary testing and costs are not
incurred.
Most toxicity reduction plans are reviewed by technical staff of the Water Quality Section. Reviewers
of these plans will judge adequacy based on information provided and the technical ability of the plans to
address the problems encountered in each specific application. Generally, those plans that follow the logic
of the EPA described toxicity reduction evaluation guidance will meet the intent of most TRE
requirements. The EPA document" Technical Support Document for Water Quality-based Toxics Control
(EPA/505/2-90-001, PB91-127415, March 1991)" includes a list of evaluation criteria for TRE plans
which could be helpfiil in designing or evaluating a plan submitted by a consultant The ideas presented
by this list include important concepts that we evaluate when reviewing the effectiveness of TRE
proposals. That list is as follows:

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• Are the objectives or targets of the TRE stated clearly and accurately?
•Are the schedule milestones for accomplishing the tacks described in the study plan**
•Are the final TRE report, progress reports, and meetings with the regulatory authority included as part
of the schedule?
•Are the approaches or methods to be used described to the extent possible prior to beginning the TRE?
•Has available EPA guidance been used in designing the TRE and developing the TRE plan (or if other
methods are proposed, are these sufficiently documented)?
•Does the TRE plan specify what results and data are to be included in the interim and final reports?
•Does the IRE plan provide for arrangements for any inspections or visits to the facility or laboratory
that are determined to be necessary by the regulatory authority?
•Are the toxicity test methods and endpoints to be used described or referenced?
•Does the approach described build on previous results and proceed by narrowing down the
possibilities in a logical progression?
•Does the plan provide for all test results to be analyzed and used to focus on the most effective
approach for any subsequent source investigations, treatability studies, and control evaluations?
•Are optimization of existing plant/treatment operations and spill control programs part of the inirial
stepsoftheTRE?
•Does the TRE plan allow a sufficient amount of time and appropriate level of effort for each of the
components of the study plan?
•Does the l it use broad characterization steps and consider quantitative and qualitative effluent
variability?
•Is toxicity tracked with aquatic organism toxicity tests throughout the analyses?
•Is the choice of tests for the TRE logical and will correlations be conducted if the species used are
different from those used for routine biomonitoring?
•Is the laboratory analytical capability and the expertise of the investigator broad enough to conduct the
various components of the evaluation?
A question which will undoubtedly arise is, "What will be the State's role in the TEE/TRE process?"
DWQ's regional office staff should be the first point of contact in these issues. Most often, they will have
the best knowledge of specific facilities and will be the hub of compliance activities. Staff of the Aquatic
Survey and Toxicology Unit in Raleigh can provide information and technical guidance on toxicity and
toxicity reduction issues. One of the responsibilities of this Unit is review of submitted TRE plans
addressing the proposed logic and direction undertaken to reduce toxicity. The comments provided by this
Unit incorporate the technical expertise of individuals whom, over the years, have evaluated TIE/TRE
plans and provided recommendations related to those plans. Additional information on waste reduction
and reuse strategies may be available from the N.C. Pollution Prevention Program in Raleigh at (919) 571-
4100. The latter is a non-regulatory program designed to provide the industrial/commercial sector with
information in this area.
Successful toxicity reduction can rely to a great extent on selection of a competent and qualified
contractor to conduct toxicity reduction activities should the services of a qualified contractor be needed to
resolve toxicity noncompliance. Should there be any questions related to this guidance document or to
toxicity reduction strategies, we would encourage you to contact Larry Ausley, Matt Matthews, or Kevin
Bowden of the Aquatic Survey and Toxicology Unit at (919) 733-2136.

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CERTIFIED MAIL
RETURN RECEIPT REQUESTED
REF: 4WM-WEB
Mr. William M. Weldon
City Operations Manager &
Utility Director
City of Starke
Post Office Drawer C
Starke, Florida 32091
SUBJ: City of Starke
NPDES Permit No. FL0028126
Administrative Order No. 96-005
Dear Mr. Weldon:
Pursuant to Section 3 09(a) of the Clean Water Act (the Act),
as amended, 33 U.S.C. §1319 (a), the Director, Water Management
Division, Region IV, United States Environmental Protection
Agency (EPA), has determined that the above named facility is in
violation of Section 301 of the Act, 33 U.S.C. §1311. As a
result, the Director has issued the enclosed Section 309(a)
Order. This order supersedes Administrative Order (A.O.) No. 94-
042 issued on March 14, 1994.
This Order is issued to enforce existing requirements under
the Act and, therefore, does not replace, modify, or eliminate
any other requirement of the Act. Notwithstanding the issuance
of this Section 309(a) Order, EPA retains the right to bring
further enforcement action under Sections 309(d) or 309(g) of the
Act, 33 U.S.C. §1319(d) or §1319(g) respectively, for the
violations cited in this Order and for any other violation of the
Act. Violations of the Act, including requirements contained in
a National Pollutant Discharge Elimination System (NPDES) permit
or a Section 309(a) Order, remain subject to a civil penalty of
1

