United States
Environmental Protection
Agency
Office of Water Enforcement
Enforcement Division
Washington, DC 20460
October 1979
430979504
Water
S-EPA
Interim NPDES
Compliance Biomonitoring
Inspection Manual
MCD - 62
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NOTES
To order this publication, MCD-62 "NPDES Compliance Samplinq
Inspection Manual", write to:
General Services Administration (8FFS)
Centralized Mailing Lists Services
Buildinq 41, Denver Federal Center
Denver, Colorado 80225
Please indicate the MCD number and title of publication.
Multiple copies maybe purchased from:
National Technical Information Service
Springfield, Virginia 22151
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INTERIM NPDES
COMPLIANCE BIOMONITORING
INSPECTION MANUAL
UNITED STATES .ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF WATER ENFORCEMENT
COMPLIANCE BRANCH
1979
U.S. Environmental Protection Agenc;
Great Lakes National Program Office
GLNPO Library
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DISCLAIMER
This manual has been reviewed by the Office of Water
Enforcement, U.S. Environmental Protection Agency, and approved
for publication. Mention of trade names or commercial products
does not constitute endorsement or recommendation for use.
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AC KNOWLE DGEMENT
This manual was developed by a special task force of U.S. EPA
and State representatives as a cooperative project in administra-
tive and technical guidance for conducting NPDES (National Pollutant
Discharge Elimination System) compliance inspections, and the
utilization of biomonitoring techniques. Task force members met in
Washington, D.C. and Athens, Georgia and incorporated suggestions
and comments from a number of persons representing EPA Headquarters
and Regional offices, approved NPDES States/ and selected Federal
agencies.
Task force personnel included:
1. Alvaro A. Yamhure, Past Chairman, EPA, Washington, D.C.
2. Stephen L. Bugbee, EPA, Washington, D.C.
3. Robert F. Schneider, EPA, NEIC
4. David I. Shedroff, EPA, Washington, D.C.
5. Lee B. Tebo, Jr. , EPA, Region IV
6. Phil Gehring, EPA, Region V
7. Cornelius I. Weber, EPA, ORD
8. William B. Horning, EPA, ORD
9. Henry Silbermann, DNR, State of Maryland
10. Fred E. Jarvis, CRWQCB, State of California
11. Ron Preston, EPA, Region III
The work group wishes to express its appreciation to all other
persons who assisted in the preparation of this manual. Special
thanks are due to Mr. Gary R. Polvi, Mr. Brian J. Maas, Ms. Barbara
A. Schick, Chief and Environmental Scientists, respectively, of the
Technical Evaluation and Support Section, and Mr. George T. Faison of
the secretarial staff of the Compliance Branch, Enforcement Division,
Office of Water Enforcement. Comments and suggestions for revision
and improvements are invited.
iii
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FOREWORD
This is one of several monitoring documents produced by EPA
to be used in assessing NPDES permit compliance and monitoring the
quality of waste water discharges into the Nation's waters.
This particular manual provides guidance to instruct NPDES
inspectors in the proper use of those biomonitoring techniques
that have been officially recognized for EPA use.
Other EPA water monitoring publications dealing with NPDES
permit compliance are:
0 NPDES Compliance Evaluation Inspection Manual, July, 1976.
0 NPDES Compliance Sampling Inspection Manual, June, 1977,
revised edition, July 1979 - 680 - 197/480.
Also available are the following publications which provide
technical information on water monitoring procedures:
0 Biological Field and Laboratory Methods for Measuring the
Quality of Surface Waters and Effluents, EPA-670/4-73-001;
July, 1973.
0 Basic Water Monitoring Program, EPA-440/9-76-025, 1977;
Revised Edition, May, 1978.
° Model State Water Monitoring Program, EPA-440/9-74-002,
June, 1974.
0 Bioassay Procedures for the Ocean Disposal Permit Program.
EPA 600/9-78-010; Revised Edition, March 1978.
The NPDES compliance inspection program represents a sig-
nificant commitment of resources by the States and EPA to verify
permit effluent limitations and to assure that compliance with
permit requirements for monitoring, reporting and compliance
schedules are being met.
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Biomonitoring conducted in a thorough, professional manner in
accordance with procedures described in this Manual, provides the
EPA, States, and permittees with a practical tool for checking on
the effectiveness of compliance with NPDES permits and the overall
National Enforcement program. Familiarity with this Manual by EPA
inspectors is essential; its use by other interested and concerned
persons (States, permittees, consultants, etc.) is encouraged.
