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EPA
100-
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United States     Office of Water
Environmental Protection Office of Water Enforcement and Permits
Agency	Washington, DC 20460	
NPDES Compliance
Monitoring  Inspector
Training

Biomonitoring
               Repository Material
               Permanent ejection
                                              August 1990
                                         Printed on Recycled Paper

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                                           Rapositoiy Material
                                         Psmianeni Coilsciion
                     NPDES Compliance Monitoring Inspector Training Module: BIOMONITORING
                    NPDES COMPLIANCE MONITORING INSPECTOR
                                TRAINING MODULE
                                 BIOMONITORING
cr
21                       U.S. ENVIRONMENTAL PROTECTION AGENCY
in
C6
d
                                                       US EPA
                                           Headquarters and Chemical Libraries
                                               EPA West Bldg Room 3340
                                                   Mailcode 3404T
                                               1301 Constitution Ave NW
                                                 Washington DC 20004
                                                    202-566-0556
                               ENFORCEMENT DIVISION
                      OFFICE OF WATER ENFORCEMENT AND PERMITS
                           ENFORCEMENT SUPPORT BRANCH
                                      JUNE 1990

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A/834.03-239-0 l/DIS
                 NPDES Compliance Monitoring Inspector Training Module:  BIOMONITORING


                                     DISCLAIMER

This module has been reviewed by the U.S. Environmental Protection Agency's (EPA's) Office of Water
Enforcement and Permits and approved for publication. This module represents EPA's introductory
training on selected topics relating to conducting NPDES compliance inspections. Failure on the part
of any duly authorized official, inspector, or agent to comply with its contents will not be a defense in
any enforcement action,  nor will failure to comply with this guidance alone constitute grounds for
rendering  evidence obtained  thereby inadmissible in a court of law.  The mention of trade names,
commercial products, or organizations  does not imply endorsement by the U.S. Government.

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                 NPDES Compliance Monitoring Inspector Training Module: BIOMONITORING


                                AKNOWLEDGMENTS

This document represents an update of an earlier module originally developed by the Enforcement
Division of the Office of Water Enforcement and Permits.  The module was revised under the direction
of Virginia Lathrop and Gary Polvi of the Office of Water Enforcement and Permits, with the review
and comment of Shiela Frace of the Office of Enforcement and Compliance Monitoring. In addition,
the  EPA Regions provided extensive reviews. Many valuable comments were provided, .most of which
have been incorporated into this module. Science Applications International Corporation prepared this
updated moduile under EPA Contract No. 68-C8-0066, WA No. C-l-2 (E).
                                           iii

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             NPDES Compliance Monitoring Inspector Traning Module: BIOMONITORING



                         TABLE OF CONTENTS
FOREWORD	  vii

1.  INTRODUCTION  	  1-1

   1.1  OVERVIEW OF THE NPDES PROGRAM   	  1-1
   1.2  CONCEPT OF TOXICITY  	  1-3
   1.3  TOXICITY TESTING IN THE NPDES COMPLIANCE
       MONITORING PROGRAM 	  1-3

2.  BASICS OF TOXICITY TESTING		  2-1

   2.1  TOXICITY TEST DESIGN	  2-1
   2.2  ACUTE AND CHRONIC TESTS  	  2-3
   2.3  FLOW-THROUGH, STATIC RENEWAL, AND STATIC TESTS  	  2-4
   2.4  ANALYSIS OF TEST RESULTS	  2-4

3.  TOXICITY TEST COMPONENTS  	  3-1

   3.1  EFFLUENT  	  3-3
   3.2  DILUTION WATER  	,	  3-4
   3.3  TEST SYSTEM  	  3-4
   3.4  TEST ORGANISMS  	  3-4
   3.5  TEST RESULTS	  3-5
   3.6  SUMMARY  	  3-6

4.  EFFLUENT	  4-1

   4.1  SAMPLING STRATEGIES  	  4-1
   4.2  SAMPLE STORAGE AND  PRESERVATION	  4-3

5.  DILUTION WATER  	  5-1

   5.1  SOURCES OF DILUTION  WATER	  5-1
   5.2  STORAGE CONDITIONS AND HOLDING TIMES	  5-2
                                   iv

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             NPDES Compliance Monitoring Inspector Traning Module: BIOMONITORING



                   TABLE OF CONTENTS (Continued)


                                                                   Page

6.  TEST ORGANISMS  	  6-1

   6.1  SPECIES USED   	  6-1
   6.2  SOURCES OF TEST ORGANISMS  	  6-5
   6.3  ACCLIMATION AND FEEDING  	  6-5
   6.4  DISEASE  	  6-7
   6.5  LOADING RATES  	  6-7


7.  TEST SYSTEM  	  7-1

   7.1  MATERIALS USED  	  7-1
   7.2  CLEANING   	  7-2
   7.3  TEST CONDITIONS  	  7-2
   7.4  TEST REPLICATES	  7-5


8.  TEST RESULTS  	  8-1

   8.1  CONTROL SURVIVAL  	  8-1
   8.2  ACCEPTABILITY CRITERIA 	  8-1
   8.3  RESULTS CALCULATION	  8-2



                             APPENDICES
APPENDIX A -QUESTIONS AND ANSWERS ON THE BIOMONITORING
            MODULE

APPENDIX B - DATA SHEETS FOR AQUATIC TOXICITY TESTS: SUMMARY
            OF RECOMMENDED TEST CONDITIONS FOR SOME COMMONLY
            USED TEST SPECIES

APPENDIX C.- HEALTH AND SAFETY PROCEDURES

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                    LIST OF FIGURES AND TABLES


Figure                                                              Page

2-1 TYPICAL EFFLUENT CONCENTRATIONS USED IN TESTING  .	  2-2

2-2 RESPONSE CURVE FOR A HYPOTHETICAL EFFLUENT CHRONIC
   TOXICITY TEST   	  2-5

2-3 RESPONSE CURVE FOR A HYPOTHETICAL EFFLUENT ACUTE
   TOXICITY TEST   	  2-6

3-1 RELATIONSHIPS BETWEEN TOXICITY TESTING COMPONENTS,
   SHOWING IMPORTANT FACTORS FOR EACH  	  3-2



Table                                                              Page

4-1 RECOMMENDED SAMPLING STRATEGY FOR CONTINUOUS AND
   INTERMITTENT DISCHARGES FOR FLOW-THROUGH, STATIC
   RENEWAL, AND STATIC TOXICITY TEST  	  4-2

6-1 FRESHWATER SPECIES FOR WHICH THERE ARE ACUTE
   TOXICITY TESTING PROTOCOLS 	  6-2

6-2 MARINE AND ESTUARINE SPECIES FOR WHICH THERE ARE
   ACUTE TOXICITY TESTING PROTOCOLS  	  6-3

6-3 SPECIES FOR WHICH THERE ARE CHRONIC TOXICITY TESTING
   PROTOCOLS, ORGANIZED BY SPECIES	  6-4
                                  vi

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A/83403-239-01/FOREWARD
                  NPDES Compliance Monitoring Inspector Training Module:  BIOMONITORING
                                      FOREWORD

      This document is one of five training modules developed by the Office of Water Enforcement
and Permits, U.S. Environmental Protection  Agency (EPA)  to introduce the National Pollutant
Discharge Elimination System  (NPDES) program  to new inspectors.  Information in each  module
provides  training  to an  inspector unfamiliar with  the NPDES program.  The  modules address the
following topics:

      •   The Overview presents an overview of the entire NPDES program and briefly summarizes
          different types of inspections conducted under this program
      •   Legal Issues discusses the legal issues which must be addressed during an inspection and
          provides legal information to assist  inspectors in performing their duties
      •   Biomonitoring outlines the principles of biomonitoring and the role of biological testing in
          the inspection program
      •   Sampling Procedures details procedures to be used when conducting a sampling inspection
      •   Laboratory Analysis outlines procedures and information necessary to perform an effective
          evaluation of a  permittee's laboratory.

      The modules are best used in a classroom setting where there is discussion between instructors
and students and where questions can be asked.  Yet, they can  also stand alone as reference  sources.
A more detailed discussion of the topics covered in these modules appears in EPA's  1988  NgDgS
Compliance  Inspection Manual.

      These training  modules were developed primarily for inhouse  training of Regional and State
NPDES Inspectors. However, they are available as well to other interested parties such as attorneys,
other program offices, facility owners and operators, and members of the general public.
                                             vii

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                 NPDES Compliance Monitoring Inspector Training Module:  BIOMONITORING
      Regional and State personnel are encouraged  to provide EPA Headquarters with changes or

information which instructors or managers believe would improve these modules. The content of the

modules will be updated and revised periodically.  Comments, information, and suggestions to improve

the modules should be addressed to:
                     Enforcement Support Branch (EN-338)
                     Office of Water Enforcement and Permits
                     U.S. Environmental Protection Agency
                     401 M Street, SW
                     Washington, DC  20460
                                           viii

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                          NPDES Compliance Monitoring Inspector Module: BIOMONITORING


                                  1.  INTRODUCTION

1.1  OVERVIEW OF THE NPDES PROGRAM
      The Federal Water Pollution Control Act of 1972, as amended by the Clean Water Act
(CWA) of 1977, specifies the objectives of restoring and maintaining  the chemical, physical, and
biological quality of the Nation's waters. The Act provides broad authority to the U.S.
Environmental Protection Agency (EPA) to undertake several activities:

      •   Establish the National Pollutant Discharge Elimination System (NPDES) program and
          the National Pretreatment Program
      •   Define pollution control techniques and establish effluent  limitations based on these
          technologies or the  level necessary to protect State water quality standards (whichever is
          more stringent)
      •   Obtain information  through reporting and compliance inspections
      •   Take enforcement actions, both civil and criminal, when violations of the Act occur.

The Act  also provides authority for the States to undertake activities  to protect their waters:

      •   Establish State water quality standards to protect designated uses
      •   Apply for authority to run the NPDES program and the National Pretreatment Program.

      The NPDES program, mandated by Section 402 of the Act, regulates the discharge of
pollutants from point sources such as municipal treatment plants, industries,  animal feedlots,  aquatic
animal production facilities and mining operations.  In order to discharge, each point source  is
required  to obtain a NPDES permit containing effluent limits, a compliance schedule, monitoring
Notes:
                                             1-1

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and reporting requirements, and any other terms and conditions necessary to protect water quality.

       To determine whether these NPDES permit conditions are being met, Section 308 of the Act
authorizes inspections and two types of monitoring of permittee facilities:  self-monitoring
conducted by the permittee and compliance monitoring conducted by the permit-issuing agency.
According to the Act, an inspection may be conducted where there is an existing NPDES permit or
where  a discharge exists or is likely to exist and no permit has been issued.

       Historically, EPA has relied on technology-based standards to control the discharges of point
sources, particularly industries.  Technology-based standards for industrial point sources are targeted
on regulated pollutants, known as  conventional, nonconventional, and toxic pollutants, and on the
industrial categories that contribute the majority of regulated pollutants.

       Technology-based limits on  conventional pollutants, the typical emphasis of many early
NPDES permits, has resulted in  improved receiving water quality. However, the CWA also requires
that permit  limits reflect State water quality standards in  the form of numeric standards on
individual toxicants and narrative standards prohibiting toxic conditions in general. For this reason,
permits may require additional limits on specific toxicants or whole effluent toxicity (WET).

       Whole effluent toxicity limits are designed to measure the toxic impact of the
effluent on biota and are a necessary addition to chemical-specific limits for several reasons:

       •   The interactions of individual constituents of wastewaters are greater than predicted by
          summing the effects of  individual toxicants
       •   The toxic effects of many compounds are unknown
Notes:
                                              1-2

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       •   The bioavailability of many compounds changes with changes in pH, temperature, or
          even the presence of oxygen.

