vvEPA
Unfed States
Environmental Protection
Agency
-ice a Water
2N-338)
331-S-92-002
March 1992
Introduction To
Water Quality-Based Toxics
Control For The
NPDESProgram
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Introduction to Water Quality-Based
Toxics Control for the NPDES Program
U.S. Environmental Protection Agency
Enforcement Division
Office of Wastewater Enforcement and Compliance
Enforcement Branch Support Branch
March, 1992
Printed on Recycled Paper
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UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
OFFICE OF
WATER
ACKNOWLEDGEMENTS
The Office of Wastewater Enforcement and Compliance (OWEC), in its constant effort to
provide current guidance, training, and methods for program priority areas to Regional
and State staff, often finds it necessary to produce "user friendly* versions of its technical
materials. In response to the need for these supplementary educational materials, the
Enforcement Support Branch (ESB) of OWEC has developed Introduction to Water
Quality-Based Toxicity Testing for the NPDES Program.
This document is written for non-biologists and other staff unfamiliar with water quality-
based (WQB) permitting and enforcement and whole effluent toxicity (WET) testing. Many
.thanks must go to those who have reviewed and commented to assure the document's
scientific accuracy, consistency with the Technical Support Document for Water Quality-.
based Toxics Control (TSD), and focus oh a layman audience:
Sheila Frace, Industrial Section, Permits Division, OWEC
Mary Reiley, Enforcement Support Branch, Enforcement Division, OWEC
N BillI Peltier, Environmental Services Division, Region IV ,
Margarete Heber, Ecological Risk Assessment Branph, Office of Science and
Technology
Ted Coopwood, Enforcement Support Branch, Enforcement Division, OWEC
Jim Pendergast, Water Quality and Industrial Permitting Branch, Permits Division,
OWEC
This document is not guidance, policy, or regulation. Its sole function is to provide the
necessary introduction to the basic concepts of the WQB - WET program and ease the
transition to technical guidance and methods. Comments and questions are welcome.
Address comments or questions to Mary Reiley, Enforcement Support Branch (EN-338),
401 M Street, S.W., Washington, D.C. 20460.
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This guide was prepared by the Cadmus Group, Inc. for the
U.S.Environmental Protection Agency, Office of Wastewater
Enforcement and Compliance through Contract No. 68-C9-009 and
Work Assignment No. S-3-7(R). The Project Manager was Michael
Dover, Ph.D., and the author was Joan S. Jolly, Ph.D.
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Table of Contents
The Scientific Necessity to Protect Water Quality ... . '.._•.. 1
The Legal Authority to Protect Water Quality .... ^,.,.,...,. S
National Pollutant Discharge Elimination System (NPDES) Peiroit Program ....... — 3
The Integrated Approach to Water 'Quality-Based Toxics Control..... ,„„-.„.. 4
Capabilities and Limitations of Whole Effluent Toxicity Testing —............ „ 5
Scientific Concepts in Toxicity Testing 6
Acute Toxicity Tests. • ••--•• - • ••••••• '...-• 6
Chronic Toxicity Tests ... '. ;..... 7
Dose-Response Curves .....8
Toxicity Units ........;.... 9
Practical Aspects of Whole Effluent Toxicity Testing ; ,.. .'10
Sample Collection , ...10
Sample Handling .". , 10
Designs for Toxicity Tests . .-. ............... 11
Test Organisms . 12
The Discharge Monitoring Report , 12
Compliance with Whole Effluent Toxicity Limits 13
Self-monitoring Reports .13
Discharge Monitoring Report/Quality Assurance....... 13
Inspections. • .... 13
Citizen Complaints •• 14
The Role of Quality Assurance in Compliance ..14
Violations of Permits Haying Whole Effluent Toxicity Limits 14
Types of Violations.. ..: ..14
The Role of the Toxicity Reduction Evaluation ..'..... 15
Case Histories of TREs ..'...,.... '. • '• • r 16
- - . • /.
Appendix A EPA's Authority to Regulate Toxicity '. • - - • 19
Appendix B Excerpts Taken From Sample NPDES Permits
and Relevant to Whole Effluent Toxicity Testing -21
..•••.' • • ' i •
-> . . . • .,.,,•.'•.
Appendix C Documents Relevant to Toxicity Testing . ... ........ ............ 35
AppendixD Glossary '. ... 43
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List of Figures
Figure 1. Btoaccumulatfen and Bfoconcentratfon 2
Figure 2. Btomagnification of a Toxic Substance .....' ., ;. . 2
Figure 3. Relationship Between Daily Concentrations, Long-term Averages, Wastetoad Allocations !
and Permit Limits'.-.. .,.......; ...;....,, 4
•\ • '. . - . , _ ' ' . •
Figure 4. Dose-Response Curve for a Hypothetical Acute Toxicity Test , 8
Figure 5. Dose-Response Curve for a Hypothetical Chronic Toxicity Test •., .9
Figure 6. , Flowchart for a Toxicity Reduction Evaluation (TRE) 15
. . - r ' •'.__' - , .
' • • . ' ' ! ' ' '
List of Tables
Table 1. . . The Integrated Approach: Advantages and Disadvantages of Each Method ; 5
Table 2. Acute vs. Chronic Toxicity Tests 6
Table 3. Species Commonly Used as Test Organisms in Chronic Toxicity Tests .. ...;.. 12
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Toxlcfty Testing
Introduction to Water Quality-Based
Toxics Control for the NPDES Program
The U.S. Environmental Protection Agency (EPA) and
designated State agencies are responsible for enforcing
the Federal Water Pollution Control Act of 1972, as amended
by the Clean Water Act (1977,1981) and the Water Quality
Act of 1987. Collectively, these are usually known as the
Clean Water Act, or CWA. The Clean Water Act's overall
objective is "to restore and maintain the chemical, physical,
and biological integrity of the nation's water." EPA is
authorized by the CWA to regulate the discharge of sub-
stances into the waters of the United States. To this end,
EPA issues permits specifying the conditions under which
a facility may, discharge effluents into a body of water. The
agency also tracks compliance with these permits by
requiring permittees to monitor their effluents. A primary
goal of this monitoring is to ensure that, in keeping with the
Jaw, no discharges contain "toxic pollutants in toxic amounts."
EPA makes use of both chemical and biological moni-
toring in evaluating compliance with permits. Chemical
monitoring checks water for the presence and concentra-
tion of substances. Biological monitoring, also called
biomonitoring,1 makes use of living organisms to moni-
tor water quality.2 Biomonitoring includes such monitoring
as toxicity testing and bioassessment {which evaluates
the condition of a body of water by studying its resident
organisms). , .
This Guide focuses on a principal means of bio-
monitorjng: whole effluent toxicity (WET) testing. The
legal definition of whole effluent toxicity is the "aggregate
toxic effect of an effluent as measured directly by a toxicity
test." In plainer language,
whole effluent toxicity test-
ing evaluates the toxic ef-
fects of effluents on living
organisms. The Guide pre-
sents whole effluent toxic-
ity in the broader context of
the scientific necessity to
regulate water quality and
EPA's legal authority to do
Whole Effluent
Toxicity Testing
evaluates the toxic
effects of effluents
on living organ-
isms.
so. The Guide then describes whole effluent toxicity in
greater detail and discusses practical aspects of toxicity
testing, from collecting samples to reporting results. Fi-
nally, the Guide covers the subject of maintaining compli-
ance with a permit that has a limit on whole effluent toxicity.
The Scientific Necessity to
Protect Water Qualify
What does it mean to say that a pollutant or effluent is
toxic? Toxicity has two characteristics. First, toxicity is a
harmful effect occurring in a human, other animal, plant, or
microbe as a result of a chemical substance. This adverse
effect can take many forms: disease, deformity, behavioral
changes, reproductive malfunction, or genetic damage.
For example, certain pesticides contain chemicals called
organophosphates. These compounds break down an
animal's neurotransmitters—substances that the animal
produces to regulate-the transmission of impulses along
the nerves. Intheabsenceof neurotransmitters, nervesfire
continuously, causing the animal to suffer convulsions and
death. Second, toxicity is a direct, rather than an indirect,
result of a chemical substance or mixture of substances.
The organophosphates in pesticides are toxic because
they cause direct harm to organisms.3
'Terms included in Appendix D, the Glossary, are extra bold
the first time that they appear in the text.
2 U.S. Environmental Protection Agency. Technical Support
Document (TSD) for Water Quality-based Toxics Control (EPA/
505/2-90-001), 1991. p. xix. - ' ,
, Suspended matter in water is an example of a substance
that is harmful but not toxic because its effect on organisms is
indirect. Suspended matter limits the amount of light that pen-
etrates water, thereby reducing the rate of photosynthesis— the
process by which plants convert carbon dioxide, water, and
energy from the sun into usable food. The reduced rate of
photosynthesis in turn adversely affects algae by lowering their
growth rate. >
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Figure 1 Bioaccumulation and Bioconcentratlon
BIOACCUMULATION » UPTAKE THROUGH FEEDING + BIOCONCENTRATION
Absorption Through The
Outermost Layer (such as
an animal's integument or
a plant's cell wall)
Uptake Through
Respiration
When toxic substances occur in water, these sub-
stances can adversely affect all the organisms that live
there: microorganisms, plants, insects, worms, shrimp,
other invertebrates, fish, amphibians, waterfowl, and such
mammals as beavers and otters. Toxic substances that
settle into the sediment can also damage organisms living
there. Some toxic substances cause immediate harm to
organisms, but others cause effects over a longer period of
time. In some cases a toxic substance brings about a long-
term effect as a result of bloaccumulatlon, the passage of
a substance from the environment into living tissues.
Bioaccumulation occurs by two routes: feeding and
btoconcentratton. (See Figure 1.) Bloconcentration is the
passage of a substance from water into an organism. For
a fish, then, bioconcentration can occur by absorption
through the skin or by uptake through the gills. A substance
that bioaccumulates reaches higher concentrations in liv-
ing tissues than occurs in the surrounding water. The
Regional Environmental Services Divisions (ESDs) can
advise regulatory personnel as to which substances tend to
bioaccumulate and which do not.
If all the members in a food chain bioaccumulate a
substance, the stage is set for biomagnification. (See
Figure 2.) Biomagnification is the process of a substance's
passing up the food chain and becoming concentrated in
the tissues of the organisms toward the top of the food
chain. For example, suppose aquatic vegetation in a
polluted river bioconcentrates a toxic substance and cray-
fish then feed on the vegetation. The crayfish too become
contaminated but at a higher concentration becayse each
crayfish consumes a quantity of vegetation. A fish that
feeds on these crayfish also becomes contaminated, and
at a still higher concentration because the fish eats several
crayfish. Hence, a chemical that bioaccumulates in each
Figure 2 Biomagnification
of a Toxic Substance .
In this picture the shading of the water
.represents a toxic substance that
vegetation, crayfish, and fish bio-
accumulate. These organisms make
up a food chain, with the'crayfish
feeding on the vegetation and the fish
feeding on the crayfish. Conse-
quently, biomagnification of this toxic
substance—represented by the in-
creasingly darker shading of the veg-
etation, the crayfish, and the fish-
occurs as the substance passes up
the food chain.
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Toxicity Testing
member of a food chain may pose more of a risk to animals
higher in the food chain than to those lower down the chain.
How do toxic substances enter bodies of water? Many
types of events can introduce toxic substances into ponds,
lakes, rivers, and streams. Pollutants can leach from
contaminated so ils. For example, if a farmer sprays insec-
ticide on a field, rain may wash the insecticide into nearby
waters, polluting them. This type of discharge constitutes
a non-point discharge, because the discharge's source is
broad. In addition, sewage treatment facilities and indus-
trial plants can pollute waters by discharging effluents into
them through pipes, ditches, channels, or tunnels. Dis-
charges from such specific locations are called point
source discharges, which are the primary target of the
toxicity testing activities described in this Guide.