Because EPA policy and specific biomonitoring requirements
and techniques have not been finalized, this manual is being
published as interim guidance. However, it is imperative that
plans and methods for biomonitoring be initiated now so that
problem areas can be discovered and addressed before final policy
is implemented.
Readers are encouraged to offer comments or suggest revisions.
These should be directed to the NPDES Compliance Biomonitoring
Manual Review Committee, Technical Evaluation and Support Section,
Compliance Branch (EN-338), Enforcement Division, Office of Water
Enforcement, U.S. Environmental Protection Agency, 401 M Street,
S.W., Washington, D.C. 20460.
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NPDES COMPLIANCE
BIOMONITORING INSPECTION MANUAL
TABLE OF CONTENTS
Page No.
Disclaimer ii
Acknowledgement iii
Foreword iv
Table of Contents vi
List of Appendices ix
I. Introduction 1-1
A. Background 1-1
B. Purpose of Manual 1-4
C. Statutory Authority 1-5
D. Biomonitoring Requirements in Permits 1-7
E. Federal and State Cooperation 1-7
II. Legal Considerations II-l
A. Access and Warrants - Constitutional
and Statutory Requirements II-l
B. Discussions with Permittees or Their
Agents - Privilege Against Self-
Incrimination II-2
C. Expert and Other Testimony II-3
D. Chain of Custody and Preservation of
Documents II-4
E. Relations with the Public II-7
III. Planning Biomonitoring Inspections III-l
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Page No.
A. Pre-inspection Planning Activities III-l
B. Coordination of Inspection Activities with
Permittees, other EPA Programs, and
Government Agencies III-5
IV Inspection Types IV-1
A. Announced and Unannounced Inspections IV-1
B. Sampling-Type Inspections IV-1
C. Evaluation Inspections IV-3
V. Quality Assurance V-l
A. Effluent Sampling and Handling V-l
B. Test Organisms V-2
C. Facilities and Equipment ............. V-3
D. Dilution Water V-4
E. Test Conditions V-4
F. Reference Toxicants V-4
G. Record Keeping V-6
VI. Health and Safety VI-1
A. General VI-1
B. Personal Conduct VI-2
C. Safety Equipment VI-2
D. General Laboratory Operation VI-2
E. Transportation VI-3
F. Emergency Health and Fire Protection VI-3
G. Accident Reports VI-3
VII. Conducting Biomonitoring Inspections VII-1
A. Facilities Access ..... VII-1
B. Conducting Sampling Type-Inspections VII-2
C. Conducting Evaluation Inspections VII-5
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VIII. Post Inspection Activities VIII-1
A. Data Evaluation VIII-1
B. Toxicity Laboratory Evaluation Form VIII-5
C. Distribution of Inspection Report VIII-5
D. Follow-up Activities VIII-5
viii
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LIST OP APPENDICES
Page
A. Methods for Measuring the Acute Toxicity of
Effluents to Aquatic Organisms, EPA-600/4-78-012 . . . A-l
B. Toxicity Test Report Outline B-l
C. Laboratory Review or Audit C-l
D. Example of Daily Activities of On-site Toxicity
Monitoring D-l
E. Sample Tags and Chain of Custody Forms E-l
F. Document Handling Procedures F-l
G. Definitions G-l
H. Factors Useful in Selecting Candidates for
Inspection H-l
I. Conduct of Inspections After the Barlow's
Decision 1-1
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SECTION I
INTRODUCTION
A. Background
The Federal Water Pollution Control Act Amendments of 1972
established the objective of restoring and maintaining the chem-
ical, physical, and biological integrity of the Nation's waters.
This objective has remained unchanged in the 1977 amendments to
the Act, commonly referred to as the Clean Water Act of 1977. To
achieve this end, the Act sets forth a series of goals, including
the goal of eliminating the discharge of pollutants into navigable
waters by 1985. The principal mechanism for reducing the
discharge of pollutants is through implementation of the National
Pollutant Discharge Elimination System (NPDES) established by
Section 402 of the Act.