1.2 CONCEPT OF TOXICITY
       By definition, toxicity is a characteristic of a substance (or group of substances) that causes
adverse effects in organisms.  Adverse effects are mortality and those effects that limit an organism's
ability to survive in nature.  Toxicity of a substance is measured by observing the responses of
organisms to increasing concentrations of that  substance.  One substance is more toxic than another
when it causes the same adverse effect at a lower concentration.

       For any given substance, toxic effects are alleviated when the concentrations to which
organisms are exposed are reduced. Thus, by reducing the toxicity of a discharge (by reducing the
concentrations of toxic constituents), the toxic  effect of that discharge on receiving waters is also
reduced.  Similarly, greater dilution of a toxic discharge will lead to lower toxic effects in receiving
waters.  The key to effective toxics control is the limitation of measured toxicity in a discharge.

       Toxicity testing is the focus of this training module.  However, this module is only a brief
introduction to toxicity testing and should not  be used as a reference for testing procedures.  Check
with the Regional biologist for advice and recent changes in  published toxicity  testing procedures.

1.3 TOXICITY TESTING IN THE NPDES COMPLIANCE MONITORING  PROGRAM
       Toxicity testing is either performed or evaluated in five NPDES inspections:

       •   Compliance Evaluation Inspection (CEI)
       •   Compliance Sampling Inspection (CSI)
Notes:
                                              1-3

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                           NPDES Compliance Monitoring Inspector Module:  BIOMONITORING


       •   Performance Audit Inspection (PAI)
       •   Toxics Sampling Inspection (XSI)
       •   Compliance Biomonitoring Inspections (CBI).

Depending on the inspection, toxicity testing accomplishes one or more of the following six
purposes:

       •   Determines compliance with State water quality standards
       •   Determines compliance with permit conditions
       •   Evaluates quality of self-monitoring data
       •   Assesses self-monitoring performance
       •   Examines facilities, equipment, records, and reports for self-monitoring
       •   Develops permit limits.

       This module is designed to introduce new inspectors to the concepts and practices of toxicity
testing as  it relates to CBIs.  It is not intended to be a definitive source of material on toxicity
testing; rather, its focus is on explaining the points pertinent to CBIs.  After mastering the material
in this module,  an inspector should be able to:

       •   Define terms used in whole effluent toxicity testing
       •   Describe whole effluent toxicity testing procedures
       •   List  key aspects of toxicity critical to interpretation of results
Notes:
                                              1-4

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       •   List guidance documents that can be used to implement CBIs.

Appendix A contains questions and answers that an inspector can  use to self-test him/herself after

completing this module.


       This module provides a basic introduction to toxicity tests.  More specific information can be

found in the following manuals:


       •   NPDES Compliance Inspection  Manual. May 1988.

       •   Methods for Measuring the Acute Toxicitv of Effluents to Freshwater and Marine
          Organisms. EPA/600/4-85/013, March 1985.

       •   Methods for Estimating the Chronic Toxicitv of Effluents and Receiving Waters to
          Freshwater Organisms. EPA/600/4-89/001, March 1989.

       •   Methods for Estimating the Chronic Toxicitv of Effluents and Receiving Waters on
          Marine and Estuarine Organisms.  EPA/600/4-87/028, May 1988.

       •   Technical Support Document for Water Quality-based Toxics Control. EPA-440/4-85-
          032, September 1985 or more recent revisions.

       •   Permit Writer's Guide to Water Quality-based Permitting for Toxic Pollutants.  EPA
          440/4-87-005, July 1987.


Before a CBI is carried out, the inspector is strongly advised to consult one or more of these

manuals for further explanation and to consult the Regional biologist.
 Notes:
                                             1-5

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                         2.  BASICS OF TOXICITY TESTING


2.1  TOXICITY TEST DESIGN

       Toxicity tests are techniques to determine the  relative toxicity of a permittee's discharge or

effluent by measuring the responses of organisms to solutions

containing various percentages of effluent and dilution water. In general, test organisms are

exposed to a series of solutions that contain a range  of effluent dilutions.  However, test designs

vary, depending on how the results are to be used. Three general types  of tests are performed:
          Range Finding Test - These are usually 24-hour tests conducted to determine the
          approximate level of toxicity of an effluent, and the concentration to be used in
          definitive tests.  The test organisms are exposed to a wide range of concentrations to
          determine the highest concentration that killed no (or few) organisms and the lowest
          concentration that killed all (or most) organisms.

          Screening Test - In this type  of test, organisms are exposed to only one dilution  of
          effluent.  If a toxic response is observed (see next section), further testing may then be
          conducted on the effluent. The use of screening tests varies from Region to Region and
          from State to State. Some Regions and States rely heavily on screening tests to  reduce
          the effort required  for detailed testing.  Other Regions and States, who feel the benefits
          of definitive data outweigh the incremental costs, insist that definitive tests be done on
          effluents requiring toxicity testing.

          Definitive Test - This test estimates the concentrations at which a certain percentage or
          significant number of organisms exhibit a  certain response.  In a definitive test, several
          groups (replicates)  of organisms are exposed for a predetermined length of time  to
          solutions of various proportions of effluent and dilution water (Figure 2-1).  The
          response of each organism in  each  test concentration is observed and recorded, and the
          number of responses are analyzed in relation to the concentration of effluent to  which
          they were exposed.
Notes:
                                              2-1

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               NPDES Compliance Monitoring Inspector Training Module:  BIOMONITORING




                                  FIGURE 2-1.


            TYPICAL EFFLUENT CONCENTRATIONS USED IN TESTING
             0%
           Control
                     6.25%
                              12.5%
                                        25%
                                                50 %*
           * Used in screening tests
100%*
Notes:
                                      2-2

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       Responses that are commonly observed in each test can be one or more of the following:

       •   Death - Number of organisms killed by a test solution
       •   Growth - Increase in body weights or sizes of test organisms (e.g., the growth of a
          female fish is directly proportional to the number and quality of eggs she will produce at
          maturity)
       •   Reproduction - Number of offspring produced per female or increase in numbers of
          organisms
       •   Terata - Number of gross abnormalities shown in early life stages.
These responses relate directly to an organism's ability to survive in nature and, thus, can indicate
whether an effluent will jeopardize the survival of the test species in receiving water.

2.2    ACUTE AND CHRONIC TESTS
       Toxicity tests are generally described as either acute or chronic tests.  Acute and chronic
refer to the length of time organisms are exposed to toxicants before adverse responses are
observed.  Acute toxicity measures short term effects with impacts usually resulting in death or
extreme physiological disorder to organisms immediately or shortly following exposure to a
contaminant.  Chronic toxicity involves long-term effects of small doses of a contaminant and their
cumulative effects over time. These effects may ultimately lead to the death of the organism or
disruption of functions such as reproduction or growth.
Notes:
                                              2-3

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2.3    FLOW-THROUGH, STATIC RENEWAL, AND STATIC TESTS
       Toxicity tests  are also described according to the way in  which organisms are exposed to test
solutions.  In a flow-through test, effluent and dilution water are mechanically renewed numerous
times a day.  This test  setup  requires specialized equipment (a serial or proportional dilutor or
syringe pumps) and is  more costly to operate than a static test.  In  a  static renewal test,  the test
solutions are replaced  periodically (usually daily) with fresh effluent and dilution water.   In a static
test, the solutions used at the start of the test are not replaced for  the test's duration.  Both static
renewal and static tests require only basic equipment.

       Toxicity tests  may be conducted on-site (at the effluent discharge) or off-site (at a central
laboratory). The major advantage of on-site testing is the guaranteed exposure of test organisms  to
volatile or  biodegradable toxic constituents.  When toxicity may be  due to volatile compounds [e.g.,
chlorine from Publicly  Owned Treatment Works (POTWs), which may be lost, on-site testing may
be more appropriate.  Generally, static renewal and static tests  are  performed off-site because the
cost of maintaining a mobile laboratory on-site is usually more expensive that shipping effluent to a
laboratory.  Flow-through testing, which requires large volumes of both effluent and dilution water,
is generally performed  on-site due to the difficulty of obtaining and shipping enough effluent (and
dilution water) to continuously replenish the water in test containers.

2.4    ANALYSIS OF TEST RESULTS
       With appropriate testing conditions, data generated by definitive toxicity testing are easily
interpreted. A sample of typical results appears in the semilogarithmic plot in  Figure 2-2. The
percentage of test organisms showing a particular response is plotted  against the percentage of
effluent in  the test solutions.  This graph is typical in that it shows a generally  increasing percent
response for increasing percentages of effluent.  The graph is also typical in that it shows a change
Notes:
                                              2-4

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               NPDES Compliance Monitoring Inspector Training Module:  BIOMONITORING





                                   FIGURE 2-2.

   RESPONSE CURVE FOR A HYPOTHETICAL EFFLUENT CHRONIC TOXICITY TEST
     100
                  Response Curve for a Hypothetical Effluent
                             Acute Toxicity Test
     90-
     80-
     70
      10
             10    20     30     40     SO     60


                             PERCENT EFFLUENT
                                                       •  MtaaurMl
                                                       *  Calculated
70    80    90    100
 Notes:
                                       2-5

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                NPDES Compliance Monitoring Inspector Training Module: BIOMONITORING





                                   FIGURE 2-3.

     RESPONSE CURVE FOR A HYPOTHETICAL EFFLUENT ACUTE TOXICITY TEST
                   Response Curve for a Hypothetical Effluent
                              Chronic Toxicity Test
                                                     m  Measured
                                                     *  Calculated
               10    20    30
                                       50     60


                                 PERCENT EFFLUENT
90    100
Notes:
                                      2-6

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                  NPDES Compliance Monitoring Inspector Training Module:  BIOMONITORING
in response of 75 percent or more between two test concentrations.  Sometimes this response is
even more exaggerated, with no responses at one concentration and  100 percent responses at the
next higher concentration as shown in Figure 2-3.
       The terms LC», EC», NOEC, LOEC, ChV and ICa refer to different measures of toricity
and are explained below. These various measures of toxicity are also illustrated graphically in
Figures 2-2 and 2-3 through the use of a hypothetical example.  In these graphs, increasing
concentrations of hypothetical effluent produce differing levels of toxic response (as measured by
species growth or mortality in these cases).

       The LC» (for lethal  concentration) is the calculated percentage of effluent (point estimate) at
which 50 percent of the organisms die in the test  period. In Figure 2-3, the LC» is indicated at the
concentration corresponding to 50 percent responses in the test solution.  While graphics such as
this can be used to determine LCyS, these methods do not allow the calculation of the error
associated with LQo estimation.  Usually the LC» is calculated statistically by computer programs
that fit the response curve to a mathematical function.  Computer-based calculation procedures
usually print an estimate of the error associated with the LC» estimate.

       The EC» is the calculated concentration (point estimate) at which 50 percent of the
organisms show a particular effect (not necessarily death).  For some species (e.g., Ceriodaphnia
dubia)  where the  point of death is not certain, immobility is often used as a surrogate  for death.
Results for responses like the immobility responses in Daphnia may be reported as an EC*>
(calculated in the same manner as the LC*,).  Often, however, no distinction is made between
and LQoS when the response  is a surrogate for death.
Notes:
                                             2-7

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      The NOEC (no observable effect concentration) is the highest tested concentration at which
the organisms' responses are not significantly different statistically from controls.  The NOEC (like
the LOEC and ChV defined in the following paragraphs) is normally determined only for chronic
tests. In Figure 2-2, the NOEC is 12.5 percent effluent since the response in the 12.5 percent
effluent is not significantly different from the control response, but the 25 percent effluent response
is significantly different from controls.

       The LOEC  (lowest observable effect concentration)  is the lowest tested concentration at
which organisms' responses are significantly  different statistically from controls.  This occurs at
25  percent effluent in Figure 2-2.  The ChV (chronic value) is the calculated geometric mean of the
NOEC and LOEC (the square root of the product of the NOEC and the LOEC).  The ChV is
 17.7 percent effluent for the data in  Figure 2-2.