However, regulators should keep in mind that CWA's
definition of point source discharges (40 CFR 122.2, also
section 122.3) includes discharges that do not have such a
highly localized source. For example, discharges from a
concentrated aquatic animal production facility constitute
point source discharges, as do the discharges from com-
bined sewer overflows and discharges of storm water. In
deciding whether a discharge is a point source or a non
point source, regulators should consult the cited federal
regulations.
The Legal Authority to Protect
Water Quality
, Since EPA's authority to protect water quality origi-
nates with the CWA, an understanding of the Act will clarify
EPA's role. (See Appendix A for excerpts from the CWA.)
The CWA has as one goal the policy"that the discharge of
toxic pollutants in toxic amounts be prohibited." The Fed-
eral regulations' 'tree froms" summarize this statement
well: water should be free from pollutants that settle to
cause objectionable deposits; float, such as debris, scum,
and oil; cause objectionable odor, color, taste, or turbidity;
cause injury ortoxic effects to humans, animals, or plants;
and cause population by undesirable or nuisance aquatic
life. To make its goal a national one, the CWA requires
States to include in their Water Quality Standards narrative
statements addressing each of the "free froms."
In addition to describing what constitutes clean water,
the CWA states that no one may discharge a pollutant into
water unless the discharge is incompliance with the Act.
The CWA protects public health, water supplies, and wild-
life in part by mandating limitations on point source dis-
charges. Toxicity constitutes one of the parameters that
CWA limits in point source discharges.4
Federal reguIattoEks state that water
should be free from pollutants resulting
in
• objectionable deposits;
• floating material, such as debris,
scum, and oil;
• objectionable odors, colors, tastes,
and turbidity;
• harmful effects to humans, animals,
or plants;
• undesirable or nuisance aquatic life.
National Pollutant Discharge Elimination
System (NPDES) Permit Program ' .
To accomplish its goals, several sections of the Clean
Water Act give EPA the authority to restrict and monitor the
discharge Of pollutants, including toxic substances, into the
nation's waters. To this end, EPA has established the
(National Pollutant Discharge Elimination System (NPDES)
permit program, which sets guidelines for issuing permits to
facilities that produce effluents they wish to discharge into
national waters.
Any facility discharging effluents into the water must
have a permit. Permits limit the concentration Of pollutants
in effluents. In addition, permits must establish limits to an
effluent's toxicity in cases where a discharge may result in
a violation of water quality .standards. When permit limits
are based on existing wastewater treatment technologies,
EPA documents refer to them as technology based. In
some cases a permit limit aims at attaining a specified level
of water quality without regard to existing treatment tech-
nologies. In such a case EPA documents refer to the
permit limit as water quality based.
In addition to the technology based limits described
above, an NPDES permit issued to a discharging facility x
aims at safeguarding the State's Water Quality Standard
(WQS). This standard establishes for a body of water the
maximum in- stream pollutant concentrations compatible
with both the water's assimilative capacity and its desig-
* In Natural Resources Defense Council, Inc. v. EPA, 859
F.2d 156 (D.C. Cir. 1988), the Court concluded that EPA has the
authority to express permit limitations in terms of toxicity provided
that the limits reflect requirements of CWA, as described in 40
CFR 125.3(c)(4). The Court held,that the CWA's broad definition
of "pollutant" in section 502(6) authorizes the use of toxicity to
regulate effluents, even though toxicity is an attribute of pollutants
rather than a pollutant itself. :
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nated use. To this end, an NPDES permit specifies an
effluent's contents, frequency, and site of discharge. (See'
Appendix B for excerpts from NPDES permits.) It also
controls the effluent's concentration and mass (in Ibs/day)
by setting a maximum daily limit, a monthly average limit,
and for publicly owned treatment works a seven-day
average limit for the effluent. The maximum dally limit
(MDL) is the highest value allowable for a discharge
during a 24-hour period. The more restrictive average
monthly limit (AML) consists of the highest value allow-
able for the average of daily discharges occurring over a
one-month period.
An NPDES permit written to protect water quality
derives the MDL and the AML from two other models: the
wastetoad allocation and the long term average. The
wastetoad allocation (WLA) is the maximum amount of
effluent thatthe receivingwatercan assimilate in a day from
a permitted facility without violating the WQS. The WLA is
set to achieve the WQS. The long-term average (LTA)
represents the acceptable mean of an effluent's pollutant
concentrations or parameters. The LTAtakes into account
the variability in a facility's effluent, so that there is 99%
probability thatthe WLA will not be exceeded. Togetherthe
five models and model outputs of concern to most NPDES
permittees—WQS, WLA, MDL, AML, and LTA—make up a
hierarchy that protects the receiving water (Figure 3). .
The Integrated Approach to Water Quality-
Based Toxics Control
As well as mandating regulation of discharges, the
Clean Water Act also specifies that EPA shall measure the
health of waters by means of three methods:
• Analyzing the chemical content of waters. This type of
monitoring, called chemical-specific testing, is ac-
complished by subjecting samples to laboratory tests
that identify chemical substances and measure their
concentrations.
• Studying organisms inhabiting the receiving water.
This is achieved through biological assessment, gen-
erally called bloassessment. Bioassessment evalu-
ates the biological condition of a body of water by
'studying its biota, which are its resident organisms,
and its chemical and physical characteristics. Specific
means of btoassessment include surveying biota and
measuring biological criteria to determine whether a
pollutant has had an adverse effect on the biota.
• Testing effluents for toxic effects on living organisms.
This is accomplished principally through whole effluent
toxlclty testing.
Figure 3 Relationship Between Dally
Concentrations, Long-term
Averages, Wastetoad
Allocations, and Permit Limits
CD
I
&
c
o
o.
Wastetoad Allocation (WLA)
Maximum Daily Limit (MDL)
Average Monthly Limit (AML)
Long Term Average (LTA)
Effluent's daily pollutant
concentration or parameter
10 20
Time (in days)
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Toxfcity Testing
,
The Integrated Approach:
Advantages and Disadvantages of Each Method
METHOD
ADVANTAGES
DISADVANTAGES
CHEMICAL-SPECIFIC
TESTING
Precise
Complete toxicology
Information about human health
May know fate of substance
and/or how to treat it
Prevent impact
Inexpensive where effluent
contains only a few toxics
Expensive where effluent contains
many toxics,
Bioavailability unknown
May not consider all toxics present
Misses interactions between toxics
Does not measure ecological effects
BIOASSESSMENT
Measures ecological effects
Shows historical trends
Impact has already occurred
Does not specify source of damage
May not evaluate effects of variation
in flow rate
No information about human health
WHOLE EFFLUENT
TOXICITY TESTING
• Total toxicity
> Does not require detailed
knowledge of chemical nature.
, of substances in an effluent
> Only bioavailable toxics measured
• Prevent impact
Specific toxics not known
No information about treatment,
persistence, or presence in sediment
Toxicity may differ in ambient conditions
No information about human health
Taken together, these three methods are referred to
as the integrated approach to monitoring water quality.
Each method contributes specific types of information to an
evaluation of water quality, and all three are required for a
complete evaluation of the biological condition of a body of
water. (See Table 1.)
An NPDES permit establishes by means of limits how
the permittee will meet WQS. When setting water quality-
based limits and determining compliance, EPA uses the
principle of independent application of WQS. This
principle states that no one of the three methods is inher-
ently .superior to the other two in evaluating the health of
water. Rather, as Table 1 indicates, each method contrib-
utes to the analysis, and each also has its limitations. As
a result, data collected using one method should not be
used to contradict or Overrule data obtained with either of
the other two. Specifically, if results of any one method
show an impairment of water quality, then EPA believes
that an impairment may exist.
Capabilities and Limitations of
Whole Effluent Toxicity Testing
Whole effluent toxicity testing measures the total toxic
effect of an effluent by means of tests that expose living
organisms to that effluent and note the effects of the
effluent on these organisms. Several studies have demon-
strated that results obtained with testing for whole effluent
toxicity are reproducible within a laboratory and also be-
tween laboratories to the same degree as is found for
chemical-specific testing.5 Studies also demonstrate that
whole effluent toxicity correlates well with the observed
impact on receiving water.6
5 A Review of Inter- and Intralaboratory Effluent Toxicity Test
Method Variability (W. J. Rue, J. A. Fava, and D.R. Grothe. 1988. Aquatic
Toxicology and Hazard Assessment: 10th Volume. ASTM STP 971).
. A Perspective on Biological Assessments (D.R: Grothe,
R.A. Kimerle, and C.D. Malloch. 1990. Water Environment and
Technology). ; '
Results: Interlaboratory Comparison of Acute Toxicity Tests
Using Estuarine Animals (S.C. Scnimmel. 1981. EPA-600/4-81-
003). .
• Biomonitoring to Achieve Control of Toxic Effluents (U.S. EPA.
1987 EPA 625/8-87/013).
ExaminingtheRelationshipBetweenAmbientToxcityandlnstream
Impact (K.L. Dickson, W.T. Waller, LP. Ammann, and J.H. Kennedy.
1991. Submitted to: Env. Toxicol. and Chem.).
Comparison of Measured Instream Biological Responses with
Responses Predicted by Ceriodaphnia Chronic Toxicity Tests (K.W.
Eagteson, D.L Lenat, L Ausley, and F. Winborne. 1990. Env. Toxicol.
and Chem. 9:1019-28).
A Comparative Ecological and Toxicological Investigation of a
Secondary Wastewater Treatment Plant Effluent and its Receiving
Stream (W.J. Binge, J.A. Black, T.M. Short, and A.G. Westerman. 1989.
Env. Toxicol. and Chem. 8:437-50).
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Testing for whole effluent toxicity provides a way*to
evaluate an effluent in the absence of detailed information
about the chemicals it contains. Using this method, then,
facility personnel or lab technicians can measure the toxic-
ity of an effluent without knowing all of its components,
which ones are toxic, and whether components interact in
ways that alter their toxicity. Because whole effluent
toxfc'rty testing uses living organisms to detect pollutants, it
also has the advantage of measuring the effects of only
those toxic substances that are bloavailable, or present in
a form that can affect organisms. On-going whole effluent
toxteity testing can alert regulators should an effluent's
effects become more damaging.
Testing for whole ef-
fluent toxicity also has its
limitations. Because this
method does not specify
which substances in an
effluent are toxic, it gives
no indication of how to
treat the effluent's toxic-
ity. Furthermore, without
knowledge of specific
Whole effluent
toxicity testing
provides a way to
evaluate an efflu-
ent in the absence
of detailed infor-
mation about the
chemicals it
contains.
chemical substances, a
toxicity test provides no
information about protect-
ing human health. Since
toxicity tests involve a lim-
ited number of species, results give only a partial toxic
profile of an effluent. In addition, whole effluent toxicity
testing does not indicate how long toxicity persists in water
or whether sediment has become toxic as well. Finally,
toxicity tests may not take into account changes in toxicity
that can result from environmental changes, such as water
temperature or acidity.
Scientific Concepts in
Toxicity Testing
Toxicotogists test for whole effluent toxicity by means
of toxicity tests. These involve exposing a designated
species of live organisms, called the test organisms, to an
effluent and to dilutions of that effluent. Toxicity tests
include two types: acute and chronic. (See Table 2.)
Acute Toxicity Tests
Acute toxicity tests last no more than 96 hours and
measure an effect occurring in this short time period.
Generally, acute toxicity tests measure an effluent's lethal-
ity. Results from an acute toxicity test indicate the effluent
concentration at which a certain percentage of the organ-
isms died. This concentration, which is referred to as the
Lethal Concentration (LC), is generally followed by the
percentage of test organisms killed! For example, if a
certain concentration of an effluent causes 50 percent of
the organisms in the test to die, then this concentration is
the effluent's LC^,.