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up to $25,000 per day for each violation, pursuant to Section
309(d) of the Act, 33 U.S.C. §1319(d).
Under Executive Order 11738 and EPA regulations issued
thereunder (40 Code of Federal Regulations, Part 15), facilities
to be utilized in contracts, grants, or loans must be in full
compliance with all standards established pursuant to the Clean
Water Act and the Clean Air Act. A violation of this
Administrative Order may result in the subject facility becoming
ineligible for participation in any work associated with a
Federal contract, grant, or loan.
2

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2
Should you have any questions concerning the enclosed Order,
please contact Lisa Spurlin at (404)347-3555, Extension 4241.
Sincerely,
Beverly Houston Banister, Acting Chief
Water Permits and Enforcement Branch
Water Management Division
Enclosure
cc: Florida Department of Environmental Protection,
Northeast District
bcc: ORC Section Chief
EPA-Headquarters
SPURLIN:2-05-96 k \user\shared\tn\starke ao
Spurlin	Lankf ord	Pf af f
3

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CERTIFIED MAIL
RETURN RECEIPT REQUESTED
REF: 4WM-WEB
Ms. Virginia B. Wetherell, Secretary
Florida Department of Environmental
Protection
Twin Towers Office Building
2600 Blair Stone Road
Tallahassee, Florida 32399-2400
SUBJ: City of Starke
NPDES Permit No. FL0028126
Administrative Order No. 96-005
Dear Ms. Wetherell:
Pursuant to Section 309(a) of the Clean Water Act, 33 U.S.C.
§1319(A), I have determined that the above referenced facility is
in violation of the Clean Water Act. As a result, I have issued
an Administrative Order (A.O.) to the facility, and have enclosed
a copy of that Order for your reference. The Order is presently
being served.
This is also to advise you that A.O. No. 94-042 issued on
March 14, 1994 has been superseded by the A.O. issued today.
Sincerely,
Robert F. McGhee, Director
Water Management Division

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Enclosure
bcc: ORC Section Chief
EPA Headquarters
SPURLIN 2-05-96 k \user\shared\cn\star)ce ao
Spurlin	Lankf ord	Pf af f	Banister.
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
REGION IV
IN THE MATTER OF
City of Starke
P. 0. Drawer C
Starke, FL 32091
NPDES PERMIT NO. FLO028126
ADMINISTRATIVE ORDER
NO. 96-005
ORDER PURSUANT TO SF.CTTON 309(a)
CLEAN WATER ACT
Pursuant to the authority of Section 309(a) of the Clean
Water Act (hereinafter, the Act), 33 U.S.C. §1319(a), which has
been delegated to the Regional Administrator of the Environmental
Protection Agency (EPA), Region IV, and redelegated to the
Director, Water Management Division, Region IV, the following
Findings of Fact and Violation are made, and Order issued:
FINDINGS OF FACT

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1.	City of Starke, (hereinafter, the Respondent) is a
person within the meaning of Section 502(5) of the Clean Water
Act, 33 U.S.C. §1562.
2.	The Respondent operates a wastewater treatment plant,
(hereinafter, the Facility), located at 100 Edwards Road, Starke,
Florida, which discharges "pollutants" into Alligator Creek,
"navigable waters" of the United States; these terms are defined
in Section 502 of the Act, 33 U.S.C. §1562.
3.	Section 301(a) of the Act, 33 U.S.C. §1311 (a),
prohibits the discharge of pollutants into waters of the United
States except as in compliance with a National Pollutant
Discharge Elimination System (hereinafter, NPDES) permit issued
pursuant to Section 402 of the Act, 33 U.S.C. §1342.
4.	The EPA issued NPDES Permit No. FL0027669 (hereinafter,
the Permit) effective on January 1, 1995, with an expiration date
of September 30, 1999, authorizing the above discharge from the
Facility.
5.	The Permit includes whole effluent toxicity limits and
requires the Respondent to conduct toxicity tests to determine
compliance with those limitations.