       The IC» (inhibition concentration) is the calculated percentage of effluent (point estimate) at
which  the organisms exhibit a 25 percent reduction in a non-quantal biological measurement such as
 fecundity or growth.

       Each of these toricity test results has units  that are concentrations of chemicals or
 percentages of effluent  As was explained in the Introduction, a substance  having an LOEC (or
 ChV, or any other reporting measure) that is  lower than that for another  substance is more toxic
 than the other substance when toricity is measured using the same test and the same species. For
 example, one effluent with an EC* of 42 percent effluent is more toxic than an effluent with an
 EQ, of 84 percent effluent
 Notes:
                                              2-8

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       There is an inverse relationship between toxicity and the effluent concentration percentage
causing a toxic response.  In other words, the same toxicity test response (e.g., LQ,) at lower
percentages of effluent (i.e., more dilution) indicate higher toxicity than test results at higher
percentages of effluent using less dilution.  A toxic unit, which is directly proportional to toxicity, is
sometimes  used to express the effluent's toxicity.  Toxic units are defined as lOO/LC*, for acute or
100/NOEC for chronic when the LC» or NOEC are expressed as percent effluent.  An effluent with
an LC,o of  50 percent effluent has an acute toxicity, TU., of 2  acute toxic units.  Similarly, an
effluent with  a NOEC of 25 percent effluent has a chronic toxicity, TUCT of 4 chronic toxic units.
The major  advantage of using toxic units to express toxicity test results is  that toxic units increase
linearly as  the toxicity of the effluent increases.  So an effluent with  a TU. of 4  is twice as toxic as
an effluent with a TU. of 2.  A  second advantage to using toxic units is that they are directly
analogous to  constituent concentrations and can be used in wasteload allocations with the same
equations as  individual constituents.
Notes:
                                              2-9

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                         3.  TOXICITY TEST COMPONENTS

      Reliable toxicity test results can only be expected when each of several key components are
handled within the minimum quality assurance requirements discussed below. It is, therefore, very
important to understand the relationships between these components and the critical factors that
determine the acceptability of each from  a quality assurance standpoint Toxicity tests consist of
five components:

      •   Effluent
      •   Dilution water
      •   Test system
      •   Test organisms
      •   Test results.

      In simple  terms (Figure 3-1), effluent and dilution water are combined in the test system
with test organisms to produce test results.  Each component must be of a specific quality for
successful toxicity testing.  If the dilution water, for example, is toxic to test organisms, it will be
impossible to determine the toxicity caused by the effluent Similarly, if inappropriate materials are
used for the test system, or if the test system is not adequately prepared, observed toxicity might
not be due to the effluent, but to the test system itself. Since  the objective of toxicity testing is to
obtain toxicity data on a given effluent, any factor that could be a source of toxicity, other than  the
effluent, must be carefully  controlled.
Notes:
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                                    FIGURE 3-1.


            RELATIONSHIPS BETWEEN TOXICITY TESTING COMPONENTS,
                    SHOWING IMPORTANT FACTORS FOR EACH
             Sampling mathod and
              storage conditions
              Sourc* and storage
                  condition*
     Tast conditions and
        protocols
 Test
system
   Test
organisms
                                     Test
                                    results
                       Sourot, aocSmatton, and
                          disaas* control
Notes:
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      Factors important for each of these test components are briefly outlined below.
Chapters 4-8 of this module then develop each component in more detail.  An inspector must keep
these considerations in mind whenever evaluating a permittee's toxicity testing procedures.  An
inspector should also be familiar with other reference sources mentioned in Chapter 1.  Appendix B
also  lists EPA recommended test conditions for the test species most commonly used in aquatic
toxicity testing.

      When examining a  permittee's laboratory  facilities, an inspector should also be aware of
standard health and safety procedures. A list of standard health and safety precautions is included
in Appendix C.

3.1    EFFLUENT
      Effluent  must be sampled at points specified in the NPDES permit and stored in such a way
that  the sample is representative of the entire effluent, does not appreciably change in char-
acteristics before it is tested, and is taken without contamination from other sources.  An effluent
of variable quality may be sampled continuously  or as a series of grab samples in order  to obtain
samples that represent the effluent as much as possible. Single grab samples are  usually adequate
for unvarying acute tests of effluents. Three or more grab samples are recommended for chronic
tests.

      Sample containers must prevent the loss of volatile components in order to preserve the
representativeness of the sample. Further, the sample must be used in the toxicity test before
significant changes occur to any toxic characteristics that may be present Toxicity can vary because
chemicals are lost due to volatilization, chemical precipitation, or biological degradation. Samples
should be tested within 36 hours of the time  taken.  The sample should also  usually be  refrigerated
or stored on ice to prevent biological degradation of organic materials.  Sample contamination is
avoided by using clean sampling gear and sample containers.
Notes:
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       The inspector-should refer to the companion training module on Sampling Procedures for
more background information on sampling.

3.2    DILUTION WATER
       Dilution water is chosen depending on the objective of the test  Either reconstituted water,
receiving water, or other natural waters are appropriate for specific purposes. Dilution water
should be specified in the NPDES permit The type of water appropriate for specific tests is
discussed in Chapter 5 of this module.

       Dilution water, by itself, should not be toxic.  Tests that have significant mortality in  dilution
water controls should be discarded.  If dilution water causes mortality in test organisms during
acclimation, the dilution water should not be used in toxicity testing.

3.3    TEST SYSTEM
       Materials used to construct the system must prevent contamination of the sample and
dilution water, test conditions must be appropriate for the test species, and effluent dilutions used
in testing must be accurate.  All containers, valves, and tubes of the test system must be of
materials that do not provide or remove toxicants and  must be thoroughly cleaned before testing.
In addition, key variables, such as temperature and dissolved oxygen, must be controlled to ensure
test reproducibility.

3.4    TEST ORGANISMS
       Test organisms used in toxicity testing must be of known  history, free of disease, and
acclimated to test conditions.  The organisms should also respond  "normally" to reference toxicants.
"Wild" organisms (Le., those taken from natural waters) are not generally appropriate for toxicity
testing unless cultured in the laboratory for several generations.  Most test organisms can be
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obtained from commercial supply houses or laboratory cultures.  Diseased organisms should not be
used in toxicity testing since the disease will affect the organisms' response to any toxicants in an
effluent.

       Organisms to be used for toxicity testing should be acclimated slowly to the temperature,
salinity, and pH conditions in dilution water prior to  the test These parameters can affect test
results substantially, unadapted organisms may exhibit responses to changes in these  parameters that
may be confused with a toxic response.

       A wide variety of organisms can  be used for toxicity testing, but acceptable test organisms for
compliance monitoring are normally specified in the discharger's permit. The same test organism
should be used for  the compliance inspection.  Two types of freshwater organisms most commonly
used in toxicity test inspections are fathead minnows  (Pimephales promelas) and water  fleas (Cerio-
daphnia dubia).  Saltwater species used  for toxicity testing include  the sheepshead minnow
(Cyprinodon variegatus), the inland silverside (Menidia beryllona), the mysid shrimp (Mysidopsis
bahia), and the sea urchin (Arbacia punctulata).

3.5    TEST RESULTS
       Test results must show clear responses to  increasing dilutions of effluent, and responses in
controls must be negligible.  The expected result  for  toxicity  tests is increasing responses with
increasing percentages of effluent  If one or more dilutions yield responses inconsistent with this
pattern, it is  likely that either those dilutions were incorrectly labeled or that contamination
occurred in one or more dilutions. The inspector should view such results as suspicious and
should require further technical evaluation of the results.
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       Organisms in controls (i.e., dilutions with no effluent) should not exhibit any significant
number of deaths or other adverse effects.  Controls are the basis to determine toxic effects of
effluents.  If appreciable responses occur in controls, results based on the data will be inaccurate.

3.6    SUMMARY
       The quality of the test results is based on proper handling and set-up of the effluents,
dilution water, test organisms, and the test system.  The results of any particular test indicate the
rigor with which these factors were considered during testing.  These factors are explained in greater
detail in the following four Chapters of this module.  If results show questionable patterns or if
there are significant responses found in controls, the inspector should not use them to determine a
permittee's compliance status.
Notes:
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                                      4.  EFFLUENT

      The first major component discussed in detail is the effluent to be tested. The way in which
effluents are sampled and stored will determine whether any given toricity test can be claimed to be
representative of the effluent or discharge.  Representativeness of the sample may be an issue
during any legal proceedings that might arise as a result of an inspection, so adherence to the
principles  and procedures in the EPA guidance manuals (see Section 1.3) is critical to avoid
problems.

      Effluent samples must be representative of the discharge.  If holding is necessary, the
samples must be stored under strict conditions and  for limited times so that no appreciable change
in toxic  characteristics occurs before testing.  All samples also should be taken at the location
specified in the permit unless the  toricity of particular wastestreams is being evaluated.

4.1   SAMPLING STRATEGIES
      The type and frequency of  samples taken (e.g., grab, composite) must be consistent with
those required in the permit.  For flow-through tests that are not done by pumping effluent directly
into dilutors, daily  sample sizes must be sufficient to supply the dilutor for 24 to 36 hours.  This
volume will depend on the type of test  being conducted and the number of dilutions being run (see
Chapter 7 on Test  System).  For static-renewal and static tests, dairy sample volumes should be
sufficient to replenish  all dilutions in the test series and to provide separate vials of the dilutions to
allow for DO, pH,  and salinity analysis  without contaminating the test dilutions.  This volume will
depend on the type of test being conducted and the dilutions being run.  Refer to Table 4-1 for
more information on sampling strategies.
Notes:
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                                           TABLE 4-1.

   RECOMMENDED SAMPLING STRATEGY FOR CONTINUOUS AND  INTERMITTANT
DISCHARGES FOR FLOW-THROUGH, STATIC RENEWAL, AND STATIC TOXICITY TESTS
                                        Continuous Discharge
       Test Type

       Row-through


       Static renewal
       Static
    Preferable

Rump effluent directly
to dilutor
    Retentoa TUM
      14 Day*

 One grab sample daily
                                                                       One grab sample daily
                                                                       One grab sample on
                                                                       flnt day
                                       Intermittent DIsdwrM
       Tot Type

       Flow-through


       Static renewal



       Static
 Continuous Dtscharft
During 1 or 2 Adjacent
    8-hoor Shifts

One grab sample
midway through shifts

One grab sample
midway through shifts
daily

One grab sample
midway through shifts
on flnt day
   Discharge tnm
   Sat
  One grab sample of
  discharge daily

  One grab sample of
  discharge daily
  One grab sample of
  discharge oa flnt
  day
Discharge to Estnary
 on Outgoing Tide

 One grab sample of
 discharge daily

 One grab sample of
 discharge daily
 One grab sample of
 discharge on first
 day
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4.2   SAMPLE STORAGE AND PRESEVATTON
      Sample containers for large volumes of effluents should be either covered fiberglass or
unsealed stainless steel tanks.  Small volumes of effluent can be stored in reusable glass jugs or
nonreusable Cubitainers or plastic "milk jugs.*

      Samples for on-site tests should be used immediately when practicable, but must be used
within 36 hours of collection.  It is generally not possible to refrigerate the large volume samples
(200 liters or more) that are required  for flow-through fish tests, but all other samples should be
either iced or refrigerated  if they are not to be used immediately.

      Samples to be used for off-site  tests should be iced for shipment and refrigerated (4'C) on
receipt by the testing laboratory.  As a minimum requirement in all cases, tests should be initiated
within 36 hours of collection.  In the case of short term chronic tests, samples taken on days one,
three, and five may be held for a slightly longer period of time (up to  48 hours) to  complete the
test In no case should any preservative be used in samples to be tested for toxicity.
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                                 5.   DILUTION WATER

      The second major component discussed in detail is dilution water.  The use of appropriate
dilution water ensures that there will be a low number of responses in the controls and that any
responses found in the solutions containing effluent are due to the effluent itself.