To conduct an acute toxicity test, lab technicians
expose groups of test organisms to different concentra-
tions of effluent. Lab technicians prepare these concentra-
tions by diluting effluent with uncontaminated water ad-
justed to meet the test organism's needs (such as for
salinity or hardness). An acute toxicity test must also
include a group of organisms subjected to the same con-
ditions as the other test organisms but exposed to diluting
water only. Since these organisms are not exposed to
effluent, any deaths that occur among them do not result
from toxic substances that might be in the effluent. Such a
group, which receives no exposure to the factor being
tested, is called a control. For data from, an acute toxicity
test to be valid, the control group must have at least 90
percent survival. (The Guide discusses quality control
more fully in the section entitled "Compliance with Whole
Effluent Toxicity Limits.")
EPA has published a manual that presents the Agency's
approved protocols for acute toxicity.tests.7
7U.S. EPA. 1991. Methods for Measuring the Acute Toxicity
of Effluents to Aquatic Organisms, 4th Edition. Office of Research
and Development, Cincinnati, OH. EPA-600/4-90-027.
Table 2.
Acute vs. Chronic Toxicity Tests
TRAIT ACUTE
Duration Up to 96 hours
Measurement Death, generally
ij , • i
CHRONIC
Partial or Full Life
Cycle of Test
Organism
Death or Sub-
lethal Effect
(such as
decrease in
growth or
reproduction)
6
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Toxlclty Testing
Chronic Toxicity Tests ~
Unlike acute tests, chronic toxicity tests may con-
tinue for as long as the entire life cycle of the test organism.
Snorter chronic tests last seven days or even (ess. Such
tests, called short-term chronic toxiclty tests, focus on
the period in a test organism's life cycle when it shows the
greatest sensitivity to its environment.
In chronic toxicity tests', lab technicians record the
death of test organisms but also monitor other effects, such
as fertilization, growth, and reproduction. Toxtoologists
use these tests to determine the lowest tested concentra-
tion at which organisms show an adverse effect from their
exposure to effluent. This concentration is called the
Lowest Observed Effect Concentration (LOEC). An-
other important concentration is the highest tested concen-
tration displaying no effect on the organism, called the No
Observed Effect Concentration (NOEC) or the No Ob-
served Effect Level (NOEL). The LOEC or NOEC (NOEL)
is determined by a statistical procedure called hypothesis
testing and can vary considerably depending on the spe-
cific series of dilutions used in the test. Chronic toxicity
tests may also report a presumably safe concentration
called the Chronic Value (ChV), which lies between the
LOEC and the NOEC. Specifically, the ChV represents the
geometric mean of the LOEC and the NOEC.
An alternative to an LOEC or an NOEiC is a parameter
called an inhibition Concentration (1C). An 1C indicates
the concentration of effluent that inhibits a biological pro-
cess, such as reproduction, by a specified percent. Since
some studies show that NOECs determined by hypothesis
testing are analogues of IC^s, regulatory agencies may
want to stipulate that permittees use IC^s rather than
LQECs or NOECs *
The basic set-«p for a chronic toxicity test is much the
same as that for an acute teioty test. Lab technicians
- e:xp0se §«»i|ps of test oipamsms to different effluent con-
centrations, which are prepared by diluting effluent with an
appropriate diluting solution. As with an acute toxi<% test,
a chronic toxicity test must include a contesl. In order for a
chronic toxicity test to be vaftd, trie organisms In ihe control
group should have a minimum survival of 80 percent and
should achieve an acceptable leva* tar the effect being
measured, such as growth or repron&ctei, The EPA
manuals that present the approved protocols forstoTt-tenn
chronic toxicity tests specify the acceptable effect levels for
each specific test. One of these manuals deals with toxicity
tests using freshwater organisms,9 and the other describes
toxicity tests employing marine and estuarine organisms.10
Generally, acute toxicity tests measure
an effluent's lethality. In chronic toxic-
ity tests, lab technicians record the
death of test organisms but also moni-
tor other effects, such as fertilization,
growth, and reproduction.
, •• TSD for Water Quality-based Toxics Control, p. 6.
9 U.S. EPA. 1991. Short-Term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater
Organisms 3rd Edition. Office of Research and Development, Cincinnati, OH. EPA-600/4-91/000.
10 U.S. EPA. 1991. Short-Term Methods for Estimating Chronic Toxicity of Effluents and Receiving Waters to Marine and Estuarine
Organisms. 2nd Edition. Office of Research and Development, Cincinnati, OH. EPA/600/4-91/003. ,
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Figure 4 Dose-Response Curve fora
Hypothetical Acute Toxlclty Test
Data
Percent Percent
Effluent Mortality;
100 100
50
25
12
6
0
100
50
0
0
0
Regular Plot of Data, showing S-shapad curve
100
612 25 50
Percent Effluent
Semi-log Plot of Data, giving a straight Una
too
100
6 12 25 50 100
Percent Effluent
Dose-Response Curves
Displaying data graphically often helps in understand-
ing results from a test. To this end, biologists frequently
make use of a type of graph called a dose-response
curve, which plots the concentration of a substance against
organisms' response to the substance. In the case of a
toxicity test, the dose-response curve plots the concentra-
tion of the effluent against the observed effect, giving an S-
shaped curve (Figure 4). To make interpretation of such
curves easier, biologists often plot the data in such a way
as to give a straight line rather than a curve. This straight-
line plot is called a semi-tog plot. For comparison, Figure
4 shows data from a toxicity test both as an S-shaped curve
and as a semi-log plot.
Figure 4 presents data from an acute toxicity test. For
this test, organisms were exposed to 6,12,25,50, and 100
percent (undiluted) effluent. The dose-response curve for
the data shows that at a 50 percent concentration all of the
test organisms died, at a 25 percent concentration half of
the organisms died, and at 12 percent none died. For this
effluent, the LC.- occurs at an effluent concentration of 25
percent.
Data from chronic toxicity tests can also be visualized
with a dose-response curve. Figure 5 shows dose-re-
sponse curves for data collected during a chronic toxicity
test using two effluents; A and B: For this test, organisms
were exposed to 6,12,25,50, and 100 percent concentra-
tions of each effluent. In order to compare the data for the
two effluents, -technicians would have had to perform the
same chronic toxicity test on both and use the same
species,to test the two effluents. Other test conditions,
such as temperature and oxygen availability, also must be
the same for both. According to the dose-response curve,
effluent A appears to have 12 percent effluent concentra-
tion for its LOEC and 6 percent effluent concentration as its
NOEC. For effluent B the LOEC appears to be 50 percent
and the NOEC 25 percent.
Comparing dose-response curves indicates the rela-
tive toxicities of effluents. The dose-response curves in
Figure 5 show effluent B as less toxic than A. Effluent A
requires considerably more dilution than effluent B to reach
an LOEC or an NOEC. In other words, toxicity is inversely
proportional to LOECs and NOECs: the more toxic an
effluent, the lower the value of its LOEC or NOEC. The
same relationship holds true for LCs.
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Toxlclty Testing
Flgur&5 Doss-Response Curve fora
Hypothetical Chronic Toxlclty Test
100
100
75
at so
£
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Practical Aspects of Whole
Effluent Toxicity Testing
Testing the toxicity of whole effluents involves a num-
ber of steps. Permittees must follow acceptable proce-
dures foreach of these steps in orderto obtain valid results.
(See Appendix 8 for examples of how an NPDES permit
specifies details of whole effluent toxicity testing.) Toxicity
testing begins with collecting and handling samples. The
tests themselves may occur at the facility, requiring it to
store samples properly and perform the tests according to
accepted protocol. In some cases, the facility may send
samples to a laboratory fortesting. Acceptable transport to
the lab and appropriate handling at the lab then become
practical matters to consider. Finally, collecting and ana-
lyzing data and reporting results to the regulatory agency
complete the testing process. To gain a better understand-
ing of the practical aspects of whole effluent toxicity testing,
let us consider each step in greater detail.
Sample Collection
A facility's NPDES permit specifies many aspects
concerning the collection of the sample. These may
include the location, timing, and method of sample collec-
tion. Unless personnel at the facility follow the guidelines
in the permit, the data from the sample will not be consid-
ered valid.
In most cases, permits specify the outfall (the site of
discharge) as the place for collecting samples. For some
facilities, however, a location between the final treatment
and the outfall may give better access to a sampling point.
In the case of a facility that chlorinates its effluent, the
regulatory agency may wish to evaluate toxicity before
chtorination. Alternatively, a permit may specify sampling
before and after chtorinatfon and after dechlorination as
well. Athird situation that may result in a sampling site other
than the outfall occurs when a regulatory agency wishes to
assess a wastewater stream before it joins otherwastewater
streams.
Several factors enter into the timing of sample collec-
tion. One of these factors is a facility's schedule for
discharging: some facilities discharge continuously and
others only intermittently. The purpose of the toxicity test
can also affect sample timing. For example, a regulatory
agency may want sampling for an acute toxicity test to
correspond with the point in a facility's operation when its
discharge is most toxic.
The actual collection of samples can occur by one of
two methods. The method specified in the permit will
depend on the object of the test and the nature of the
facility's operation. A grab sample, as the name implies,
is a single sample. This type of sampling requires little time
and a minimum of equipment. It can prove useful for
sampling an effluent with toxicity that changes little over
time.
The othertype of sample, called a composite sample,
is a mixed sample collected over a specified period of time.
Although composite sampling can be performed manually,
devices exist to accomplish this task automatically. A
composite sample may prove ideal for a chronic toxicity
test. However, this sampling method may mask periods of
peak toxicity, which are relevant to acute toxicity tests.
Whichever sampling method a facility uses, personnel
collecting samples should minimize aeration. Aeration
results in the loss of volatile chemicals, which are sub-
stances that readily pass from the liquid to the gaseous
sjate. When an effluent sample loses such substances, a
toxicity test may indicate less toxicity than the effluent
actually contains.
Sample Handling
As with sample collection, permittees must handle
samples appropriately in order fortoxicity tests to give valid
results. First, permittees need to store samples in suitable
containers and, as with sample collection, they need to
avoid aeration during transfer to storage containers. Be-
cause stainless steel is easily decontaminated, it makes
good containers for tests conducted on-site. Glass or
disposable plastic containers are recommended forsamples
shipped to laboratories for off-site testing.
Once samples are in suitable containers, permittees
must provide proper storage. For on-site tests, permittees
should store on ice any sample not used immediately.
However, a sample should not be stored for longer than 24
hours before being used for a test. Some tests require such
large volumes that storing the sample on ice becomes
impractical. Permittees may store such samples at ambi-
ent temperatures. During cold weather, personnel should
use heat tapes to prevent large samples from freezing.
If a facility sends a sample to a laboratory for testing,
personnel at the facility should store the sample on ice and
ship it on ice as soon as possible after collection. When the
sample reaches the lab, technicians there should store it at
refrigerator temperature (4°C). Laboratories are required
to initiate tests within 36 hours of receiving a grab sample
or upon completion of a composite sample.
In some cases, permittees must also make chemical
adjustments to samples. The pH, which indicates how acid
or basic a liquid is, may require adjustment before the start
of a toxicity test. A permittee may also need to alter a
sample's hardness, which is principally the amount of
calcium carbonate in water. When a sample requires
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Toxictty Testing
adjustment, the permittee shouid allow a portion
sample to remain unadjusted. This unadjusted portion &
used in a parallel study, which consists of conducing tie
toxicity test on a full dilution series. The parallel study
reveals whether the adjustment contributes to, masks, or
has no effect on the observed toxicity.
Designs for Toxicity Tests
To monitor a facility's compliance with its limit, the
permit specifies which types of .tests a facility needs to
perform on its effluent. These may include toxicity tests-
acute, chronic, of both. A permit that specifies toxicity tests
will also indicate the test design, which varies with the site
and operation of the discharging facility.
Static tests are tests that use the same effluent
sample throughout the test or in which only limited re-
placement of the sample effluent occurs. Static tests,
which are generally conducted in a laboratory, have the
advantage of being simple and inexpensive to perform.
They require little space, manpower, and equipment. Gen-
erally, they use only small effluent volumes, one to 20 liters.