NPDES permits have been issued to approximately 55,000 muni-
cipal and industrial point sources. Permits contain four primary
elements: (1) final effluent limitations reflecting statutorily
required treatment levels; (2) interim effluent limitations until
the attainment of final effluent limitations; (3) construction
schedules for the achievement of final effluent limitations; and
(4) reporting requirements relating to compliance with effluent
limitations established for each parameter limited in the permit
for both interim or final effluent limitations and compliance with
construction schedules.
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Compliance with these effluent limitations and self-
monitoring requirements of NPDES permits is assessed by the
regulatory agency through a combined program of self-monitoring
data review and facility inspections.
Historically, the major focus of final effluent limits
contained in NPDES permits has been the control of conventional
pollutants such as BOD, pH, fecal coliform, etc. At the time of
first round permitting, these pollutants were considered the
parameters which most urgently needed controls.
In second round permitting, however, Agency emphasis is
shifting from the conventional pollutants to the control of toxic
pollutants. This is due in part to the NRDC Consent Decree which
identified 65 families of toxic pollutants for which both ambient
and effluent guidelines must be developed. Furthermore?, this
shift is the next logical step in water pollution control as the
potential for environmental harm from conventional pollutants
decreases due to compliance with effluent and water quality
standards.
Traditionally, NPDES permits have contained specific
numerical limits for individual chemicals. Similarly, effluent
guidelines which are established for Consent Decree toxic
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pollutants under Section 307 (a), will also be chemical specific
numerical limits. However, this approach has several drawbacks.
It does not take into account synergistic toxicity resulting from
a combination of two or more chemicals in an effluent, nor can it
be expected to always provide an adequate margin of safety for
chemicals which lack specific toxicity data. In addition,
periodic sampling and analysis for these compounds during NPDES
self-monitoring activities is extremely resource intensive and
many times the methods for analysis are not completely reliable.
In general, since the effluent guidelines will be technology
based, they will not always be able to guarantee water quality
sufficient for the protection of indigenous aquatic life.
Because of this, the Act provides for methods of developing more
stringent water pollution controls, such as water quality
standards which are based on specific biological toxicity data.
It is because of these issues and Section 308, which specifically
provides for biological monitoring, that the NPDES Biomonitoring
Program has been developed. Since biomonitoring is a measure of
the direct acute toxicity of the effluent, it provides a good
estimate of the effluent's potential for environmental harm and
its potential effect on indigenous biological communities. In
addition, biomonitoring provides a method for assessing the
adequacy of and assuring compliance with State water quality
standards.
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Simply stated the objectives of the biomonitoring program
are as follows.
1) To serve as a screening mechanism, isolating toxic
conditions in effluents which may not have been detected through
routine chemical analyses;
2) To evaluate effluents that are in compliance with State
water qualtiy standards;
3) To provide a monitoring function which will act as an
early warning system for toxics which may or may not be
controlled through BCT/BAT and;
4) To serve as a surrogate test for toxic conditions as a
prerequisite and priority setting process for the more resource
intensive chemical analysis for toxic pollutants.
B. Purpose of Manual
The NPDES Biomonitoring Inspection Manual is primarily
designed to aid in the conduct of NPDES compliance inspections by
EPA and State agency personnel and will also be of use in the
orientation and training of any technical personnel. Definitions
of selected terms used in this manual are presented in Appendix H.
This manual is intended to assist EPA in meeting its
statutory obligations under section 308 and other pertinent
parts of the Clean Water Act, of 1977 (see also section I C. of
this manual). In addition, this manual will be of assistance in
fulfilling the following Agency needs:
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1. To provide Agency-wide policy and a uniform procedural,
administrative, legal and technical framework which all of EPA's
Regional Surveillance and Analysis (S&A) personnel and State
agency employees can use as guidance. This uniform reference is
an important step in making biomonitoring data and admihistrative
information more valid and of greater use to enforcement offices.
Furthermore, data obtained in this manner can greatly contribute
to the continued refinement and development of biomonitoring
techniques.
2. To simplify NPDES biomonitoring activities and standardize
techniques for performing toxicity tests.
3. To provide administrative and procedural support to the
Permits Biomonitoring Policy Guidance manual for the NPDES
Permits Program which establishes guidelines for the
inclusion of biomonitoring requirements in NPDES permits.
For the purpose of addressing the technical aspects of aqua-
tic acute toxicity testing this manual has incorporated in its
entirety the EPA report: "Methods for Measuring the Acute
Toxicity of Effluents to Aquatic Organisms", EPA-600/4-78-012.
Revised July 1978.