      The choice of dilution water is  generally  specified in the NPDES permit and depends on the
purpose of the toxicity test.  Standard  dilution water should be used to evaluate the inherent
toxicity of the effluent.  Dilution water from the receiving stream or a nontoxic equivalent should
be used to test for interactions after discharge.  Under most circumstances, however, the dilution
water should  not cause any toxic responses in test organisms.  A lack of toxic responses in controls
in the toxicity test is evidence of the suitability of the dilution water.

5.1   SOURCES OF DILUTION WATER
      To determine the inherent toxicity of an  effluent (e.g., for testing compliance with a toxicity
limit), the source  of water should be a standard dilution water made up from distilled water and
known chemical compounds.  The inspector should review the EPA manuals for details of obtaining
dilution waters with various characteristics.

      To determine the effects of an  effluent on saltwater organisms, sea salts must be added to a
freshwater effluent to attain the proper salinity. Dilution water could be hypersaline brine, an
artificial  mix  (such as the commercial  brands: 40  Fathoms* or Hawaiian Mix11), or natural seawater,
depending on the specific recommendations for  the protocol being used.

      To determine the toxicity of an effluent relative to the receiving water, the dilution water
should be taken from the receiving water as close as possible to, but outside the influence of, the
Notes:
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outfall. If the test's objective is to determine the toxic contribution of the effluent to receiving
waters, receiving waters are appropriate as dilution water - whether they are contaminated or not.
If the additive toxicity of the effluent to the receiving water  is being established, receiving waters
should be sampled daily as the source of dilution water.

       With the estuarine (saline) receiving water, dilution waters of the same salinity as the
receiving waters at the outfall should be used. If uncontaminated dilution water is required, waters
from an adjacent estuary can be used or dilution water can be created with artificial sea salts.
Alternatively, more saline waters (hypersaline brine) can be diluted to obtain dilution waters of the
proper salinity.

       If receiving waters are not available as dilution water, standard test waters,  other surface
waters, or ground water may be used.   Waters other than receiving waters should be free from toxic
effects (controls should have less than  10 percent mortality for acute  tests and 20  percent mortality
for chronic tests). Dechlorinated tap water should not generally be used as dilution water unless
extensive precautionary steps are taken before its use.  The EPA manuals describe techniques for
treating tap water, but also discourage  its use in favor of a standard dilution water or receiving
waters.

5.2    STORAGE CONDITIONS AND HOLDING TIMES
       Dilution water obtained from receiving waters should be immediately used  for testing.  If it
is not used within 24 hours, it should be refrigerated  (4'C) as soon as  it  is collected until it is
used.  In any case, it should be used within 36 hours of collection.
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                                 6.  TEST ORGANISMS

      The third major component discussed in detail is the test organism.  Since test organisms are
the "analytical detection tool" to determine toxicity levels and since they can vary in their responses
for reasons that are difficult to  understand, special care must be devoted to their handling and
treatment.  Organisms used for toxicity testing are limited to certain species for which there are
testing protocols.  The life stage, source, acclimation and feeding procedures, presence of disease,
and the number of organisms placed in test chambers all affect the degree to which organisms
respond to toxicants.  It is, thus, important that these factors are standardized as much as possible.

6.1   SPECIES USED
      Tables 6-1 and 6-2 present the common name, scientific name, test temperature, and age
range or life stage for those species for which there are acute toxicity testing protocols. Any of
these species may be used in toxicity testing, but if a monitoring species is specified in a permit,
that species must be used for all compliance monitoring for that effluent.  If a toxicity limit is being
determined  for an effluent, EPA recommends that at least three species be used to determine which
is most sensitive to the effluent.

       Table 6-3 presents the common name, scientific name, test temperature, age range or life
stage, and responses measured  for all species for which there are short-term chronic toxicity testing
protocols.  Any of these species may be used in toxicity testing, but if a monitoring species is
specified in a permit, that species must be used for all compliance monitoring for that effluent.
 Notes:
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                                                    TABLE 6-1.

                                      FRESHWATER SPECIES FOR WHICH
                            THERE ARE ACUTE TOXICITY TESTING PROTOCOLS
                                                       FISH
rjM>Tn F«"t
Brook trout
Coho salmon
Rainbow trout
COLD (U-C)
A
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                                                    TABLE 6-2.

                              MARINE AND ESTUARINE SPECIES FOR WHICH
                           THERE ARE ACUTE  TOXICITY TESTING PROTOCOLS
Common Name

English sole
Sand dab

Winter flounder
COLD 
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TABLE 6-3.
SPECIES FOR WHICH THERE ARE
CHRONIC TESTING PROTOCOLS, ORGANIZED BY SPECIES
Common Nime
Fathead minnow
Fathead minnow
Cladocenn
Algae
Inland stenUe
*+H*
Sheepshead
Mywds
Macroalgae
SeaUrchim
Gree* Set Urctta
Pupte Set Urchia
Sand Dollar
Response Measured
Larval survival and
growth
Embryo/larval survival
and teratogenieity test
Survival and
reproduction
Growth
Larval survival and
growth
Embryo survival and
teratogenieity test
Larval survival and
growth
Survival aad growth
Reprods)ciiosi
Ftrtflizatioa
FenfltoatJoa
FcrtttiaUoft
!•*»— jflf- -«ln •
rCf2JliZtiUOB
T< HI ixra t ore Test Length
25'C 7 days
25-C 8 days
2S*C 7 days
24'C 96 hours
25-C 7 days
25-C 9 days
25'C 7 days
26«C 7 days
23'C 7 days
20*C 1-2 hotn
12*C 1-2 hows
12'C 1-2 horn
12-C 1-2 hous
Age of Organism
Newry hatched larvae
2-24 hour embryos
>24 hour adults
4-7 day culture
7 day*
>24 hour larvae
> 24 hour larvae
7 days
egj ajM spcfM cdb
egg aad spem celb
egg aad spent oefli
egg aad tpen ceOs
egg and sperm cells
Scientific Name
Pimephales promelas
PimepHates prwnetas
Cfnotuxpnitut spp*
Selenastrum
capncarnuaim
MenitHa berytSna
Cyprinodtm varitgatus
Cypmodon iiirifgnnti
Mysidopnt batiia
~* mm
M*d* puncttil*
S. 4 me tacMfliitt
A pufpunttcf
OoMaVtuttr ccccnmcf
Notes:
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6.2    SOURCES OF TEST ORGANISMS
       Although it might seem desirable to use organisms residing in the receiving water as test
organisms, it is usually not practical for several reasons:  (1) sensitive species might not be pres-...:t;
(2) it is usually difficult to find a sufficient number of organisms of the right age and condition;
(3) special permits may be required in order to collect  and use these species; (4) the exposure
history to toxic chemicals  is unknown; (5) they may be  infested by parasites or diseases that can
affect responses; and (6) they may  have difficulty acclimating to test conditions.

       Species most commonly used for test organisms  are easily cultured.  Laboratory cultures are,
therefore, the best supply  of organisms. However, the  sensitivity of laboratory cultures must be
tested periodically (once each month) with reference toxicants to ensure that the response of
laboratory stocks is typical of other individuals of the same species.

       If the organisms  are not in  culture, they may be obtained from commercial supply houses  or
government  sources.  However, each batch of organisms received from an outside source must be
tested with reference toxicants to ensure that their responses are typical of other individuals  of the
same species.

       If it is decided to collect organisms from the wild, they must be observed over a period of
time to ensure that they do not become diseased or lose condition due to being held in the
laboratory.   Wild organisms should be tested with reference toxicants to ensure their responses are
typical of other individuals of the same species, where  known.

6.3    ACCLIMATION AND FEEDING
       Test organisms are normally kept in culture in water and at temperatures that may be
different from the condition and temperature of the dilution water for a test. Culture waters are
chosen because they are readily available at a laboratory at low cost; temperatures are chosen to
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ensure maximum perpetuation of stocks in the chosen water.  Since changes in temperature and
changes in key water parameters can have an apparent toxic effect on test organisms, organisms
must be acclimated to test conditions prior to testing.

6.3.1  Acclimation
       Acclimation is achieved by slowly adding dilution water over 24 to 48 hours to the water in
which organisms are cultured or held.  When organisms are in waters substantially different from
the dilution water, the rate of change of conditions between the storage water and dilution water
should be limited. Organisms commonly are stressed by changes in temperature of greater than
3'C or changes of salinity of greater than 3 ppt in any 12-hour period so changes during
acclimation should not exceed these rates. In addition, changes  in pH or alkalinity should be made
slowly to allow organisms to adapt  If mortality of the organisms exceeds 5 percent in the 24 hours
immediately preceding initiation of the  test (even if this includes the acclimation period), a new
batch of test organisms should be obtained.  If a new batch of organisms has 10 percent  mortality
in the 24 hours preceding a test in the  same dilution water, a different dilution water should be
used.   Acclimation for each of the EPA chronic tests is discussed in the chronic manuals.
       Feeding test organisms during testing is sometimes necessary, depending on the species used
and the test's length.  Recommended feeding procedures for each test and species are outlined in
the EPA manuals previously cited in Chapter 1.  With static tests, excess food must be removed as
soon as possible after feeding to ensure that bacterial decomposition does not foul the water or
reduce oxygen concentrations in the test solutions, except in the case of Ceriodapnia. which must
be fed after the adult is transferred to the fresh test solution.  With static renewal tests, feeding
should occur just prior to renewal  of the test solutions.  Feeding during flow-through tests does not
usually pose significant problems, but adding much more food than is necessary to maintain the
health of test organisms is not recommended.  Whenever possible, adequate amounts of nutritional
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food must, be provided to maintain normal growth rates of test organisms during testing.

6.4    DISEASE
      An obviously diseased or discolored organism should not be used in toxicity tests since either
of these conditions may cause the  organism to be more sensitive to toxicant stress. If disease
develops during the course of a toxicity test (particularly if it develops in the controls as well as
lower concentrations of the effluent), the test should be terminated and the results discarded.

6.5    LOADING RATES
      Organisms in test chambers consume oxygen and excrete potentially toxic materials during a
toxicity test.  It is important to control the number of organisms so that effects caused by their
presence can be minimized. For this reason, it is recommended that  the weight of organisms in test.
chambers should not exceed 5 g/1  of test solution for flow-through tests  at 20* C or colder, and
2.5 g/1 for flow-through tests above 20*G  In static and static renewal tests, organism weight should
not exceed 0.8 g/1 of test solution at 20-C or less, and 0.4 g/1 above 20*C.  Loading rates for each
of the chronic tests are listed  in the chronic manuals.

      Further, the recommended  loading rate should not cause the dissolved oxygen concentration
in the test chambers to be  reduced below 40 percent saturation1 for test  temperatures above 20* C
or 60 percent saturation for test temperatures at 20* C or less.  However, if oxygen concentrations
approach these limits due to bacterial action in  the test solutions, aeration may be necessary to
prevent  low oxygen levels from adversely affecting the test organisms.  Aeration should  be
minimized to prevent loss of volatile toxics.
i
   For saltwater tests with Menidia sp., oxygen concentrations may need to exceed 40 percent
   saturation.
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                                    7. TEST SYSTEM

      The fourth component discussed in detail is the test system. This component is the most
complicated to explain because it is closely tied to the procedures used in toxicity testing.  The test
system includes a number of specific items, such as:

      •   Equipment used for storing effluents and dilution water prior to use
      »   Chambers where effluent and dilution water are mixed in the appropriate concentrations
      •   Test chambers where organisms are exposed to the effluent/dilution water mixtures
      •   Tubing, valves, or pumps through which effluent or dilution water passes
      •   Test conditions.

The first four items listed above are discussed  together since the important considerations for these
items are the materials of which they are constructed and their cleanliness during a toxicity test.
Test conditions are dealt with separately at the end of this section.