However, static tests do not show .changes in the effluent
over time. Also, over the course of the test, dissolved
oxygen can become depleted. As a resu It, organisms could
suffer adverse effects not caused by toxicity.
Aquatic lexicologists have developed two types of
static tests. Static nonrenewal tests use the same
effluentsamplefortheduratfonofthetest. The nonrenewal
test can provide some measure of how long toxicity persists
in an effluent. On the other hand, the adsorption of toxics
onto the test chamber and the degradation of toxics may
occur, resulting in a decrease in the apparent toxicity. An
effluent can also lose some of its volatile toxics over the
course of a static nonrenewal test. Finally, organisms
release sulasteces, sucn as wastes and carbon dioxide,
asJSmey metateofes. Over time, inese accumulating sub-
stances, called metabolites, may interact with toxics, result-
ing in increased, decreased, or otherwise afteredttsxiGSy,
In static renewal tests, fresh eSiuerfi replaces all or
part of the effluent In Jest dhsmfeess at specified intervals.
For example, a static renewal test lasting SB tews rasay
specify fresh effluent at 24 hours, 48 hours, and 72 hours.
Lab technicians accoiuplish renewal either by transferring
test organisms to fresh effluent at the same dilution or by
replacing all or part of the effluent in test chambers. Static
renewal tests reduce the loss of toxics associated with the
nonrenewal static test.
The flow-through test provides adJ#erej£set-up for
a toxicity test by continuously pumping fresh effluent or
effluent dilution through test chambers. Fresh effluent
can come directly from the outfall. Alternatively, person-
nel can place grab samples or composite samples in a
holding tank to provide fresh effluent for pumping through
sample chambers. Flow-through tests are conducted on-
site. •
The flow-through test has several advantages overthe
static test. First, it gives information about fluctuations in
the toxicity of a facility's effluent. Second, dissolved oxygen
levels remain higher in a flow-through test than in a static
test. Third, this method of testing reduces the loss of toxic
substances to adsorption and volatilization. Finally, the
continuousftow method prevents metabolitesfrom building
up and interacting with toxics.
Although flow-through tests have many advantages
over static tests, they also have some disadvantages. The
flow-through test does not provide information about the
persistence of toxicity. Such tests require large volumes of
effluent and dilution water. They also entail complex and
expensive equipment, which requires more maintenance
than the equipment used for static tests.
Static tests are tests that use the same effluent sample
throughout the test or in which only limited replacement of
the sample effluent occurs. The flow-through test provides a
different set-up for a toxicity test by continuously pumping
fresh effluent or effluent dilution through test chambers.
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Test Organisms
Toxicotogists generally choose as test organisms spe-
cies that biologists have studied thoroughly and that are
known to be sensitive to many substances. In addition,
species used as test organisms must be readily available
as well as easy to maintain and culture under laboratory
conditions. Table 3 lists the species commonly used in
chronte toxicity tests. Acute toxicity tests encompass a
larger number of species.
Several factors determine which species a protocol
requires for a given test. These factors include both the
pollutants present in the effluent and the sensitivity of test
organisms to these pollutants. Whether a facility dis-
charges into freshwater or seawater may also influence the
choice of test organism. If a permit specifies a toxicity limit
lor the eff fuent itself, then toxicity tests may use freshwater
organisms even if the facility discharges into salt water. If
a permit specifies a facility's toxicity limit in the water that
receives the effluent, then tests will use freshwater organ-
isms for a freshwater receiving water and marine organ-
isms for a salt water receiving water.
The species chosen for a test may not be a major
inhabitant in the discharging facility's location. In many
cases, the test species may not inhabit the facility's locale
at all. .Such circumstances frequently cause regulators and
permittees to wonder why tests do not employ local spe-
cies. EPA has several reasons for discouraging the use of
resident organisms:11
• Studies show that species commonly used as test
organisms represent the range of sensitivity shown by
resident species in ecosystems currently subject to
testing.
Receiving waters may lack sensitive species as a
result of previous exposure to pollutants.
Many States require collecting permits, which may
prove difficult and time-consuming for facilities or
testing laboratories to obtain.
Using resident species imposes additional burdens for
quality control to ensure that all organisms belong to
the same species, fall within the appropriate age
range, and do not vary in condition as a result of
handling procedures or seasonal environmental
changes.
A facility or laboratory using a resident species would
need to develop protocols for culturing and testing the
, species and for assessing inter- and intra-laboratory
variability. Such additional tasks might well prove
time-consuming and expensive.
The Discharge Monitoring Report
The report containing the results of tests, including
toxicity tests, performed on effluents is called a discharge
monitoring report (DMR). For tests performed on-site,
the permittee prepares the DMR, following the specifica-
tions outlined inthe permit. Fortests performed off-site, the
contracted laboratory prepares the report. The laboratory
sends the report to the facility, and in some cases sends a
copy directly to the regulatory agency as well.
11 TSD for Water Quality-based Toxics Control, p. 17.
Tables.
Species Commonly Used as Test Organisms
in Chronic Toxicity Tests
TYPE OF ORGANISMS FRESHWATER SPECIES
VERTEBRATE • fathead minnow, Pimephales
promelas
INVERTEBRATE • acladoceran, or water flea,
Ceriodaphnia dubia
ALGA • a green alga, Selanastrum
capricornutum
MARINE SPECIES
• sheepshead minnow,
Cyprinodon variegatus
• mysid, or shrimp,
Mysiodopsis bahia
• sea urchin, Arbacia
punctulata
»
• a red macroalga, Champia
parvula
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Toxiclty Testing
What information should a DMR provide? The DMTV
must show the test result, such as the LC, LOEC, NOEC,"
or 1C. These results should represent appropriate statisti-
cal analysis of the "raw data*—the unanalyzed numbers
obtained from each test chamber and entered on a data
sheet. Many States and EPA Regional Offices require
DMRs to include these raw data along with the test results.
As an example, the data sheet for an acute toxicity test
measuring LC50 would include the number of test organ-
isms alive in each test chamber at different times after the
start of the experiment, ending at 96 hours. The data sheet
would also report on additional parameters, such as the
amount of dissolved oxygen, the pH, and the temperature.
Appendix C outlines the information that a DMR should
contain. In addition to the test results and, in some States,
raw data, the DMR also includes a sheet detailing quality
assurance of the test. Quality assurance is discussed fully
in the following section.
Compliance with Whole Effluent
Toxicity Limits
To determine whether the facility's effluent remains
within the limits of the permit, the permit specifies the.
means for monitoring compliance, these include, but are
not limited to, self monitoring reports, a quality assurance
(QA) summary of the WET test, and inspections. Regu-
latory agencies also monitor compliance by investigating
citizens' complaints about a facility. Quality assurance
has a major role in compliance, assessing the validity of
test results. ': - .
Self-monitoring Reports
Self-monitoring reports include DMRs and also re-
ports on progress in maintaining compliance schedules.
The regulatory agency receives these reports and enters
data from them into the Permit Compliance System
(PCS), a national computerized database. PCS flags
violations of permit limits, compliance schedules, and re-
porting schedules. PCS has additional functions, including
automating the preparation of the Quarterly Noncompli-
ance Report (QNCR). This report lists facilities that have
violations requiring attention.
.Because the PCS is a computerized system, monitor-
ing compliance involves having the acceptable limits forthe
DMR accurately reflect the permit writer's intention. To
ensure this close correspondence, the permit writer should
- record the acceptable test limits in a sample DMR. PCS
personnel can then enter these limits into the database.
Dischmg® Monitoring Report/Quality
Assurance
Because sett-momtomg constitutes such a large part
of ooropfianoe rajGriitenng, is® EPA has instituted a pro-
gram called Discharge itonttorJng Bepon/Quallty As-
surance (DMR/QA) to track the quality of self-monitoring..
This program determines whether a permittee can analyze
and report data accurately. The permittee receives a
sample containing the substances occuFringtoiVhetacitity's
effluent. Using the appropriate tests, the permittee must
analyze the sample and Tepoit results.
Regulatory agencies note whether these results fal
within an acceptable range of the actuaipoJfufarrt concen-
tration in the sample. The regulatory agency then follows
up with the permittee on poor test results or late submittal
of test results. In the past, this program has applied only to
chernteal analyses, but as of 1991 the EPA has extended
it to include toxicity testing.
Inspections
A third aspect of compliance monitoring involves on-
site inspections conducted bytheregulatory agency. These
include inspections that focus on records and facility opera-
tion and others that collect samples for independent analy-
sis. The Performance Audit Inspection and the Compli-
ance Evaluation Inspection concern records and facility
operation. A Performance Audit Inspection (PAI) evalu-
ates a permittee's self-monitoring program, verifying re-
ported data and compliance by checking records. A PAI
also includes an inspector's observing the self-monitoring
process from the collection of samples through laboratory
analysis and reporting. Like the PAI, the Compliance
Evaluation Inspection (CEI) examines records and makes
observations, butthe emphasis differs from a PAI. The CEI
concentrates on records and only briefly observes the
facility, its effluent, and its receiving water.
Another inspection, the Compliance Biomonttoring
Inspection (CBI) requires the collectionof effluent samples
for analysis by acute and chronic toxicity tests. In addition,
the CBI includes the examination of records and the brief
observations of the CEI.
Discharge Monitoring
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Major facilities receive annual inspections. These
inspections serve five functions:
• Verification of permittee compliance,
• Development of enforcement information,
• Response to citizen complaints,
• Collection of information for permit development, and
• Maintenance of a regulatory presence.
The NPDES Compliance Inspection Manual (May
1988) details different types of inspections and procedures
for carrying them out.
.Citizen Complaints
Citizens'concerns form part of the monitoring process.
When citizens make complaints about af acility's operation,
these are registered with a State or EPA Regional Office.
The regulatory agency then follows up the complaints with
a review of the facility's self-monitoring data, an inspection,
or both. -
The Role of Quality Assurance
In Compliance
NPDES permits place responsibility for quality assur-
ance with the permittee. In fact, some States and Regions
have developed QA forms, which permittees must submit
along with their self-monitoring reports. To monitor quality
assurance, then, regulators can examine self-monitoring
reports and QA forms to determine whethera permittee has
had tests performed and data analyzed according to the
terms and schedule set out in the permit and whether
controls forthese tests fall within acceptable limits. Appen-
dix C contains a sample of this form. '
Only valid testing can give valid results.
If the permittee has met QA standards, then test-
results and analyses are considered valid. If the permittee
has failed to meet these standards, the test results and
analyses are unacceptable. The regulator should spend no
more time studying them, and the permittee must repeat
the test. Only valid testing can give valid results. Invalid
tests constitute permit violations and leave a permittee
open to enforcement action.
Documents called chain of custody form an impor-
tant part of quality assurance. On these documents the
permittee and the laboratory record details about the col-
lection, handling, transport, and analysis of a sample.
Regulators can study the chain of custody to determine
whether permittees and contracting laboratories have fol-
lowed acceptable protocol. When a facility violates its
permit, the chain of custody may provide valuable informa-
tion about the source of the problem. Appendix C contains
a sample chain of custody form.
Violations of Permits Having
Whole Effluent Toxicity Limits
, What happens when a facility violates a permit limit or
requirement that relates to whole effluent toxteity? For
permits with water-quality based limits, regulators must
review any violation for potential impact on water quality.
The EPA guidelines set out in the Enforcement Manage-
ment System forthe National Pollutant Discharge Elimination
System (September 1986) suggest that regulators gear
responses to the level of the violation.
Minor violations may require no more than a telephone
call or a letter. A violation that results from improper
analytical methods may result in a warning letter, called a
Notice of Violation (NOV) or a Section 308 letter, which
requires the permittee to repeat the tests using acceptable
procedures. In tracking a permit that has "monitor only"
requirements, qualified regulatory personnel must deter-
mine whether to establish a discharge limit.