C. Statutory Authority
Biological monitoring (biomonitoring) is the logical and
appropriate method for ascertaining the "...biological integrity
of the Nation's waters." No other method is likely to accomplish
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the monitoring needed to check on the desired restoration and
maintenance of biological integrity.
Section 308(a) of the Act states in part:
(A) the Administrator shall require the owner or
operator of any point source to (i) establish and
maintain such records, (ii) make such reports, (iii)
install, use and maintain such monitoring equipment or
methods (including where appropriate, biological
monitoring methods), (iv) sample such effluents (in
accordance with such methods, at such locations, at such
intervals, and in such manner as the Administrator shall
prescribe), and (v) provide such other information as he
may reasonably require; and
(B) the Administrator or his authorized representative,
upon presentation of his credentials —
(i) shall have a right of entry to, upon or through
any premises in which an effluent source is located
or in which any records required to be maintained
under clause (A) of this subsection are located,
and
(ii) may at reasonable times have access to and
copy any records, inspect any monitoring equipment
or method required under clause (A), and sample any
effluents which the owner or operator of such
source is required to sample under such clause.
The Administrator is specifically authorized by the
provisions of section 308 to install, use, and maintain monitoring
equipment and methods, including biological monitoring, to
determine "...whether any person is in violation of any effluent
limitation, or other limitation, prohibition or effluent standard,
pretreatment standard, or standard of performance..."
Thus, the use of biomonitoring techniques for NPDES
compliance is clearly recognized and authorized by statute.
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D. Biomonitoring Requirements in Permits
At the option of the permitting agency, NPDES permits may
contain specific enforceable toxicty limits or biological self-
monitoring requirements. In many cases, permitting authorities
may elect to include biological self-monitoring requirements
without toxicity limitations, such as when data on the potential
toxicity of the effluent has not been well documented. While data
obtained during biological self-monitoring from facilities which
do not have specific permit toxicity limitations may not be
directly used as the basis for enforcement, it can be used 1) in
enforcement case development, 2) to identify eminent health
hazards, 3) to flag possible permit violations of chemical
parameters and/or identify potential toxic conditions in the
receiving waters. For these reasons enforcement offices may also
elect to perform biomonitoring inspections at any time regardless
of whether there are biomonitoring requirements in a NPDES
permit.
E. Federal and State Cooperation
Congress has directed that the EPA seek, encourage, and
support cooperation with the States (Sections 101, 102 and 103 of
the Clean Water Act of 1977).
EPA personnel involved in planning and conducting biomonitor-
ing inspections for NPDES compliance should at all times, be aware
of the value and need of coordinating Federal inspections and
other activities and programs with the appropriate State officials
and agencies. This should be a reciprocal arrangement, with State
Agencies also coordinating inspections with EPA. This essential
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coordination depends on a continuing commitment to inform the
appropriate EPA State representatives of plans and intended
activities concerning NPDES compliance monitoring in a particular
State.
Cooperation between EPA and the States must be promoted on a
continuing basis to be successful, and if done will reap the
benefits of more effective control and management of the NPDES
program.
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SECTION II
LEGAL CONSIDERATIONS
A. Access and Warrants - Constitutional and Statutory
Requirements
Inspectors must gain entry to a permittee's private premises
and perform all subsequent inspection activities in a manner
consistent with the right of privacy of the permittee as guar-
anteed by the U.S. Constitution. Legal entanglements may result
if entry or access to any type of evidence is obtained through
direct or implied threats, or trickery. Any information obtained
in this fashion may not be admissible as direct evidence in
administrative or court enforcement proceedings.
The "unreasonable search and seizure" requirements of the
U.S. Constitution apply to the activities of all government
employees. These requirements mean that entry into a permittee's
private property for inspection purposes must be done only with
the consent of a person authorized to give it, or by the auth-
ority of an administrative or criminal search warrant issued by a
judge or magistrate, having jurisdiction.
In almost every case, EPA and State personnel will attempt to
obtain entry and perform their inspection or monitoring responsi-
bilities on the basis of consent of the owner, operator or person
in charge of the facility, that is, a person who has authority to
give consent. In the case of announced inspections, this can be
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the person the inspector has been directed to ask for, even if
they are not the plant manager.
Federal inspectors must also comply with the requirements of
section 308 of the Clean Water Act. That section requires Federal
inspectors to present their credentials at the time they seek
access to a permittee's premises.