7.1   MATERIALS USED
      Any material that comes into contact with either effluent or dilution water must not release,
absorb, or adsorb toxicants. A number of different choices  for this material is available. Glass and
No. 304 or 306 stainless steel are generally acceptable for freshwater holding, mixing, and test
chambers.  Stainless steel, however, is not acceptable for saltwater systems.  Square-sided glass
aquaria should be held together with small beads of silicone adhesive, with any unnecessary adhesive
removed from inside the aquaria.   If stainless steel containers are used, they must be welded, not
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soldered. -Other specialized containers of NITEX or TEFLON are also acceptable. Tanks for
holding effluents and dilution water may also be made of fiberglass. All containers or tubes made
of these materials are reusable with appropriate cleaning (see next section).

       Polyethylene, polypropylene, polyvinyl chloride, polystyrene, and TYGON may  also be used
for containers or tubing, but should be checked for  toricity before being used.  Because these
materials may absorb toxicants during a test, their reuse is discouraged to prevent  absorbed
toxicants from leaching into new effluent or dilution water.

       Copper, galvanized metal, brass, lead, and rubber must not contact the testing  solutions at
any time.

7.2    CLEANING
       New plasticware (from a known nontoxic source) can be used after rinsing with dilution
water.  New glassware should be soaked overnight in dilute (20 percent V:V) nitric or hydrochloric
acid, rinsed in tap water, and then rinsed with dilution water before use.

       Glassware and stainless steel components should be soaked in  detergent and scrubbed (or
washed in a laboratory dishwater), rinsed twice with tap water, rinsed with dilute acid, rinsed twice
with tap water, rinsed with full-strength acetone, rinsed twice with tap water, and then rinsed with
dilution water before use. Glassware for algae tests should also be neutralized in sodium
bicarbonate before use.

73    TEST CONDITIONS
       There are several physical/chemical measurements which are done in conjunction with whole
effluent toxicity tests to ensure the conditions of the test are within acceptable ranges for
Notes:
                                             7-2

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maintenance of the test organisms.  Three key parameters must be measured during a test to ensure
comparability of results between tests:

      •   Temperature
      •   Oxygen levels
      •   Salinity (for marine tests only).

Other parameters are measured because of their relationships to effluents and toxicants:

      •   pH
      •   Total alkalinity (fresh-water tests only)
      •   Total hardness (fresh-water tests only)
      •   Conductivity (fresh-water tests only)
      •   Total residual chlorine (fresh-water tests only).

Each of these parameters is discussed in the following subsections.

7.3.1 Temperature
      Each toxicity test has a nominal temperature and a range of temperatures (usually t 1* or
2*C) over which the  test should be  run.  Since organisms may be more sensitive to toxicants at
temperatures approaching their tolerance limits, these temperature ranges are critical in
standardizing responses.  The results of tests with ambient temperatures outside of the acceptable
range (in either direction) should be carefully reviewed for acceptability.
Notes:
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       Temperatures for static tests can be controlled by placing the test apparatus in a controlled
environment room or incubator at the appropriate temperature.  Temperature control is also
achieved using circulating water baths.  The latter provides more constant temperatures and  is less
likely to be affected by external factors such as lights and variations in air temperatures.
Temperatures of environment rooms or water baths should be calibrated with reference
thermometers periodically (when changes are suspected or at least once  per month).

7.3.2  Oxygen Levels
       Because low dissolved oxygen (DO) levels produce stress in test organisms, DO levels should
not decline below 4 mg/1 DO for tests above 20*C or 6 mg/1 DO for tests below 20»C  Generally,
it is best to run flow-through tests if low DO is expected to occur because flow-through tests allow
continuous replenishment of the test solutions before DO can decline in the test chambers.
However, at higher concentrations of effluent, oxygen may decline even in flow-through systems, so
aeration may be required.

       Aeration may affect the toxicity of effluents and should be used only as a last resort
Aeration, if applied, should be uniform across all test vessels.  If aeration is required in a flow-
through system, it should initially be provided in the dilution water, and if low DO still occurs in
test chambers, each test chamber should also be aerated. Aeration for individual test chambers
should be provided through a pipette, not an air stone.  Air flow through the bubbler should be at
the lowest rate necessary to maintain DO levels, but should not exceed 100 bubbles per minute.
Agitation caused by more rapid bubble rates can drive off volatile toxicants in the test system.

733 Salinity
       Salt-water tests require strict adherence to allowable salinity ranges.  Test dilutions which fall
outside of the specified  range may give inaccurate test results. Likewise, salinity should vary by no
more than ±2 parts per thousand among  the chambers (all dilutions) on a given day.
Notes:
                                              7-4

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      Salinity levels may be achieved by the use of dilution water which is natural sea water,
hypersaline brine prepared from natural sea brine, or (if recommended in the specific protocol)
artificial seawater prepared from sea salts. The selection of dilution water may limit the maximum
concentration of effluent that can be used in the test

7.3.4  pH
      Because of the effect pH has on the test organisms and the effect that it has on the toxicity
of some compounds (e.g., ammonia toxicity increases as the pH rises above 7), pH should be
monitored daily. If necessary, tests can be run in corked vessels in which gaseous CO2 has been
introduced above the test solution to control pH rise.

7.3.5  Total Alkalinity. Total  Hardness. Conductivity, and Total Residual Chlorine
      Several chemical measurements are recommended for the first day of the test to aid in the
interpretation of results, or at least to provide leads into further investigations if toxicity exists.
Total alkalinity will indicate the buffering capacity of the waste; hardness and conductivity can
indicate the magnitude of dissolved solids in the waste, and potentially indicate variability in effluent
quality.  Total Residual Chlorine is a recommended analysis because of the prevalence of this
toxicant in POTW wastewaters  and its lethality in low concentrations.

7.4 TEST REPLICATES
      In order to determine  statistically the significance of the results for any concentration of
effluent, it is necessary to test a sufficient number of organisms with each  concentration and the
control. The protocol manuals specify the minimum number of replicates and organisms required
for each method. This  information is also summarized in the summary tables in Appendix B.
Notes:
                                              7-5

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                                    8.  TEST RESULTS

      The final component of toxicity testing discussed in detail is the test result.  The valid
interpretation of test results requires that mortality in controls is limited, the conditions specified in
the previous sections are met, and the results are consistent with response patterns normally
observed in toxicity tests (e.g., increasing mortality with increasing concentrations).  If any of these
three general conditions are not met,  the inspector should not calculate summary statistics and
should ignore test results.

8.1   CONTROL SURVIVAL
      In general, survival in controls must exceed survival in all other test chambers for both acute
and chronic tests.  If it does not, calculation of the toxicity due to increasing effluent concentration
is at best an approximation of effluent toxicity.   In any case, mortality in  controls should not exceed
10 percent for acute toxicity tests and 20 percent for chronic tests (or other values as required by
States through their regulations).  If control survival does not  meet 90 or 80 percent for an  acute
or chronic test, respectively, then results should  not be used for calculating summary statistics, and a
determination of compliance using the test results cannot be made.

8.2   ACCEPTABILITY CRITERIA
      Each protocol has specified criteria  for acceptable ranges of control survival, temperature,
dissolved oxygen concentration, salinity, pH, light intensity and duration of photoperiod, organism
loading (numbers or weight per volume), feeding, and  cleaning procedures.  Summary tables of each
of the EPA methods is  in Appendix B of this module.  Tests not meeting the control criteria for
survival, growth, or reproduction are not valid.   Tests not meeting the other acceptability criteria in
these tables should be reviewed with caution and referred to the Regional Biologist.  The inspector
should review the EPA methods manual for a more extensive discussion of each of these factors.
Notes:
                                              8-1

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8.3    RESULTS CALCULATION
       The expected result in all toxicity tests is a greater number of organism responses with
increasing effluent concentrations.   On many occasions, this increasing response is observed as one
concentration eliciting no responses and the next higher concentration having 100 percent responses.
 This pattern is particularly obvious with acute tests.  In other cases, the test organisms in more
than one effluent dilution may exhibit a partial response  (between 0 and 100 percent).

       When test results do not meet the expected pattern, the test may be invalid.  Questionable
results  in an acute test include:

       •   Higher mortalities  in lower concentrations than in higher concentrations of effluent
       •   100 percent mortality in all effluent dilutions
       •   Greater percent mortality  in the control than  in the lower dilutions of effluent


Questionable results in a chronic test include:
      •   Greater growth or reproduction or fewer terata at higher concentrations of effluent than
          at lower concentrations
      •   No growth or reproduction or 100 percent terata at all effluent concentrations
      •   Less growth or reproduction or more terata in controls than in lower effluent
          concentrations.
      When any of these results occur (outside of experimental error), the results and test
conditions should be reviewed by the Regional biologist  It should be recognized, however, that
Notes:
                                             8-2

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often there will be minor variations in test results. For example, Ceriodaphnia dubia reproduction
may be higher at intermediate concentrations which are not toxic but provide a greater food
resource than lower concentrations. Thus, variations should not always be used to eliminate
otherwise valid results.  However, if the normally expected pattern  is not found, summary statistics
calculated on the results should be assessed with caution.

      Under some circumstances, compliance may still be determined with abnormal test results.
If, for example, 100 percent responses were found in  all effluent dilutions but the control was within
the acceptable response range, the appropriate toxicity measure must be below the most dilute
solution tested. Similarly, if no responses are found  in the toxicity test, the effluent can be deemed
nontoxic at 100 percent effluent

      Methods and computer programs by which summary statistics can be calculated are listed in
the manuals.  Each of the methods or programs can  be used only under limited circumstances.  If
these circumstances are not met, the results calculated will be erroneous.  Make sure that the
assumptions specified for each analysis are appropriate for the data being analyzed before using
these programs.
Notes:
                                              8-3

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             NPDES Compliacnce Monitoring Inspector Training Module: BIOMONITORING
                             APPENDIX A

                      QUESTIONS AND ANSWERS
                 ON THE BIOMONITORING MODULE

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               QUESTIONS ON THE BIOMONITORING MODULE
1.    A toxicity test in which the effluent and dilution water are continuously replenished in test
     chambers is called a	test.
2.    A	toxicity test estimates the concentration at which a predetermined toxic response
     occurs.
3.   The	is the concentration at which 50 percent of the test organisms die in a
     specified length of time.
4.   The highest concentration at which test organisms show no responses in a chronic test is
     called the       	.
5.   Toxicity is
6.   The ChV,	, is calculated by
7.   Effluent samples should be taken	.  (where)


8.   If an effluent sample is not to be used immediately, how should it be preserved?
9.   Small volume effluent samples should be stored in	,	, or
     containers.
 10.  Tanks for storing large volume effluent samples should be made of	or
 11.  Effluent samples should be used within	hours for on-site testing and	
     hours for off-site testing.


 12.  Effluent samples shipped to a laboratory for off-site testing should be preserved by being
     shipped	and	on receipt by the lab.
                                            A-l

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13.  Nonpersistant effluent samples must be used within	hours of sampling.


14.  Effluent samples for flow-through and static renewal tests are taken at least	for
     the duration of the  test.
15.  Continuous discharges with retention times of less than 14 days must be sampled
     for the duration of the test.
16.   What should be used as dilution water for a test to determine the inherent toxicity of an
     effluent?  	
17.   What should be used as dilution water for a test to determine the relative toxicity of an
     effluent in relation to receiving waters?	
18.   Mortality in controls must be less than	percent for acute tests and less than
                 for chronic tests.
19.   Test organisms are suitable for use in a toxicity test when there is less than
     percent mortality during acclimation.
20.  Receiving water that is used as dilution water should be refrigerated if not used in a test
     within 	hours.
21.  A new sample of receiving water should be taken if the sample is not used as dilution water
     within	hours of sampling.


22.  The two species most commonly used in toxicity testing are	 and	.


23.  The species to be used in testing for compliance monitoring is	.
24.  How frequently should the response of organisms receiving from supply houses be tested with
     reference toxicants?  	