Types of Violations
As mentioned earlier, the QNRC generates a list of
violations that require attention. These include, but are not
limited to, violations regarding compliance schedule mile-
stones, DMRs, and effluent limits. Compliance schedule
. milestones concern actions required of a facility to meet or
return to compliance standards. When a permittee misses
one of these milestones by more than 90 days, the permit-
tee is in violation. Failing to submit a DMR withing 30 Days
of its due date also constitutes a violation. In addition,
effluent violations constitute permit violations. Any effluent
violation, but particularly one with a potential for adversely
affecting water quality, requires review or action. Regula-
tors should consult their regional Environmental Services
Division to determine whether a particular violation has
such a potential.
14
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Toxlcity Testing
When a regulator becomes aware of an effluent viola-
tion, he or she should gather as much informatioir as
possible about the violation. The regulator should examine
the DMR to determine whether the test was run according
to protocol. Nextthe regulatorwill send aSection308 letter,
requesting information about processing at the facility and
inquiring about any problems occurring at the time of the
violation. The regulator may also request additional test-
ing. The regulator should remind the permittee of the
responsibility to report within 24 hours any violation that
may endanger health or the environment and to submit in
writing within five days a noncompliance report of the
circumstances of such a violation. If the violation does not
pose a threat to health or the environment, the permittee
can submit the report at the time of the next DMR. A
noncompliance report should address the cause of non-
compliance, the anticipated duration, and the steps taken
or planned to correct the situation. When the violation
concerns a water quality-based toxicity limit, the permittee
must also examine plant management to see whether
changes will reduce the impact on the environment.
When a permittee realizes that the facility is going to
violate a permit limit, he or she must take steps to prevent
or minimize the violation and its potential impact. These
include additional monitoring, a review of in-house pro-
cesses, and a review of self-monitoring QA to determine
whether the facility's personnel can solve the problem.
The Role of the Toxicity Reduction
Evaluation
If the permittee cannot correct the violation immedi-
ately or if such violations occur frequently, a study called a
Toxicity Reduction Evaluation (TRE) follows. A TRE
investigates a specific site in a stepwise fashion to discover
the cause of the toxicity, locate its source, determine
effective measures for reducing the toxicity, and evaluate
these measures. (See Figure 6.) The ultimate goal of the
TRE is to return the facility to compliance with its effluent
limit. Some NPDES permits require a TRE when a violation
of the limit occurs. If a permit has no such requirement, the
regulatory agency will seek a formal enforcement action to
initiate a TRE and to establish a schedule for implementing
the plan.
While regulators should provide oversight during the
TRE, the permittee has responsibility for carrying out the
study. To assist permittees' in this task, the regulatory
agency may wish to refer them to the relevant EPA
publications:
• Generalized Methodology for Conducting Industrial
Toxicity Reduction Evaluations (EPA/600/2-88/070)
Figure 6 Flowchart lor a Toxicity
Reduction* Evaluation (TRE)
Violation that Facility
Cannot immediately correct
TRE Plan
Compiling Information and Data
Evaluation of In-house Processes
Toxicity Identification Evaluation
Treatment of
Effluent
Identification of
Sources) of
Effluent Toxicity
Alteration of
In-house
Processes
Removal of Toxicity
at its Source
Implementation of
Toxicity Reduction
Confirmation of Toxicity Reduction
Toxicity Reduction Evaluation Protocol for Municipal
Wastewater Treatment Plants (EPA/600/2-88/062)
Methods for Aquatic Toxicity Identification Evaluations:
- Phase 1 Toxicity Characterization Procedures
(EPA/600/6-91/003)
- Phase 2 Toxicity Identification Procedures
(EPA/600/3-88/035)
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- Phase 3 Toxicity Confirmation Procedures
(EPA/600/3-88/036)
* Toxlcity Identification Evaluation: Characterization of
Chronically Toxic Effluents, P/7ase/(EPA/600/6-91/005).
Preparing a THE plan constitutes the first step in
conducting a TRE. This plan needs to include a description
of the study, the contractor who will perform the study, and
relevant background information on the facility. The TRE
plan also includes a schedule for conducting specif ic tasks,
such as final toxicity reduction and confirmation, and for
reporting the results of these tasks to the regulator. The
regulator then evaluates the TRE plan and notifies the
permittee of any shortcomings. Regulators should evalu-
ate the schedule as well as the plan. Though a state may
approve TIE/TRE plans, EPA does not recommend doing
so. Approval of a plan could imply that the regulator
accepts liability if carrying out the plan fails to return the
facility to compliance.
When the facility has submitted the plan to the regula-
tor, the facility must then put the plan into action. This
entails compiling all the available data on the plant's
processes, self- monitoring results, and operating guid-
ance. With this information, the facility conducts an evalu-
ation of its in-house processes.to determine whetherthese
contribute to the violation. In-house processes include
cleanup of spills, maintenance of machinery, and the
operation of treatment systems. If any of these has
contributed to the violation, the facility should institute steps
to correct these areas. Note that these "self- examination"
tasks are among the steps that a facility should automati-
cally follow when it has violated its permit. In addition, a
facility should include results of these tasks in its noncom-
pliance report, which becomes due at the latest with the
next DMR.
If these areas have not caused the violation, the facility
then may begin a Toxlcity Identification Evaluation
(TIE), an evaluation of the waste product at each step in
production or waste treatment. The first phase of a TIE
consists of additional testing, which starts as soon as
possible after a violation has occurred and even before it is
known whether a TIE will be necessary. The additional
testing establishes the effluent's variability and indicates
the severity, frequency, and duration of toxicevents. Sepa-
ration of the constituents of the waste stream into volatile
compounds, metals, pesticides, biocides, and other poten-
• tially toxic components should identify the class of sub-
stances causing toxicity.
With the toxics' classes identified, the permittee can
determine a means for resolving the problem. The permit-
tee may decide to treat the waste so as to render it nontoxic.
Alternatively, the permittee can identify the source of toxic-
ity and eliminate it at the source. An industrial facility may
elect to accomplish this by substituting a different chemical
in a particular process, substituting the process, or elimi-
nating the process alto-
gether. To bring about a ,
reduction at the source of
toxicity, a municipal facility
can impose local limits or a
pretreatment requirement.
Public education may also
provide a solution for a
municipal facility.
Once a permittee has
The ultimate goal
of a TRE is to
return the facility
to compliance with
its effluent and
limit.
determined a method for
reducing toxicity, he or she
must implementthe method
and confirm to the regulatory agency that this method
reduces toxicity to the limits set forth in the permit. When
the permittee has achieved this, the facility has returned to
compliance, and the TRE is complete.
If tests do not confirm an adequate reduction in toxicity,
the TRE process continues until it achieves its goal. In
some cases, the permittee does not succeed in achieving
source identification and reduction. In such an instance the
permittee must apply the other means for returning to
compliance: treating its waste to eliminate toxicity or ceas-
ing to discharge.
Case Histories of TREs
The following cases provide a brief glimpse of the TRE
process.
Municipal
In New Jersey, a small community of 100 homes had
an unmanned treatment plant. When regulatory authorities
inspected the plant, they discovered a toxicity problem.
Since the plant had no industrial input and its in-house
processes were running according to design standards, the
toxicityhadtoenterthesystemfromahousehold. Concen-
tration and separation of the waste stream revealed high
levels of silver. The TRE traced the entry point of the silver
to a householder's basement darkroom. The owners of the
home were asked either to limit the quantity of film that they
developed or to pay for an upgrade to the plant so that it
could treat the toxicity.
Industrial
A facility that manufactured pantyhose violated its
whole effluent toxicity limit. As with the waste-treatment
plant in New Jersey, in-house operations and professes
functioned adequately. The TIE revealed high levels of
copper and other metals as well. Rather than treat ther
16
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Toxlclty Testing
wastestream with chelators to tie up the metals, the facility
opted to reduce the source of toxicity. To this end they
limited the volume and altered the mix ratio of dyes in their
vats.
Municipal
When a municipal treatment plant violated its whole
effluent toxicity limits, personnel cleaned up in-house pro-
cesses. This effort reduced the level of toxicity but did not
eliminate it. A TIE revealed high levels of pesticides in the
effluent, and a pretreatment review showed that a pesticide
manufacturer on the influent wastestream was not operat-.
ing at full efficiency. The industrial plant had two problems.
First, while its settling pond was designed to have a
retention time of 4 to 7 days, dye studies determined the
actual retention tfme as only 1.5 hours. Second, when
workers rinsed the storage terels for the pesticides, they
tlumped ttse rinse wafer iirecUy into the wastestream.
From the wastestream the rinse water flowed into the pond
without pretreatment. Large slugs of pesticide passed
through me settling pond and entered the municipal teal-
ment plant's influent. The TRE estabSShecf teo control
strategies. First, the pesticide nrarajfacawmgfacifity dredged
its settling pond and installed a baffling system in it to
increase retention time. Second, waters rinsed storage
barrels at a different site.
Though these accounts provide only simpified ver-
sions of the actual handling of the toxicity violations in-
volved, they do suggest the scope of problems that occur
and the methods that resolve them.
17
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Toxlclty Testing
Appendix A
ERA'S Authority to Regulate Toxicity ,
Authority to Regulate Toxic Pollutants
Several sections of the Clean Water Act give the EPA authority to regulate for toxic chemicals:
Sec. 101(3)
"The objective of this Act is to restore and maintain the chemical, physical and biological integrity of the Nation's
waters."
Sec. 101(aM3)
Declaration of Goals and Policy - "it is the national policy that the discharge of toxic pollutants in toxic amounts be
prohibited...." .
" \ - '
Sec.301(a)
"Except as in compliance with this section and sections 302,306,307,318,402, and 404 of this Act, the discharge
of any pollutant by any person shall be unlawful."
Sec! 301(b)(i)(C)
"In order to carry out the objective of this Act there shall be achieved not later than July 1,1977, any more stringent
limitation, including those necessary to meet water quality standards...."
Sec. 302(a)
'• • • ' • ' , • • •' • " • '
provides the authority to establish water quality-based effluent limitations on discharges that interfere with the
attainment or maintenance of that waterquality which shall assure protection of public health, public water supplies,
and the protection and propagation1 of a balanced population of shellfish, fish and wildlife.
Sec. 303(c)(2)(B)
authorizes the adoption of numeric water quality criteria that are based upon biological monitoring or assessment
methods and the use of effluent limitations or other permit conditions based on or involving biological monitoring
or assessment methods or previously adopted numeric criteria. "Nothing in the 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...."
, . s
Sec. 304{a)(8)
requires EPA to develop and publish information on methods for establishing and measuring water quality criteria
for toxic pollutants including biological monitoring and assessment methods.
Sec. 308(a)
authorizes the installation, use and maintenance of biological monitoring methods by point sources, where
appropriate, for the development of effluent limitations or the determination of compliance with such limitations,
prohibitions, or effluent standards.
'-•'''•' ••'''•-'." .' 19
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Sec. 402
authorizes issuance of a permit for the discharge of any pollutant, or combinations of pollutants upon the condition
that the discharge meet all applicable requirements and provisions of the CWA.
40 CFR 122.44(d)
"In addition... each NPDES permit shall include conditions meeting the following requirements when applicable...
Water Quality Standards and State requirements: any requirements in addition to or more stringent than
promulgated effluent limitations guidelines or standards.., necessary to: (1) achieve water quality standards
established under section 303 of the CWA, including State narrative criteria for water quality."
(i) "Limitations must control ail pollutants or pollutant parameters... which... 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...." ,
(iv) (Numeric criterion for whole effluent toxicity)'
(v) "...has the reasonable potential to cause, or contributes to an in-stream excursion above a narrative.
criterion..., the permit must contain effluent limits for whole effluent toxicity...[except] where chemical-
specific limits for the effluent are sufficient to attain and maintain applicable numeric and narrative State
water quality standards."
Definition of the Regulated Community
The Clean Water Act (CWA) requires that every point source discharger have a permit. The regulated community,
then, could include the entire NPDES program. The Water Quality Act of 1987 provides criteria to reduce the regulated
community for toxicity limits to a more manageable level.