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3
6. On March 14, 1994, the Respondent was issued
Administrative Order (A.O.) No. 91-048 because the effluent was
revealed to be toxic from January 1990 through October 1993 to
the Ceriodaphnia dubia. The A.O. required the Respondent to
conduct a Toxicity Reduction Evaluation and to be in compliance
with toxicity effluent limits by September 30, 1995. The
Respondent's consultant concluded that organo-phosphate
pesticides were the cause of the toxicity. However, no
confirmation was conducted using the EPA/600/R-92/081 TIE Phase
III protocol. Also, the effluent continues to exhibit acute and
chronic toxicity to the Ceriodaphnia dubia.
VIOLATION
The Respondent has violated Section 301(a) of the Act, 33
U.S.C. §1311(a), and the Permit, issued pursuant to Section 402
of the Act, 33 U.S.C. §1342, in that the Respondent failed to
comply with the whole effluent toxicity limits of the Permit.
ORDER
Based upon the foregoing Findings of Fact and Violation, and

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4
pursuant to the provisions of Section 309(a)(5) of the Act, as
amended, 33 U.S.C. §1319 (a) (5), it is hereby ordered that:
1.	Within sixty (60) days after receiving this Order, the
Respondent shall begin conducting a Toxicity Identification
Evaluation, Phase III (hereinafter, TIE), to confirm that organo-
phates are the cause of toxicity to Ceriodaphnia dubia. The TIE
confirmation shall be consistent with the procedures and
protocols in "Methods for Aquatic Toxicity Identification
Evaluations, Phase III Toxicity Confirmation Procedures for
Samples Exhibiting Acute and Chronic Toxicity" (EPA/600/R-92-
081). All TIE toxicity tests will be performed using
Ceriodaphnia dubia.
2.	The TIE Phase III shall be completed no later than
March 31, 1997.
3.	Within sixty (60) days after receiving this Order, and
each 60 days thereafter until completion of the TIE, the
Respondent shall submit a report of progress made toward
completion of the TIE.
4.	Respondent shall continue toxicity testing as required

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5
by the Permit for the duration of this Order. However, no
additional testing will be required for any "routine" test
failure which occurs while the TIE is underway. After completion
of the TIE (no later than March 31, 1997), the Respondent must
resume additional testing for "routine" test failures.
5.	The Respondent shall develop and implement an
"Environmental Public Education Program" which targets organo-
phosphate pesticides and any other identified or suspected
pollutant(s). The Respondent shall develop (at a minimum)
quarterly publications (e.g. brochures, fliers, newsletters
and/or notices) for city wide distribution to inform and educate
the public on use, disposal, potential environmental impacts and
treatment issues associated with pesticides and any other
identified or suspected pollutant(s).
6.	Within ninty (90) days after receiving this Order, and
each 90 days thereafter until the compliance date, the Respondent
shall submit a report summarizing all activities conducted in
accordance with the Environmental Public Education Program.
7.	Within one year of receiving this Order,
the Respondent shall also conduct a comprehensive city wide

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5
survey to identify user groups and sources of organo-phosphate
pesticides and any other identified or suspected pollutant(s).
8.	The Respondent shall evaluate this information and
determine needed follow up actions which will effectively reduce
or eliminate organo-phosphate pesticides and/or other identified
or suspected pollutant(s) entry into the sanitary sewer system.
9.	The Respondent shall submit a summary report to describe
the survey results and subsequent programs developed and
implemented to address the survey results. The report should
also describe benefits from implementing these programs.
10.	The summary report shall be completed and compliance
with effluent toxicity limits attained no later than March 31,
1998.
11.	Information required to be submitted by this Order
shall be sent by registered mail, or its equivalent, to the
following addressees:
Beverly Houston Bannister, Acting Chief
Water Permits and Enforcement Branch
Water Management Division
U.S. EPA, Region IV

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7
34 5 Courtland Street, NE
Atlanta, Georgia 30365
ATTN: FL/TN Unit
Ernest E. Frey, Director
Florida Department of Environmental
Protection
Suite B200
7825 Baymeadows Way
Jacksonville, Florida 32256-7577
12.	Failure to comply with the requirements of this Order
shall constitute a violation of this Order and may subject the
Respondent to penalties as provided in Section 309 of the Act, 33
U.S.C. §1319.
13.	This Order does not operate as an NPDES permit and does
not replace, modify, or eliminate any requirement of the Act.
Nothing contained herein shall be construed as prohibiting
prosecution for the violations cited in this Order, for
violations of the Order itself, for violation of the Permit, or
for any other violations of the Act.
This Order is effective upon receipt.

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Robert F. McGhee, Director
Water Management Division

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