25.  How frequently should the response of organisms raised in laboratory culture be tested with
     reference toxicants?    	
                                             A-2

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26.   Organisms whose responses in a test with a reference toxicant are outside the limits specified
     for tha't toxicant	.
27.  Adding dilution water to the water that organisms were raised in prior to a test is called
28.  Test organisms should be fed types of food and in amounts that are	for each
     species and test protocol.
29.  Test organisms showing signs of disease should
30.  Only (weight1) of test organisms should be added per liter of test solution for warm water
     static tests.
31.  Test chambers may be made of	,	, and


32.  Plastic containers and tubing that are used in a toxicity test _
 33.  No materials containing	,	,	,	,  and
     should come in contact with any solution to be used in toxicity testing.
 34.  New glassware should be	before use in toxicity testing.


 35.  Before use in another toxicity test, stainless steel containers and glassware should be
 36.  Test results obtained when temperatures were outside the ranges specified by the test protocol
 37.   DO should be above	percent saturation for tests run at more than 20* C or above
      ^^__<_ percent saturation for tests run at 20* C or lower.


 38.   Aeration should be provided only by	.
 39.   Typical toxicity tests show a higher number of responses with	concentrations of
      effluent
                                              A-3

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                 NPDES Compliance Monitoring Inspector Training Module:  BIOMONITORING








                ANSWERS ON THE BIOMONITORING MODULE






1.    Flow-through





2.    DeQnitive





3.    LQ,





4.    NOEC (no observable effect concentration)





5.    A characteristic of a substance that causes adverse responses in organisms





6.    The chronic value, taking the geometric mean of the NOEC and LOEC





7.    At the point specified in a permit






8.    It should be refrigerated at 4*C or placed on ice





9.    Cubitainers, plastic milk jugs, or glass containers






10.   Fiberglass or stainless steel





11.   24 hours on-site, 76 hours off-site



12.   On ice, refrigerated






13.   36





14.   Dairy






15.   Twice daily
                                          A-4

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                  NPDES Compliance Monitoring Inspector Training Module:  BIOMONITORING








16.   Standard dilution water (artificial water)






17.   Receiving water






18.   10 for acute, 20 for chronic






19.   5






20.   24






21.   96






22.   Ceriodaphnia dubia and Pimephales promelas or the water flea and the fathead minnow






23.   The species identified in the permit



24.   On receipt of each batch






25.   At least once each month






26.   Should not be used in toricity testing






27.   Acclimation






28.   Recommended in the manuals






29.   Not be used in toricity testing






30.   0.4 g






31.   Plastic that has been tested for toxicity, glass, and stainless steel
                                             A-5

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                  NPDES Compliance Monitoring Inspector Training Module:  BIOMONITORING



32.  Should be discarded


33.  Copper, brass, galvanized metal, rubber, or lead


34.  Soaked in acid and rinsed thoroughly
35.  Rinsed with acid, rinsed with tap water, rinsed with acetone, rinsed with tap water then rinsed
     with dilution water
36.  Should be carefully evaluated for acceptability


37.  40, 60


38.  Bubbling air through a pipette at less than 100 bubbles per minute


39.  Higher.
                                             A-6

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            NPDES Compliance Monitoring Inspector Training Module: BIOMONITORING
                           APPENDIX B
           DATA SHEETS FOR AQUATIC TOXICITY TESTS:
         SUMMARY OF RECOMMENDED TEST CONDITIONS
            FOR SOME COMMONLY USED TEST SPECIES

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                  NPDES Compliance Monitoring inspector Training Module:  BIOMONITORING
                                         TABLE  1.

           SUMMARY OF RECOMMENDED TEST CONDITIONS FOR DAPHNID
               (Daphnia pulex AND Daohnia maenad ACUTE TOXICITY TEST
1.   Temperature:

2.   Light quality:

3.   Light intensity:

4.   Photoperiod:

5.   Test chamber size:

6.   Test solution volume:

7.   Age of test organisms:

8.   Neonates/test chamber:

9.   Replicate
    chambers/concentration:

10.  Total number of organisms
    per concentration:

11.  Feeding regime:


12.  Aeration:


13.  Dilution water:



14.  Test duration:


IS.  Effects measured:


16.  Test acceptability:
20 t 2°C

Ambient laboratory illumination

10-20 ME/mVs (50-100 ft-c) (ambient lab levels)

8-16 hours light/24 hours

100 ml beaker or equivalent

50ml/replicate (loading and DO must be met)

1-24 hour (neonates)

10
20
Feeding not required during first 48 hour.  For longer
tests, feed every other day beginning on the third day.

None, unless DO falls below 40% of saturation, at
which time start gentle, single-bubble, aeration.

Receiving water or other surface water, ground water, or
synthetic water: hard water for Daphnia magna;
moderately hard or soft water for Daphnia pulex

Screening test - 24 h (static test)
Definitive test - 48 h (static test)

Mortality - no movement  of body or appendages on
gentle prodding
Control survival of 90% or greater
                                           B-l

See Methods for Measuring the Acute Toxicitv of Effluents to Freshwater and Marine Organisms.
EPA/600/4-85/013, March 1985.

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                  NPDES Compliance Monitoring inspector Training Module: BIOMONITORING



                                        TABLE 2.

            SUMMARY OF RECOMMENDED TEST CONDITIONS FOR MYSID
                        fMvsidopsis bahia) ACUTE TOXICITY TEST
1.   Temperature:

2.   Light quality:

3.   Light intensity:

4.   Photoperiod:

5.   Test chamber size:

6.   Test solution volume:


7.   Age of test organisms:

8.   OrganismsAest chamber:

9.   Replicate
     chambers/concentration:

10.   Total number of organisms
     per concentration:

11.   Feeding regime:


12.   Aeration:


13.   Dilution water


14.   Test duration:
20±2°C

Ambient laboratory illumination

10-20 ^E/rnVs (50-100 ft-c) (ambient lab levels)

8-16 hours light/24 hours

250 ml beaker or equivalent

200ml/replicate (loading and DO restrictions must be
met)

1-5 days

10
20
Two drops of concentrated brine shrimp nauplii
suspension twice daily (approx.  100 nauplii/mysid)

None, unless DO falls below 40% of saturation, at
which time start gentle, single-bubble, aeration.

Natural seawater, or synthetic salt water adjusted to 20
ppt salinity

Screening test - 24 h (static test)
Definitive test - 48 h (static test);
  48-% h (now-thru)
                                           B-2

See Methods for Measuring the Acute Toxicitv of Effluents to Freshwater and Marine Organisms.
EPA/600/4-85/013, March 1985.

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                 NPDES Compliance Monitoring inspector Training Module:  BIOMONITORING
15.  Effects,measured:                   Mortality - no movement of body or appendages on
                                      gentle prodding (LC»)

16.  Test acceptability:                   Control survival of 90% or greater.
                                          B-3

See Methods for Measuring the Acute Toricitv of Effluents to Freshwater and Marine Organisms.
EPA/600/4-85/013, March 1985.

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                 NPDES Compliance Monitoring inspector Training Module:  BIOMONITORING



                                        TABLE 3.

     SUMMARY OF RECOMMENDED TEST CONDITIONS FOR FATHEAD MINNOW
                      (Pimephales promelas) ACUTE TOXICITY TEST
1.   Temperature:

2.   Light quality:

3.   Light intensity:

4.   Photoperiod:

5.   Test chamber size:

6.   Test solution volume:


7.   Age of test organisms:

8.   Number of fish/test chamber

9.   Replicate
    chambers/concentration:

10.  Total number of organisms
    per concentration:

11.  Feeding regime:

12.  Aeration:


13.  Dilution water
20±2«C

Ambient laboratory illumination

10-20 ME/mVs (50-100 ft-c) (ambient lab levels)

8-16 hours light/24 hours

1 L beaker or equivalent

0.75 L/replicate (loading and DO restrictions must be
met)

1-90 days

10
20
Feeding not required first % h

None, unless DO falls below 40% of saturation, at
which time start gentle, single-bubble, aeration.

Receiving water, other surface water, ground water, or
soft synthetic water
14. Test duration:
Screening test - 24 h (static test)
Definitive test - 48 h (static test);
48-96 h (flow-thru)
                                           B-4

See Methods for Measuring the Acute Toxicitv of Effluents to Freshwater and Marine Organisms.
EPA/600/4-85/013, March 1985.

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                 NPDES Compliance Monitoring inspector Training Module: BIOMONITORING



15.  Effects .measured:                   Mortality - no movement (LC*,)

16.  Test acceptability:                   Control survival of 90% or greater.
                                          B-5

See Methods for Measuring the Acute Toricitv of Effluents to Freshwater and Marine Organisms.
EPA/600/4-85/013, March 1985.

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                                        TABLE 4.

         SUMMARY OF RECOMMENDED TEST CONDITIONS FOR SILVERSIDE
                          (Menidia spp.) ACUTE TOXICITY TEST
1.  Temperature:


2.  Light quality:

3.  Light intensity:

4.  Photoperiod:

5.  Test chamber size:

6.  Test solution volume:


7.  Age of test organisms:

8.  OrganismsAest chamber:

9.  Replicate
    chambers/concentration:

10.  Total number of organisms
    per concentration:

11.  Feeding regime:

11  Aeration:


13.  Dilution water


14.  Test duration:
20 ± 2°C (northern latitudes)
25 +. 2°C (southern latitudes)

Ambient laboratory illumination

10-20 nE/m2/s (50-100 ft-c) (ambient lab levels)

8-16 hours light/24 hours

1 L beaker or equivalent

0.75 L/replicate (loading and DO restrictions must be
met)

1-90 days

10
20
Feeding not required first 96 h

None, unless DO falls below 40% of saturation, at
which time start gentle, single-bubble, aeration.

Natural seawater, or synthetic salt water adjusted to 25-
30 ppt salinity

Screening test - 24 h (static test)
Definitive test - 48 h (static test);
48-96 h (flow-thru)
                                            B-6

See Methods for Measuring the Acute Toxicitv of Effluents to Freshwater and Marine Organisms.
EPA/600/4-85/013, March 1985.

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15. Effects measured:                   Mortality - no movement (LC»)


16. Test acceptability:                   Control survival of 90% or greater.
                                          B-7

See Methods for Measuring the Acute Toricitv of Effluents to Freshwater and Marine Organisms.
EPA/600/4-85/013. March 1985.

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                                             TABLE 5.

      SUMMARY OF RECOMMENDED TEST CONDITIONS FOR FATHEAD MINNOW
             fPimephales promelasl LARVAL SURVIVAL AND GROWTH TEST
1.   Test type:

2.   Temperature:

3.   Light quality:

4.   Light intensity:

5.   Photoperiod:

6.   Test chamber size:

7.   Test solution volume:


8.   Renewal of test concentrations:

9.   Age of test organisms:

10.   Larvae/test chamber:

11.   Replicate
     chambers/concentration:

12.   Larvae/concentration:

13.   Feeding regime:
14.  Cleaning:

15.  Aeration:
Static renewal

25-C * 1'C

Ambient laboratory illumination

10-20 <£/mVs (50-100 ft-c) (ambient lab levels)

16 hours light, 8 hours darkness

500 ml beakers or equivalent

250 ml/replicate (loading and DO restrictions must be
met)

Daily

Newly hatched larvae (less than 24 hours old)

15 larvae/chamber (minimum 10)


4 (minimum of 3)

60 larvae/concentration (minimum 30)

Feed 0.1 ml newly hatched (less than 24-h old) brine
shrimp nauplii  three times dairy at 4-h intervals or, as a
minimum,  0.15 ml twice daily, 6 h between feedings (at
the beginning of the work day prior to renewal, and at
the end of the  work day following renewal). Sufficient
larvae are  added to provide an excess. Larvae are not
fed during the  final 12 h of the test

Siphon daily, immediately before test solution renewal

None, unless DO falls below 40% of saturation.  Rate
should not exceed 100 bubbles/min.
                                           B-8

See Methods for Measuring the Acute Toxicitv of Effluents to Freshwater and Marine Organisms.
EPA/600/4-85/013, March 1985.