40CFR122.44(d)(1)(IH)
NPDES permits must include effluent limitations for every pollutant that causes, has the reasonable
potential to cause, or contributes to an excursion above a numeric water quality criterion.
40CFR122.44(d)(1)(lv)
NPDES permits must include whole effluent toxicity limitations when a discharge causes, has the
reasonable potential to cause, or contributes to an excursion above a State numeric criterion for whole
effluent toxicity.
40 CFR 122.44(d){1)(v)
When a discharge causes, has reasonable potential to cause, or contributes to an excursion above a State
narrative water quality criterion, the permit must contain limitations on whole effluent toxicity. An exception
exists where chemical-specific limitations achieve all applicable water quality standards.
/ .
40CFR122.44(d)(1)(vi)
Where an actual or projected excursion above a water quality criterion is attributable to a particular pollutant
for which the State has not adopted water quality criterion, the permit must.contain water quality-based
effluent limitations to control the pollutant 01 concern.
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Toxicity Testing
Appendix B
Excerpts Taken From Sample NPDES Permits
and Relevant to Whole Effluent Toxicity Testing
Excerpts from an NPDES permit issued to a wastewater treatment facility and requiring
acute toxicity limits ...... , ,„ J „ 23
Excerpts from an NPDES permit issued to a wastewater treatment facility and requiring
both acute and chronic toxicity limits .!' 27
Excerpt from an NPDES permit requiring acute toxicity testing '.: ; .„„; ..31
Excerpt from an NPDES permit requiring chronic toxicity testing ...„ :.... ..33
21.
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Toxlctty Testing
Excerpts from an NPDES Permit issued to
a Wastewater Treatment Facility
and Requiring Acute Toxicity Limits
PERMIT NO. ABOOXXXXX
Major POTW
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
REGION IV
AUTHORIZATION TO DISCHARGE UNDER THE
NATIONAL POLLUTANT DISCHARGE ELIMINATION SYSTEM
In compliance with the provisions of the Clean Water Act, as amended (33 U.S.C. 1251 et seq.; the "Act"),
County, Any State
Public Works Department
Office of Environmental Services
Road
. Anv State
is authorized to discharge from a facility located at
' ' Wastewater Treatment Plant
Road
_, Any State
to receiving waters named
The Atlantic Ocean
in accordance with effluent limitations, monitoring requirements and other conditions set forth herein. The permit consists
of this cover sheet, Part I fi pages, Part II J£ pages, Part III 5 pages, and Part IV g pages.
This permit shall become effective on January 1,1991
This permit and the authorization to discharge shall expire at midnight, March 31,1994
September 2$. 1990 . ' ---
Date Issued
John Doe, Director
Water Management Division
2S
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Permit No. ABOOXXXXX
Parti
EFFLUENT LIMITATIONS AND MONITORING REQUIREMENTS - Final
During the period beginning on the effective date and lasting through the expiration date of this permit, the permittee is
authorized to discharge from outfall(s) 001, sanitary wastewater.
Such discharges shall be limited and monitored by the permittee as specified below:
PARAMETERS
Flow, MOD
DISCHARGE LIMITATIONS
MONITORING REQUIREMENTS
Annual Monthly Weekly Measurement Sample Sampling
Average Average Average Frequency Type Point
Report Report Continuous Recording Effluent
, flowmeter.
& totalizer
Carbonaceous
Biochemical
Oxygen Demand
(5-Day)
25.0 mg/l 25.0 mg/l
Total Suspended 30.0 mg/l 30.0 mg/l
Solids-
Fecal Conform
Bacteria, N/1 00 ml
Total Residual
Chlorine
pH (standard units)
Acute Whole
Effluent Toxicity
Total Nitrogen,
as N (mg/l)
Total Phosphorus,
as P (mg/l)
See Item 3
See Item 9
See Item 5
See Item 12
Report
Report
40.0 mg/l 7days/week 24hr.composite Influents
Effluent
45.0 mg/l 7days/week 24 hr. composite Influents
Effluent'
7 days/week Grab
7 days/week Grab
Continuous Recorder
See Part IV Grab
Effluent
Effluent
Effluent
Effluent
1/month 24 hr. composite Effluent
1/month 24 hr. composite Effluent
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Toxicity Testing
12.
- Permit No. ABOOXXXXX
Lethality to more than 50% of any test species in 100% effluent in a test of 96 hours duration or less wSJ constitute
a violation of Any State's Administrative Code and the terms of this permit The testing forthis reqyfreime^ must
conform with Part IV of this permit.
, Part IV
Whole Effluent Toxicity Testing Program
As required by Part_of this permit, the permittee shall initiate the series of tests described below beginning in January 1991
to evaluate whole effluent toxicity of the discharge from outfall __. AH 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/600/4-90/027, orthe most current edition. The control water and effluent used will be adjusted to asatiniiy
of 20 parts per thousand using artificial sea salts as described in EPA/600/4-90/027, Section 5 (orthe most cunert edition).'
In addition, for the inland silverside test, feeding and solution renewal shall be done at 48 hours with a portion of the original
sample thathas been kept refrigerated. A standard reference toxicant quality assurance test shalfbe conducted concurrently
with each species used in the toxicity tests and the results submitted with the discharge monftoraig report (DMR).
Alternatively, if monthly QA/QC reference toxicant tests are conducted, these results must be submitted with the DMR.
The permittee shall conduct 48-hour acute static toxicity tests using the mysid shrimp (Mysidopsis bahia) and 96-
hour acute static-renewal toxicity tests using the inland silverside (Menidia beryllina). All tests will 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 (under full dilution series) in order to catch any peaks of toxicity and to account for daily variations
in effluent quality^
1. a.
b.
2. a.
b.
3. a.
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. If, in
any separate grab sample test, 100% mortality occurs prior to the end of the test, and control mortality is less than
10% at that time, that test (including the control) shall be terminated with the conclusion that the sample
demonstrates unacceptable acute toxicity.
The toxicity tests specified above shall be conducted once every two months until 6 valid bimonthly tests have been
completed, and once every 6 months thereafter for the duration of the permit, unless notified otherwise by EPA.
These tests are referred to as "routine" tests.
Results from "routine" tests shall be reported according to EPA/600/4-90/027, section 12, Report Preparation (or
the most current edition), and shall be submitted as an attachment to the DMR. Such results are to be entered on
the DMR in the following manner: the LC^ shall be reported as the % effluent that killed or would kill 50% of the test
organisms.
If unacceptable acute toxicity (greater than 50% lethality of either test species in any of the four separate grab
sample tests within the specified time) is found in "routine" test, the permittee shall conduct three additional acute
toxicity tests on the species indicating unacceptable toxicity. For each additional test, the sample collection
requirements and test acceptability criteria specif ied 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 u ntil three 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" tests, shall be submitted
in a single report prepared according to EPA/600/4-90/027, Section 12, Report Preparation (or the most current
edition) and submitted within 45 days of completion of the third additional, valid test.
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4. All tests shall be conducted using a full dilution series. For those tests conducted prior to the effective date of the
total residual chlorine limit, samples of effluent which have been artificially dechlorinated must be used. For those
tests conducted after this date, samples of final effluent must be used.
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Toxlctty Testing
Excerpts from an NPDES Permit Issued to
a Was tewater Treatment Facility
and Requiring Both Acute and Chronic Toxicity Limits
PERMIT NO, ABOQYYYYY
Major POTW
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
REGION IV
AUTHORIZATION TO DISCHARGE UNDER THE
NATIONAL POLLUTANT DISCHARGE ELIMINATION SYSTEM
In compliance with the provisions of the Clean Water Act, as amended (33 U.S.C. 1251 et seq.; the "Act"),
. City, Any State .
P.O. Box 00000
• Any State
is authorized to discharge from a facility located at "
' • • • Water Reclamation Plant
Parkway
.Any State
to receiving waters named
River
in accordance with effluent limitations, monitoring requirements and other conditions set forth herein! The permit consists
of this cover sheet, Part IS pages, Part II Ifi pages,'Part III a pages, and Part IV 2 pages.
This permit shall become effective on January 1,1992
this permit and the authorization to discharge shall expire at midnight, September 30, .1996
September 26. 1990
Date Issued
John Doe, Director
Water Management Division
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Permit No. ABOOYYYYY |
Parti
EFFLUENT LIMITATIONS AND MONITORING REQUIREMENTS - Final
During the period beginning on the effective date and lasting through the expiration date of this permit, the permittee is
authorized to discharge from outfall(s) 001, sanitary wastewater.
Such discharges shall be limited and monitored by the permittee as specified below:
PARAMETERS
Flow, MGD
DISCHARGE LIMITATIONS
MONITORING REQUIREMENTS
Annual Monthly Weekly Measurement Sample
Average Average Average Frequency Type
^„K ^5«»%^»^ - ^3Av%A*4 /^*^r*4««» *4t* i ft D
Report
Report Continuous Recording
Flowmeter
* & Totalizer
Sampling
Point
Effluent
Biochemical
Oxygen Demand
(5-Day)
Total Suspended
Solids
Fecai Coliform
Bacteria, N/100 mr
Total Residual
Chlorine
pH (standard units)
Acute Whole
Effluent Toxidty
Chronic Whole
Effluent Toxidty
20.0 mg/l 30.0 mg/l 45.0 mg/l 5 days/week 24 hr.
Report Report 5 days/week composite
20.0 mg/l 30.0 mg/l 45.0 mg/l 5 days/week 24 hr.
Report Report 5 days/week composite
See Item 3 5 days/week Grab
See Item 8 7 days/week Grab
See Item 4
See Hem 10 (a)
See Item iO(b)
Continuous
See Part IV
See Part IV
Recorder
24 hr.
composite
24 hr.
composite
Effluent &
Influent
Effluent &
Influent
Effluent
Effluent
Effluent
Effluent
Effluent
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Toxicity Testing
~ •_.'.' Permit ABOOYYYYY
10 a. Lethality to more than 50% of any test species in. any percentage effluent in a test oi 96 hours duration or less will
constitute a violatfoqpf Any State's Administrative Codeanditie termsof Ihispermit. Thetestingforthis requirement
shall conform with Fsart IV of this permit.
b. 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 15%foranytestspetieswleonstitute
... a violation of the terms of this permit The testing for this requirement shall conform with Part IV of this permit.
Part IV
Whole Effluent Toxicrty Testing Program
As required by Part _ of this permit, the permittee shall initiate the series of tests described below bepMHisg In January-
1992 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 Toxicfty of Effluents and Receiving
, Waters to Marine and Estuarine Organisms, EP A/600/4-87/028, and Methods for Measuring the Acute Toxicity of Effluents
to Freshwater and Marine Organisms, EPA/600/4-90/027, orthe 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/600/4-87/028, Section 7 (orthe most recent 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. V
1'. a The permittee shall conduct a 7-day Mysid shrimp (Mysidopsis bahia) Growth and fecundity test and an Inland
silverside (Menidia beryllina) Larval Survival and Growth test. These tests shall be conducted using a full dilution
series including one which is equivalent to the Receiving Water Concentration (RWC) of the effluent in the receiving
water at critical conditions. Unacceptable chronic toxicity will be demonstrated if either test results in a no
observable effect concentration (NOEC) less than 15% effluent. All test results shall be statistically analyzed
according to Appendix H,EPA/600/4-91/000 (orthe most current edition).
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 8.1.4.2, EPA/600/4-91/000, or the most current edition.
All test solutions shall be renewed daily. If test results do not meet the acceptability criteria of either Section 13.12
or Section 14.12, EPA/600/4-87/028 (orthe 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 100% mortality Occurs in the RWC test concentration prior to the end of the test and control mortality is acceptable
at that time, that test.(including the control) shall be terminated with the conclusion that the sample demonstrates
unacceptable chronic toxicity.
2. a. The permittee shall also conduct a 48-hour acute static test on the Mysid shrimp (Mysidopsis bahia) arid a 96-hour
acute static^renewal test on the inland silverside (Menidia beryllina) using a full dilution series. Unacceptable acute
toxicity will be demonstrated if more than"50% lethality of either test species occurs in any dilution of the effluent
s'ample priorto the end of the test. All test results shall be statistically analyzed according to Appendix H, EPA/600/
4-91/000, orthe most current edition.
b. For each set of acute tests conducted, a fresh 24-hour composite sample of final effluent shall be used at Day 1
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of both the Mysid shrimp and the inland silversTde tests and at Day 3 of the inland silverside test. The composite
samples collected under Section 1 (b) above shall be used in the acute tests. For the inland silverside test, feeding
and solution renewal shall be done at 48 hours with a portion of the original sample that has been kept refrigerated.
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. If 100%
mortality occurs prior to the end of the test, and control mortality is less than 10% at that time, that test (including
the control) shall be terminated with the conclusion that the sample demonstrates unacceptable acute toxicity.
3. a. The toxicity tests specified above shall be conducted once every two months until 6 valid bimonthly tests have been
completed, and once every 6 months thereafter for the duration of the permit, unless notified otherwise by EPA.-
These tests are referred to as "routine" tests.
b Results from "routine" tests shall be reported according to EPA/600/4-87/028, Section 10, Report Preparation (or
the most current edition), and shall be submitted as an attachment to the DMR. Such results are to be entered on
the DMR in the following manner:
1. For the acute test results, if less than 50% survival of a test species occurs, the LC should be entered on the
DMR forthat species. If 50% or greater survival occurs, the LC that would have less than 50% survival should
be entered.
2. For the chronic test results, the NOEC should be entered on the DM R for that species.
4 a If unacceptable chronic toxicity (a NOEC less than 15% effluent.in either test) and/or unacceptable acute toxicity
(greater than 50% lethality of either test species in 100% effluent) 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 species indicating unacceptable toxicity. For each additional test, the test acceptability
criteria specified in section. V(b) 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 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 "routine1 test is still present.
1 For "routine" tests with unacceptable chronic toxicity, the permittee shall conduct additional Mysid shrimp
(Mysidopsis bahia) Growth and Fecundity and/or inland silverside (Menidia beryllina) Survival and Growth
multi-concentration tests, as appropriate. The tests will be conducted on a control, 100% effluent, and the
following % effluent concentrations: 0.25 x the RWC, 0.5 x the RWC, the RWC, and [the RWC +100]/2. The
sample collection requirements specified in section 1 (b) above shall be met.
2 For "routine" tests with unacceptable acute toxicity, the permittee shall conduct additional 48-hour acute static
toxicitytests using the Mysid shrimp (Mys/ctops/s bahia) and/or96-houracutestatic-renewaltoxicitytests using
the inland silverside (Menidia beryllina) with a full dilution series, per EPA/600/4-90/027. Four separate grab
• samples shall be collected at evenly-spaced (6-hr) intervals over a 24-hour period and used in four separate
tests.
b Results from additional tests, required due to unacceptable acute and/or chronic toxicity in the "routine" test, shall
' be submitted in a single report prepared according to EPA/600/4-91/000, Section 9, Report Preparation (orthe most
current edition) and submitted within 45 days of completion of the second additional, valid test.
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Toxicity Testing
Excerpt from an NPDES Permit
Requiring Acute Toxicity Testing
D. ACUTE BIOASSAY REQUIREMENTS
The Water Quality Standards of Any State require that ail waters be free from substances in concentmtfens ©rcara^jraaSons
which are harmful to humans, animals, or aquatic life (Any State, Water Quality Criteria for Intrastate, Interstate, and Coastal
Waters, Section 11.4. Minimum Conditions Applicable to All Waters, page 3, adopted March 22,1990) In accordance with
such requirements, the permittee is authorized to discharge from the combined outfall(s) 001 and 002 only in accordance
with the following conditions:
1.
2.
3.
4.
a.
b.
The permittee shall perform 48-hour static definitive toxicity tests in accordance with Methods for Measuring the
Acute Toxicity of Effluents to Freshwater and Marine Organisms Fourth Edition (EPA/6004-90/027). Static tests
will.be conducted on a 24-hour composite sample of effluent. Less than 36 hours will elapse between sampling and
the use of the sample.
The permittee must use both the following organisms:
(1) Pimephales promelas '(fathead minnows) ' .
(2) Ceriodaphnia dubia (water fleas) , •
Dilution water used for these tests shall consist of reagent grade water, defined as distilled or deionized water that
does not contain substances which are toxic to the test organisms. Dilution water shall consist of reagent grade
water to which the appropriate reagent grade salts have been added to make moderately hard dilution water
according to Short-Term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to
Freshwater Organisms Third Edition (EPA/600/4-91/000). These dilution waters will be deemed acceptable if the
control organisms in the toxicity tests meet the minimum EPA criteria for mortality.
The permittee shall conduct the first series of tests specified in part I above within 90 days of the issuance of the
permit. The test shall be conducted at a frequency of once per quarter for the first two years from the date of
issuance. Based on results of the eight tests, the Permit may be modified to include further testing requirements.
Following the first year of testing, the permittee may petition the State Environmental Quality Permit Board for
permission to use the most sensitive organism for the toxicity tests to be performed for the remainder of the permit
life, if a clear trend in the toxic response exists m the test data. The results of the 48-hour static definitive bioassay •
tests shall be reported to the State Environmental Quality Permit Board on the next monthly discharge monitoring
report.
If a 48-hour definitive toxicity test results in an LC^ value of less than 10.7%, the permittee shall immediately after
the first 48-hour definitive toxicity test results are finalized perform a second 48-hour definitive toxicity test. The LCM
determinations from these tests shall be reported to the State Environmental Quality Permit Board within 10 working
days after finalization of the results of each test.
In the event that the results of any 48-hour definitive toxicity test reveal that the LC^ of the permittee's effluent is
less than 10.7%, than this finding will constitute a violation of Part I of this permit, and the permittee shall:
a. Provide a schedule for the implementation of a Toxicity Reduction Evaluation Plan to reduce the toxicity of the waste
discharge to safe levels. (Safe levels will be determined by the State Pollution Control Permit Board).
In addition to the specific conditions of this permit, the permittee shall comply with all applicable conditions of 40 CFR 122.7
and 40 122.61 (2/5/90).
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Toxlctty Testing
Excerpt from an NPDES Permit
Requiring Chronic Toxicity Testing
E. CHRONIC'BIOASSAY REQUIREMENTS . , .
The Water Quality Standards of Any State require that all waters be free from substances in concentrations or contwnafes
which are harmful to humans, animal, or aquatic life (Any State, Water Quality Criteria for Intrastate and Coasfa? Waters,
section 11.4., Minimum Conditions Applicable to All Waters, page 3, adopted March 22, 1990). In accordance with s.ucri
requirements, the permittee is authorized to discharge from outfall(s) 001 only fn accordance with the following conditions:
1 . The permittee shall submit any existing toxicity data for review by the State Office of Pollution Control within 30 days
of the effective date of this permit. .'••».'
2. The permittee shall perform 7-day chronic toxictty tests in accordance with Short-Term Methods for Estimating the
Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms (EPA/600/4-91/000). Thesechronic
toxicity tests shall be initiated within 60 days of the effective date of issuance of the permit to evaluate wasiewater
toxicity.
a. Dilution water used for these tests shall consist of reagent grade water, defined as distilled ordeionized water that
does not contain substances which are toxic to the test organisms. Dilution water shall consist of reagent grade
. water to which the appropriate, reagent grade salts have been added to make moderately hard dilution water
according to Short-Term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to
Freshwater Organisms (EPA/60Q/4- 91/000). These dilution waters will be deemed acceptable if the control
organisms in the toxicity tests meet the minimum EPA criteria for mortality, growth, and fecundity.
b. The permittee shall conduct a Ceriodaphnia dubia Survival and Reproduction Test, and a Pimephales promelas
Larval Survival arid Growth Test on serial dilutions of effluent to determine if the discharge from outfall(s) 001 is
chronically toxic. Such testing will determine if the water affects the survival, growth, and fecundity of the test
organisms. Static renewal tests will be conducted on three 24-hourcomposite samples of effluent. Thefirst of these
composite samples will be used to set up the tests for the day 1 and day 2 renewals, the second of these composite
samples will be used to renew the tests on days 3 and 4, and the third composite sample will be used to renew the
tests on days 5 and 6. Not more than 36 hours will elapse between sampling and the first use of any of the composite
samples. Chronic toxicity will be demonstrated if: 1) there is a 20% or more difference in survival between test
organisms exposed to appropriate control water and any serial dilution of effluent; or 2) there is a statistically
significant difference at the 95% confidence level in reproduction between Ceriodaphnia exposed to an appropriate
control water and any serial dilution of the effluent; 3) there is a statistically significant difference at the 95%
confidence level in growth between Pimephales promelas exposed to an appropriate control water and any serial
dilution of the effluent.
c.
Such chronic toxicity tests shall be conducted once per quarter for the life of the permit, provided that each 48-hour
LC^ isgreaterthanorequalto 100%, and each chronic value is greater or equal to 100%. Thepermittee maypetition
the State Environmental Quality Permit Board to use only the most sensitive species if a cleartrend is demonstrated
in the toxic response of the test organisms.
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d. If any one chronic toxicrty test indicates the 48-hour LC^ is less than 100% or that the chronic value is less thari I
100%, the provisions in section 2(e) shall apply, and the permittee shall conduct another set of the two chronic
toxicity tests within two weeks. The results of each toxicity test shall be submitted to the State Office of Pollution |
Control within 2 weeks of completion of testing.
e. If the chronic value of any test is less than 100%, or if the acute 48-hour LC^ of any test is less than 100%, then
the effluent will be considered unacceptably chronically toxic and this result will constitute a violation of Part I of this
permit. The permittee will then be subject to the provisions of section 3.
3. In the event that after review of the above studies, the State Environmental Quality Permit Board determines the
waste stream is toxic to the receiving stream, the permittee shall provide a schedule for the implementation of a
Toxicity Reduction Evaluation Plan to reduce the toxicityof the effluent to safe levels. Safe levels will be determined
by the State Environmental Quality Permit Board. '
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Toxlcfty Testing
Appendix C §
1 i
Documents Relevant to Toxicity Testing
Report Preparationas described in Methods for Measuring the Acute Toxicity of Effluents
to Freshwater and Marine Organisms, EPA/6QQ/4-3Q/027 „ , 37
Chain of Custody Record : „.,.'..,..— 39
Quality Control Fact Sheet for Self-Biompnitoring Acute/Chronic Toxicity Test Data 41
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ToxjcHy Testing
Report Preparation
as described in Methods for Measuring the Acute Toxicity
of Effluents to Freshwater and Marine Organisms, EPA/600/4-90/027
The following general format and content are recommended for the report:
12.1 INTRODUCTION
1. Permit number
2. Toxicity testing requirements of permit
3. Plant location
4. Name of receiving water body ,
5. Contractor (if contracted)
a. Name of firm
b. Phone number '
c. Address
12.2 PLANT OPERATIONS
1. Product(s)
2. Raw materials
3. Operating schedule
.4. Description of waste treatment . "
5. Schematic of waste treatment
'6. Retention time (if applicable)
7. Volume of discharge (MGD, CFS, GPM)
8. Design flow of treatment facility at time of sampling
12.3 SOURCE OF EFFLUENT, RECEIVING WATER, AND DILUTION WATER
1. Effluent Samples
a.
b.
c.
d.
e.
f.
g-
Sampling point
Sample collection method
Collection dates and times
Mean daily discharge on sample collection date
Lapsed time from sample collection to delivery
Sample temperature when received at the laboratory
Physical and chemical data
2.
Receiving Water Samples
a. Sampling point
b. Sampling collection method
c. Collection dates and times
d. Sfreamflow at time of sampling and 7Q10
e. Lapsed time from sample, collection to delivery
f. Sample temperature when received at the laboratory
g. Physical and chemical data
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3. Dilution Water Samples
a. Source
b. Collection date(s) and time(s) (where applicable)
c. Pretreatment
d. Physical and chemical characteristics (pH, hardness, salinity, etc.) .