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16.  Dilution water:



17.  Effluent concentrations:

18.  Dilution factor:

19.  Test duration:

20.  Endpoints:

21.  Test acceptability:


22.  Sampling requirement:
23.  Sample volume required:
Moderately hard synthetic water is prepared using
Millipore Milli-Q" or equivalent deionized water and
reagent grade chemicals or 20% DMW

Minimum of 5 and a control

Approximately 0.3 or 0.5

7 days

Survival and growth (weight)

80% or greater survival in controls; average dry weight
of surviving controls equals or exceeds 0.25 mg

For on-site tests, samples are collected daily, and used
within 24 h of the time they are removed from the
sampling device.  For off-site tests, a minimum of three
samples are collected, and used on days 1-2, 3-4, and 5-
7.

2.5 L/day
                                             B-9

See Methods for Measuring the Acute Toxicitv of Effluents to Freshwater and Marine Organisms.
EPA/600/4-85/013, March 1985.

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                 NPDES Compliance Monitoring inspector Training Module:  BIOMONITORING
                                       TABLE 6.

     SUMMARY OF RECOMMENDED TEST CONDITIONS FOR FATHEAD MINNOW
    fPimephales promelas^ EMBRYO-LARVAL SURVIVAL AND TERATOGENICITY TEST
1.   Test type:

2.   Temperature:

3.   Light quality:

4.   Light intensity:

5.   Photoperiod:

6.   Test chamber size:

7.   Test solution volume:


8.   Renewal of test concentrations:

9.   Age of test organisms:

10.  Larvae/test chamber

11.  Replicate chambers/
    concentration:

12.  Larvae/concentration:

13.  Feeding regime:


14.  Aeration:


IS.  Dilution water
Static renewal

25'C ± l-C

Ambient laboratory illumination

10-20 ;iE/m'/s (50-100 ft-c) (ambient lab levels)

16 hours light. 8 hours darkness

150-500 ml beakers or equivalent

70-200 ml/replicate (loading and DO restrictions must
be met)

Daily

Less than 36 hour old embryos

15 larvae/chamber (minimum 10)


4 (minimum of 3)

60 larvae/concentration (minimum 30)

Feeding not required
None, unless DO falls below 40% of saturation.  Rate
should not exceed 100 bubbles/min.

Moderately hard synthetic water is prepared using
Millipore Milli-Q* or equivalent deionized water and
reagent grade chemicals or 20% DMW.  The hardness
of the test solutions must equal or exceed 25 mg/L
(CaCO,) to ensure hatching.
                                          B-10

See Methods for Measuring the Acute Toxicitv of Effluents to Freshwater and Marine Organisms.
EPAy600/4-85/013, March 1985.

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16.  Effluent concentrations:

17.  Dilution factor:

18.  Test duration:

19.  Endpoint:

20.  Test acceptability:

21.  Sampling requirement:
22.  Sample volume required:
Minimum of 5 and a control

Approximately 0.3 or 0.5

7 days

Combined mortality (dead and deformed organisms)

80% or greater survival in controls

For on-site tests, samples are collected daily, and used
within 24 h of the time they are removed from the
sampling device. For off-site tests, a minimum of three
samples are collected, and used on days 1-2, 3-4, and 5-
7.

2.5 L/day
                                            B-ll

See Methods for Measuring the Acute Toricitv of Effluents to Freshwater and Marine Organisms.
EPA/600/4-85/013, March 1985.

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                 NPDES Compliance Monitoring inspector Training Module:  BIOMONITORING



                                        TABLE 7.

                SUMMARY OF RECOMMENDED TEST CONDITIONS FOR
                   Ceriodaphnia SURVIVAL AND REPRODUCTION TEST
1.   Test type:

2.   Temperature:

3.   Light quality:

4.   Light intensity.

5.   Photoperiod:

6.   Test chamber size:

7.   Test solution volume:


8.   Renewal of test concentrations:

9.   Age of test organisms:

10. Neonates/test chamber:

11. Replicate
    chambers/concentration:

12. Neonates/concentration:

13. Feeding regime:


14. Aeration:

IS. Dilution water:



16. Effluent concentrations:

17. Dilution factor
Static renewal

25-C ± 1-C

Ambient laboratory illumination

10-20 »iE/m2/s (50-100 ft-c) (ambient lab levels)

16 hours light,  8 hours darkness

30 ml beakers or equivalent

IS ml/replicate (loading and  DO restrictions must be
met)

Daily

Less than 24 hour, all released within a 8-h period

1
10

10
Feed 0.1 ml each of YCT and algal suspension per test
chamber dairy

None

Moderately hard synthetic water is prepared using
Millipore Milli-Q* or equivalent deionized water and
reagent grade chemicals or  20% DMW.

Minimum of 5 and a control

Approximately 0.3 or O.S
                                           B-12

See Methods for Measuring the Acute Toxicitv of Effluents to Freshwater and Marine Organisms.
EPA/600/4-85/013, March 1985.

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                  NPDES Compliance Monitoring inspector Training Module:  BIOMONITORING
18.  Test duration:                       Until 60% of control females have three broods (may
                                        require more  or less than 7 days).

19.  Endpoint:                           Survival and reproduction

20.  Test acceptability:                    80% or greater survival and an average of 15 or more
                                        young/surviving female in controls.  At least 60% of
                                        surviving females in controls should have produced their
                                        third brood.

21.  Sampling requirement:               For on-site tests, samples are collected daily, and used
                                        within 24 h of the time they are removed from the
                                        sampling device.  For off-site tests, a minimum of three
                                        samples are collected, and used on days 1-2, 3-4, and 5-
                                        7.

22.  Sample volume required:             1 L/day
                                           B-13

See Methods for Measuring the Acute Toricitv of Effluents to Freshwater and Marine Organisms.
EPA/600/4-85/013, March 1985.

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                 NPDES Compliance Monitoring inspector Training Module: BIOMONITORING
                                       TABLES.

         SUMMARY OF RECOMMENDED TEST CONDITIONS FOR THE ALGAL
                        (Selenastrum capricornutum) GROWTH TEST
1.   Test type:

2.   Temperature:

3.   Light quality:

4..  Light intensity:

5.   Photoperiod:

6.   Test chamber size:

7.   Test solution volume:

8.   Renewal of test concentrations:

9.   Age of test organisms:

10.  Initial cell density:

11.  Replicate

12.  Shaking rate:

13.  Dilution water.


14.  Effluent concentrations:

15.  Dilution factor

16.  Test duration:

17.  Endpoint:
Static

25-C ± l«C

"Cool white" fluorescent lighting

86 t 8.6 ME/mVs (400 t 40 ft-c)

Continuous illumination

125 or 250 ml beakers or equivalent

50 or 100  ml/replicate

None

4 to 7 days

10,000 cells/ml

Shambers/conc.:

100 cpm continuous, or twice daily by hand

Algal stock culture medium without EDTA or enriched
surface water

Minimum  of 5 and a control

Approximately 0.3 or 0.5

96 h

Growth (cell counts, chlorophyll fluorescence,
absorbance, biomass)
                                          B-14

See Methods for Measuring the Acute Toxicitv of Effluents to Freshwater and Marine Organisms.
EPA/600/4-85/013, March 1985.

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                   NPDES Compliance Monitoring inspector Training Module:  BIOMONITORING
 18.  Test acceptability:                   2 X IP cells/ml in the controls; variability of controls
                                        should not exceed 20%

 19.  Sample volume required:             i L (one sample for test initiation)
                                           B-15

See Methods for Measuring the Acute Toxicitv of Effluents to Freshwater and Marine Organisms.
EPA/600/4-85A)13, March 1985.

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                 NPDES Compliance Monitoring inspector Training Module:  BIOMOMTORING



                                       TABLE 9.

    SUMMARY OF RECOMMENDED TEST CONDITIONS FOR SHEEPHEAD MINNOW
            rCvprinodon varieeatusl LARVAL SURVIVAL AND GROWTH TEST
1.   Test type:

2.   Salinity:

3.   Temperature:

4.   Light quality:

5.   Light intensity:

6.   Photoperiod:

7.   Test chamber size:

8.   Test solution volume:


9.   Renewal of test concentrations:

10.  Age of test organisms:

11.  LarvaeAest chamber

12.  Replicate
    chambers/concentration:

13.  Source of food:

14.  Feeding regime:



15.  Cleaning:

16.  Aeration:
Static renewal

20ppt to 32ppt * 2ppt

25-C t 2-

Ambient laboratory illumination

10-20 ME/mVs (50-100 ft-c) (ambient lab levels)

14 hours light, 10 hours darkness

300ml - 1L beakers or equivalent

250-750ml/replicate (loading and DO restrictions must
be met)

Daily

Newly hatched larvae (less than 24 hours old)

15 larvae/chamber (minimum 10)


4 (minimum of 3)

Newly hatched Anemia nauplii (less than 24 hours old)

Feed once a day O.lOg wet weight Anemia nauplii per
replicate on Days 0-2; feed 0.15g wet weight Anemia
nauplii per  replicate on Days 3-6

Siphon daily, immediately before test solution renewal

None, unless DO falls below 60% of saturation, then
aerate all chambers. Rate should be less than 100
bubbles/minute
                                          B-16

See Methods for Measuring the Acute Toxicitv of Effluents to Freshwater and Marine Organisms.
EPA/600/4-85/013, March 1985.

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                  NPDES Compliance Monitoring inspector Training Module:  BIOMONITORING
17.  Dilution water:


18.  Effluent concentrations:

19.  Dilution factor:

20.  Test duration:

21.  Effects measured:

22.  Test acceptability:
Uncontaminated source of natural seawater, hypersaline
brine, or artificial seawater mixed with deionized water

5 and a control

Approximately 0.3 or 0.5

7 days

Survival and growth (weight)

Control survival of 80% or greater, and average control
average dry weight of 0.6 mg or greater or, if preserved,
0.5 mg or greater.
                                             B-17

 See Methods for Measuring the Acute Toxicitv of Effluents to Freshwater and Marine Organisms.
 EPA/600/4-85/013, March 1985.

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                 NPDES Compliance Monitoring inspector Training Module:  BIOMONITORING
                                       TABLE 10.

    SUMMARY OF RECOMMENDED TEST CONDITIONS FOR SHEEPHEAD MINNOW
   (Cvprinodon variegatus) EMBRYO LARVAL SURVIVAL AND TERATOGENICITY TEST
1.  Test type:

2.  Salinity:

3.  Temperature:

4.  Light quality:

5.  Light intensity:

6.  Photoperiod:

7.  Test chamber size:

8.  Test solution volume:

9.  Renewal of test concentrations:

10.  Age of test organisms:

11.  EmbryosAest chamber

12.  Replicate chambers/
    concentration:

13.  Embryos per concentration:

14.  Feeding regime:

IS.  Aeration:

16.  Dilution water


17.  Effluent concentrations:

18.  Dilution factor
Static renewal

5ppt to 32ppt i 2ppt

25'C t 2-

Ambient laboratory illumination

10-20 vE/mK (50-100 ft-c) (ambient lab levels)

14 hours light, 10 hours darkness

500ml

400 ml (minimum of 250 ml)

Daily

less than 24 hours old

15 embryos/chamber (minimum 10)


4 (minimum  of 3)

60 (minimum of 30)

Feeding not required

None, unless DO falls below 60% of saturation

Uncontaminated source of natural seawater, hypersaline
brine, or artificial seawater mixed with deionized water

5 and a control

Approximately 03 or 0.5
                                         B-18

See Methods for Measuring the Acute Toricitv of Effluents to Freshwater and Marine Organisms.
EPA/600/4-85/013, March 1985.