12.4 TEST CONDITIONS
1. Toxfcrty test method used (title, number, source)
2. Endpo'intlsioftest
3. Deviations from reference method, if any, and reason(s)
4. Date and time test started
5. Date and time test terminated
6. Type and volume of test chambers
7. Volume of solution used per chamber
8. Number of organisms per test chamber
9. Number of replicate test chambers per treatment
10. Feeding frequency, and amount and type of food
11. Acclimation temperature of test organisms (mean and range)
12. Test temperature (mean and range)
12.5 TEST ORGANISMS ,
1. Scientific name
2. Age
3. Life stage .
4. Mean length and weight (where applicable)
5. Source
6. Diseases and treatment (where applicable)
12.6 QUALITY ASSURANCE
1. Reference toxicant used routinely; source
2. Date and time of most recent reference toxicant test;
test results and current cusum chart
3. Dilution water used in reference toxicant test
4. Physical and chemical methods used
12.7 RESULTS
1. Provide raw toxfcity data in tabular form, including daily records of affected organisms in each concentration
(including controls)
2. Provide table of endpoints: LC50, NOEC, Pass/Fail
3. Indicate statistical methods used to calculate endpoints
4. Provide summary table of physical and chemical data
5. Tabulate QA data
12.8 CONCLUSIONS AND RECOMMENDATIONS
1. Relationship between test endpoints and permit limits
2. Action to be taken
3d
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Toxlctty Testing
. CHAIN OF C
Client Mama
Address
Attention
Telephone
Dish No.
c«
'Sample I.D.
Date
Collection
Time Grab Como
i
-
USTODV RECORD
Priority Numt
Container,
Vol #,of Tvt»
-
Preservative
Sample
TVDS
, -- ~-.--_... '••• -'" :
Page oT
Analysis required/Remarks
Analysis List 1: . . '""•', :
Analysis List 2: ' ,
Analysis List 3: ' ,
Collected and relinquished by:
Date/Time:
Relinquished by:
Date/Time;
Method of Shipment:
Date/Time:
Relinquished by:
Date/Time:
Received by: Date/Time
Received by: Date/Time
Received by: Date/Time
Received for n,.«./r-.—
Laboratory: Date/Time
Hazards Associated with Samples:
Comments:
pH Temp Res.Chlorine Flow
DO Comp
ositer Start/Stof
i
> / Other
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Toxicity Testing
Quality Control Fact Sheet for Self-BiomonStoring
Acute/Chronic Toxicity Test Data
Permit No..
Facility Name
Facility Location
Laboratory Investigator.
Permit Requirement
Sampling Location -_
Limit
Type of Test '
Test Flesults
' LC50/EC50/NOEC/IC25-
Qualitv Control Summary
Date of sample
Control Mortality.
Type of Sample.
Test Duration
Test Organism Age
95% Confidence Interval.
Statistical Method .
Dates of Test
Control Mean Dry Weight
. Temperature Maintained within,+/-1° of test temperature? .
* Yes No \ '•',
Dissolved oxygen levels always greater than 4 Omg/L?
Yes. No_
Loading factor for all exposure chambers less than or equal to maximum allowed for the test type and temperature?
, Yes No.
Do the test results indicate a direct relationship between effluent concentration and response of the test organism
(i.e., more deaths occur at the highest effluent concentrations)?
Yes 1 No , '.-•''"•..'
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Toxicity Testing
Appendix D
Glossary
Acute-to-chronIc ratio (ACR)
A ratio that compares the concentration of an
effluent or a toxic substance that causes acute
toxicity in a species with the concentration of an
effluent or a toxic substance that causes chronic
toxicity to the same species. The ACR makes
possible interconversion of acute toxicity units and
chronic toxicity units: TUg - (ACR)(TUC).
Acute toxicity test
A test that uses living organisms and measures an
effect that occurs within 96 hours. Acute toxicity
tests commonly, but not exclusively, measure the
death of organisms.
Acute toxicity unit (TUa)
The reciprocal of the LC5Q multiplied by 100. That
is,TUa = (1/LC50)(100).
Average monthly limit (AML)
In an NPDES permit, the highest value allowable
for the average of daily discharges occurring over
a one-month period. ,
Bioaccumulatlon
The passage of substances from the environment
into a living organism's tissues by means of ab-
sorption, respiration, or feeding. Also called bio-
logical accumulation.
Bloassessment
Evaluation of the biological condition of a body of
water based on studies of the organisms living
there and on the chemical and physical character-
istics of the body of water. Also called biological
assessment.
BioavailabilHy
The presence of a substance in a form that can
affect organisms. Also called biological availability.
Bloeoneentratfon
The passage of a substance from water into an
Organism by absorption through its skin or during
respiration. '
Biomagniflcation
, The process of a substance's passing up the food
chain and becoming more concentrated in organ-
isms toward the top of the food chain. Also called
biological magnification.
Blomonltoring
The use of living organisms to monitor water qual-
ity by such means as toxicity testing and
bioassessment. Also"called biological monitoring.
Biota
The organisms occurring in a specified area or
during a specified period.
Chemical-specific
Concerning a limit or test that involves specific
.chemical substances.
Chain of custody
A document for recording information concerning
the collection, handling, transfer, and analysis of
an effluent sample.
Chronic toxicity test
A test that measures a sublethal effect on test
organisms over an extended period of time, gener-
ally the life cycle of the test organisms.
Chronic toxicity unit (TUC)
The reciprocal of the NOEC multiplied by 100.
Thatis.TU =(1/NOEC)(100).
.w
Chronic Value (ChV)
In a chronic toxicity test, a presumably safe con-
centration between the LOEC and the NOEC. The
ChV is a point estimation calculated as the geo-
metric mean of the LOEC and NOEC.
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Compliance Blomonitoring inspection (CBI) "
At a facility with -an NPDES permit, an inspection
conducted by the regulatory agency or its contrac-
tor for the purpose of collecting effluent samples
for analysis by acute and chronic toxicity tests. In
addition, the CBI involves the examination of
records and the brief obseivations of the CEI.
Compliance Evaluation Inspection (CEI)
At a facility with an NPDES permit, an inspection
conducted by the regulatory agency or its contrac-
tor to evaluate a permittee's self- monitoring by
examining records and making brief observations
of the facility, its effluent, and its receiving water.
Composite sample
A mixed sample collected over a specified period
of time.
Control
In a toxicity test, a group of organisms exposed to
the same conditions as other groups of organisms
but not exposed to effluent.
CWA Clean Water Act.
• Discharge monitoring report (DMR)
A report of the results of tests that have been
' conducted on an effluent.
Discharge monitoring report quality assurance
(DMR/QA)
An EPA program that tracks the quality of self-
monitoring.
Dose-response curve
A graph that plots the concentration of a substance
against the test organisms' level of response.
Flow-through test
A test in which fresh effluent or freshly diluted
effluent is pumped through test chambers
continuously.
Food Chain
A series of organisms that sequentially feed on one
another.
Grab sample
A sample collected at one time.
Independent Application
The principle that no one of the three methods of
monitoring water quality—chemical analysis,
biomonitoring, or bioassessment—is inherently
superior to the other two.
Inhibition Concentration (1C)
In a chronic toxicity test of an effluent or substance,
the concentration causing an inhibition of a biologi-
cal function, such as reproduction, in test organ-
isms. ICs are reported as the concentration at
which test organisms show a specified level of
inhibition: IC2g is the concentration at which the
biological function shows 25 percent inhibition in
test organisms.
Integrated Approach
In monitoring water quality, a threefold approach
that consists of chemical analysis, biomonitoring,
and bioassessment.
Lethal Concentration (LC)
In an acute toxicity test of an effluent or substance,
the concentration causing death in test organisms.
LCs are reported as the.concentration proving
lethal to a percentage of the test organisms: LC5Q
is the concentration at which 50 percent of the
organisms die.
Long-term average (LTA)
In an NPDES permit, the acceptable mean of an
effluent's pollutant concentrations or parameters
over the life of the permit or facility.
Lowest Observed Effect Concentration (LOEC)
In a chronic toxicity test of an effluent orsubstance,
• - the lowest concentration causing an observable
effect in test organisms.
Major facility
Either 1) a municipal permittee that has a design
flow of one million gallons per day or greater, a
service population of 10,000 or greater, or signifi-
cant impact on water quality, 2) any non-municipal
permittee that has an industrial rating of. 80 or
higher, or3) any discretionary permittee evaluated
as necessary to be declared a major.
Maximum daily limit (MDL)
In an NPDES permit, the highest value allowable
for a discharge during a 24-hour period.
No Observable Effect Concentration (NOEC)
In a chronic toxicity test of an effluent orsubstance,
the highest concentration at which no observable
effect occurs in test organisms.
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Toxicity Testing
No Observable Effect Level (NOEL)
Same as No Observable Effect Corvsentration. '
Notice of Violation (NOV)
A notice from the EPA region to the delegated
State stating that a violation of a facility's NPDES
permit has occurred and that if the State does not
take action, the Federal government will. The
permittee receives a copy of the NOV.
Performance Audit Inspection (PAI)
At a facility with an NPDES permit, an inspection
conducted by the regulatory agency or its contrac-
tor to evaluate the permittee's self-monitoring pro-
gram. Specifically, the inspection verifies reported
data and compliance by checking records and in
addition observes the self-monitoring process from
the collection of samples through laboratory analy-
sis and reporting. .
Permit Compliance System (PCS)
A national computerized database that contains
information about NPDES permits—including lim-
its, monitoring schedules, frequency of discharge,
inspections, permit date, and self-monitoring re-
ports—and that flags violations of permit limits,
' - compliance schedules, and compliance reporting.
Point source discharge
Generally, a discharge that originates at a specific,
identifiable location, such as a pipe, ditch, channel,
or tunnel. Point source discharges also include
discharges having less localized sources. CWA
(40 CFR 122.2) defines point source discharge,
and section 122.3 provides further clarification.
QA Quality assurance.
Quarterly Noncompliance Report (QNCRJ
A report issued quarterly and listing facilities that
have violations requiring attention.
Short-term chronic toxicity test
A chronic toxicity test that focusses on the period
in an organism's life cycle when it shows the
greatest sensitivity to its environment. Short-term
chronic toxicity tests, which must include at least
one-tenth of an organism's life cycle, generally last
7 days or less.
Static nonrenewal test
A static test that uses the same effluent for the
duration of the test.
Static renewal test
A static test in which fresh effluent replaces all or
part of the effluent at specified intervals.
Statte test
A test that uses the same effluent for the duration
of the test or in which only limited replacement of
the effluent occurs.
Technology based
Concerning a standard, requirement, or method
having existing technologies as its bass.
Test organism
A species of organism used in a biological test.
Toxicity
' Harmful effects occurring in a human, other ani-
mal, or plant as a direct result of a chemical
substance. - - •
Toxicity Identification Evaluation (TIE)
An evaluation of the waste product at each step in
production or waste treatment.
Toxicity Reduction Evaluation (THE)
A study that investigates a specific site in a stepwise
fashion in order to uncover effective measures for
controlling effluent toxicity and for resolving a
toxicity problem.
Wasteload allocation (WLA)
The maximum amount of pollutants that a body of
water can assimilate in a day from a specific facility
without violating the State's Water Quality
Standards.
Water quality based
Concerning a standard, requirement, or method
aimed at attaining a specified level of water quality
without regard to what technologies exist to achieve
this level.
Water Quality Standard (WQS)
A law or regulation that states the use of a body of
water, the numeric and narrative water quality
criteria necessary to protect this use, and an
antidegradation statement. , ' •
Whose effluent toxicity (WET)
A form of bfomonitoring that considers the total
toxic effect of an effluent as measured by a test
that uses living organisms.
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