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                   NPDES Compliance Monitoring inspector Training Module:  BIOMONITORING



 19.  Test Duration:                       9 days

 20.  Effects measured:                    Percent hatch; percent larvae dead or with debilitating
                                         morphological and/or behavior abnormalities such as:
                                         gross deformities, curving spine, disoriented, abnormal
                                         swimming behavior; surviving normal larvae from
                                         original embryos

 21.  Test acceptability:                    Control survival of 80% or greater.
                                            B-19

See Methods for Measuring the Acute Toxicitv of Effluents to Freshwater and Marine Organisms.
EPA/600/4-85/013, March 1985.

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                 NPDES Compliance Monitoring inspector Training Module: BIOMONITORING



                                       TABLE 11.

  SUMMARY OF RECOMMENDED TEST CONDITIONS FOR THE INLAND SILVERSIDE
               (Menidia bervllina) LARVEL SURVIVAL AND GROWTH TEST
1.  Test type:

2.  Salinity:

3.  Temperature:

4.  Light quality:

5.  Light intensity:

6.  Photoperiod:

7.  Test chamber size:

&  Test solution volume:


9.  Renewal of test concentrations:

10.  Age of test organisms:

11.  LarvaeAest chamber

12.  Replicate chambers/
    concentration:

13.  Source of food:

14.  Feeding regime:



IS.  Cleaning:


16.  Aeration:
Static renewal

5ppt to 32ppt t 2ppt

25'C t 2'

Ambient laboratory illumination

10-20 itE/mYs (50-100 ft-c) (ambient lab levels)

14 hours light, 10 hours darkness

300ml - 1L containers

250-750ml/replicate (loading and DO restrictions must
be net)

Daily

7-11 days post hatch

15 larvae/chamber (minimum 10)


4 (minimum of 3)

Newly hatched Anemia nauplii

Feed O.lOg wet weight Anemia nauplii per replicate on
days 0-2; Feed 0.15g wet weight Anemia nauplii per
replicate on days 3-6

Siphon daily, immediately before test solution renewal
and feeding

None, unless DO falls below 60% of saturation,  then
aerate all chambers.  Rate should be less than 100
bubbles/minute
                                          B-20

See Methods for Measuring the Acute Toxicitv of Effluents to Freshwater and Marine Organisms.
EPA/600/4-85/013, March 1985.

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                   NPDES Compliance Monitoring inspector Training Module:  BIOMONUORING
 17.  Dilution water:


 18.  Effluent concentrations:

 19.  Dilution factor:

 20.  Test duration:

 21.  Effects measured:

 22.  Test acceptability:
Uncontaminated source of natural seawater or
hypersaline brine mixed with deionized water

At least 5 and a control

Approximately 0.3 or 0.5

7 days

Survival and growth (weight)

Control survival of 80% or greater, control average dry
weight (for 7 day old larvae) of 0.5 mg or greater, or, if
preserved, 0.43 mg or greater.
                                             B-21

See Methods for Measuring the Acute Toxicitv of Effluents to Freshwater and Marine Organisms.
EPA/600/4-85/013, March 1985.

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                 NPDES Compliance Monitoring inspector Training Module:  BIOMONITORING
                                       TABLE 12.

     SUMMARY OF RECOMMENDED TEST CONDITIONS FOR THE Mvsidopsis bahia
                 7-DAY SURVIVAL GROWTH, AND FECUNDITY TEST
1.  Test type:

2.  Salinity:

3.  Temperature:

4.  Light intensity:

5.  Photoperiod:

6.  Test chamber.

7.  Test solution volume:

8.  Renewal of test solutions:

9.  Age of test organisms:

10.  Organisms/test chamber

11.  Replicate chambers/
    treatment:

12.  Source of food:

13.  Feeding regime:


14.  Cleaning:

15.  Aeration:


16.  Dilution water


17.  Number of treatments/study:
Static renewal

ZOppt to 30ppt t 2ppt

26--27-C

10-20 nE/mVi (50-100 ftc.) (ambient lab levels)

16 h light, 8 h darkness, with phase in/out period

8oz plastic disposable cups or 400ml glass beakers

ISOml/replicate cup

Daily

7 days

5


8

Anemia naupfu

Feed 150 24-h old Anemia nauplii per mysid daily, half
after test solution renewal and half after 8-12 hours

Pipette excess food from cups daily

None, unless DO falls below 60% of saturation, then
gentry in all cups

Uncontaminated source of natural seawater or
hypersaline brine

Minimum of 5 and a control
                                          B-22

See Methods for Measuring the Acute Toxicitv of Effluents to Freshwater and Marine Organisms.
EPA/600/4-85AH3, March 1985.

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                  NPDES Compliance Monitoring inspector Training Module:  BIOMONITORING



18.   Dilution factor:                      Approximately 0.3 or 0,5

19.   Test duration:                       7 days

20.   Effects measured:                    Survival, growth, and egg development

21.   Test acceptability:                    Control survival of 80% or greater, control dry weight
                                        of 0.2 mg/mysid or  greater.
                                            B-23

See Methods for Measuring the Acute Toricitv of Effluents to Freshwater and Marine Organisms.
EPA/600/4-85/013, March 1985.

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                  NPDES Compliance Monitoring inspector Training Module: BIOMONITORING
                                        TABLE 13.

                SUMMARY OF RECOMMENDED TEST CONDITIONS FOR
                         Arbacia punctulata FERTILIZATION TEST
1.  Test type:

2.  Salinity:

3.  Temperature:

4.  Light quality:

5.  Light intensity:

6.  Test vessel size:


7.  Test solution volume:

8.  Number of sea urchins:
9.   Number of egg and sperm cells
    per chamber

10.  Replicate chambers/
    treatment:

11.  Dilution water
11  Dilution fecton

13.  Number of treatments/study:

14.  Test duration:

IS.  Effects measured:

16.  Test acceptability:
Static

30ppt *2ppt

20'tl-C

Ambient laboratory light during test preparation

10-20 ME/m'/s (50-100 ftc.) (ambient lab levels)

Disposable (glass) liquid scintillation vials (20ml
capacity), not pre-cleaned

5ml

Pooled sperm from four males and pooled eggs from
four females per test


About 2000 eggs and 5,000,000 sperm cells per vial


4 (minimum of 3)

Uncontaminated source of natural seawater or deionized
water mixed with hypersaline brine or artificial  sea salts

0.3 or 0.5

Minimum of 5 effluent concentrations and a control

1 hour and 20 minutes

Fertilization of sea urchin eggs

Control fertilization spermregg ratio of 70-90%.
                                           B-24

See Methods for Measuring the Acute Toxicitv of Effluents to Freshwater and Marine Organisms.
EPA/600/4-85/013, March 1985.

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                  NPDES Compliance Monitoring inspector Training Module: BIOMONITORING
                                        TABLE 14.

                SUMMARY OF RECOMMENDED TEST CONDITIONS FOR
                     Champia parvula SEXUAL REPRODUCTION TEST
 1.   Test type:

 2.   Salinity:

 3.   Temperature:

 4.   Photoperiod:

 5.   Light quality:

 6.   Light intensity:

 7.   Test vessel size:

 8.   Test solution volume:

 9.   Dilution water:
10.  Dilution factor:

11.  Number of dilutions:

12.  Number of replicate Chambers
     per treatment:

13.  Number of organisms
     per test chamber

14.  Test duration:
 Static

 30ppt ±2ppt

 22-24-C

 16 h light, 8 h dark

 Cool white fluorescent lights

 100 ^E/mVs (500 ft.c.)

 200 ml polystyrene cups, or 250 ml Erlenmeyer flasks

 100ml

 30 parts per thousand salinity natural seawater, or a
 combination of 50% (30 part per thousand salinity)
 natural seawater and 50% (30 pan per thousand)
 salinity artificial seawater

 0.3 or 0.5

 At least 5 and a control


 4 (minimum of 3)


 5 female branch tips and 1 male plant

 2-day exposure to effluent, followed by 5 to 7 day
 recovery period in control medium for cystocarp
development
                                           B-25

See Methods for Measuring the Acute Toxicitv of Effluents to Freshwater and Marine Organisms.
EPA/600/4-85/013, March 1985.

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                  NPDES Compliance Monitoring inspector Training Module:  BIOMONITORING



15.  Effects, measured:                   Reduction in cystocarp production compared to controls

16.  Test acceptability:                   Control survival of 80% or greater, control average
                                       cystocarp production of 10 or greater per plant
                                       (NOTE: plants fragmenting in lower  concentrations may
                                       indicate undue stress)
                                          B-26

See Methods for Measuring the Acute Toxicitv of Effluents to Freshwater and Marine Organisms.
EPA/600/4-85/013, March 1985.

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             NPDES Compliacnce Monitoring Inspector Training Module:  BIOMONITORING
                             APPENDIX C

                 HEALTH AND SAFETY PROCEDURES

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                  NPDES Compliance Monitoring Inspector Training Module: BIOMONITORING


                      HEALTH AND SAFETY PROCEDURES1



1.0  GENERAL PRECAUTIONS

1.1  Collection and use of effluents in toxicity tests may involve significant risks to personal safety
     and health.  Personnel collecting effluent samples and conducting toxicity tests should take all
     safety precautions necessary for the prevention of bodily injury and illness which might result
     from ingestion or invasion of infectious agents, inhalation or absorption of corrosive or toxic
     substances through the skin, and asphyxiation due to  lack of oxygen or the presence of
     noxious gases.

1.2  Prior to sample collection and laboratory work, personnel should determine that all necessary
     safety equipment and materials have been obtained and are  in good condition.


2.0  SAFETY EQUIPMENT

2.1  Personal Safety Gear

     Personnel should  use safety equipment as required, such as rubber aprons, laboratory coats,
     respirators, gloves, safety glasses, hard hats, and safety shoes. Plastic netting on glass beakers,
     flasks, and other glassware minimizes breakage and subsequent shattering of the glass.

2.2  Laboratory Safety Equipment

     Each laboratory (including mobile laboratories) should be provided with safety equipment such
     as first aid kits, fire extinguishers, fire blankets, emergency showers, and eye fountains.


3.0  GENERAL LABORATORY AND FIELD  OPERATIONS

3.1  Work with effluents should be performed in compliance with accepted rules pertaining to the
     handling of hazardous materials (see safety manuals listed in Paragraph 5.0).  It is
     recommended that personnel collecting samples and performing  toxicity tests should not work
     alone.

3.2  Because the chemical  composition of effluents is usually only poorly known,  they should be
     regarded as potential health hazards and  exposure to them should be minimized.

3.3  It is.advisable to  cleanse exposed parts of the body immediately  after collecting effluent
     samples.
        'Adapted from "Short-term Methods for Estimating Chronic Toxicity of Effluents &
    Receiving Waters for Marine and Estuarine Organisms," EPA, May 1988.
                                             C-l

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                  NPDES Compliance Monitoring Inspector Training Module:  BIOMONITORING


3.4  All containers should be adequately labeled to indicate their contents.

3.5  Good -housekeeping contributes to safety and reliable results.

3.6  Electrical equipment or extension cords not bearing the approval of Underwriter Laboratories
     must not be used.  Ground-fault interrupters must be installed in all Vet" laboratories where
     electrical equipment is used.

3.7  Mobile laboratories should be properly grounded to protect against electrical shock.


4.0  DISEASE PREVENTION

4.1  Personnel handling samples which are known or suspected to contain human wastes should be
     immunized against tetanus, typhoid fever, and polio.


5.0  SAFETY MANUALS

5.1  For further guidance on safe  practices when collecting effluent samples and conducting toxicity
     tests, check with the permittee and consult general safety manuals, including the USEPA
     Occupational Health and Safety Manual (1977), and Health and Safety for Toxicitv Testing by
     D.B. Walters and CW.  Jameson, Butterworth Publishers, Woburn, Massachusetts (1984).


6.0  WASTE DISPOSAL

6.1  Wastes generated during toxicity testing must be properly handled and disposed of in an
     appropriate manner.  Each testing facility will have its own waste disposal requirements based
     on local, State, and Federal rules and regulations.  It is extremely important that these be
     known, understood, and complied with by all persons responsible for performing toxicity tests.
                                            C-2

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