Appendix J of this manual includes copies of memoranda from
EPA's Assistant Administrator for Enforcement which provide guide-
ance in the conduct of inspections in light of the recent Supreme
Court decision in Marshall v. Barlow's Inc.
Both the constitutional and statutory requirements apply to
individuals and corporations.
B. Discussions with Permittees or Their Agents - Privilege
Against Self-Incrimination
During the course of an enforcement effort, sufficient in-
formation may become available to have reason to believe that a
particular person has violated a Federal law. A likely pos-
sibility is the determination that a particular person may have
knowingly made a "false statement in [a] .. .document filed or
required to be maintained under [the Clean Water Act]", such as
in a logbook or report to the permitting agency; or has falsified,
tampered with, or rendered inaccurate a monitoring device or
method required to be maintained under the Act, such as a
biomonitor ing device or an ancillary measuring device. Such
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declarations or actions constitute Federal crimes under section
309(c) of the Act. Persons (but not a corporation) who have
violated section 309(c) may not be required to testify against
themselves. In addition, any enforcement leads obtained from
information developed from statements made by suspects who have
not given notice of and voluntarily given up their Constitutional
right to remain silent may likewise not be available as evidence
against these persons in a criminal trial.
Further, section 308 of the Act does not give inspectors au-
thority to do anything other than enter, inspect and copy records,
inspect monitoring equipment and sample effluents. The inspectors
must limit their discussions with permittees' personnel to request-
ing basic information required to enable the inspectors to perform
their duties. This admonition applies to State inspectors, if State
law includes State offenses equivalent to those in section 309 of
the Act.
C. Expert and other Testimony
Facts and opinions derived from inspections often constitute
testimony before administrative bodies and courts. Not everything
that is seen or heard, however, may be admitted. The two excep-
tions which may lead to the exclusion of testimony of inspectors
are relevance and hearsay. Any information which will not aid the
finder of fact to determine an issue in a case is not relevant,
and is therefore inadmissible. Thus, the results obtained from
the bioassay of samples taken at a point other than the specified
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sampling point referred in an NPDES permit would probably be held
by a judge to be insufficiently related to a determination of tox-
icity at the designated point, and would therefore not be allowed
in evidence. Similarly, with few exceptions, direct testimony
may only be given by a witness on facts based on personal
knowledge. Thus, in most situations, a statement like "I know
it's true because Henry told me so" constitutes hearsay and will
not be admissible to prove the truth of whatever Henry said.
In testifying, inspectors who may be acting as ordinary
witnesses, are authorized not only to state facts known to them,
but may also testify as to their opinions in their area of
expertise. Technical matters, such as the propriety of sampling
or an opinion as to the results obtained from a particular biomon-
itoring experiment are examples of expert opinions which are
admissible evidence. In order to qualify as experts, persons must
show that their opinions on scientific matters will assist the
trier of fact, and that they are qualified as experts in that
field by virtue of their knowledge, skill, experience, training or
education.
D- Chain of Custody and Preservation of Documents
Results obtained by persons having expertise in conducting
sampling and biomonitoring (see Subsection 3, above) are, in fact,
valid. However, an additional effort in custodial care is abso-
lutely required to assure the admission of that information in
enforcement proceedings. A recent paper authored by expert EPA
biologists described this issue as follows:
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The only difference between normal scientific
prudence and legalistic practice is an added stress
upon written accountability.*
This "written accountability" requires the maintenance of
chain of custody records, detailed recording of all aspects of
sampling and biomonitoring, and the preservation of that
documentation.
In the context of biomonitoring done on-site, chain of
custody records are those which show the source of the materials
tested and which indicate that test results are not rendered
inaccurate by deliberate tampering or unintentional error.
In terms of biomonitoring done off-site, chain of custody
consist of records and/or labels, field data sheet and/or field
log book showing where, when and by whom a sample was taken and
the persons to whom custody was relinquished throughout the
sampling and testing process, together with appropriate notations
in the laboratory log book listing the names of sample custodians
as well as notations on the security measures taken to protect the
integrity of the sample during testing. Appendix E contains
samples of a chain of custody record and sample labels similar to
those used in one of EPA's laboratories but modified for specific
use with bioassay samples.
*Prager, Flemer and Browne. "Some U.S. Concerns in Obtaining
and Using Non-human Tissue Samples," 1979
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Both on-site and off-site testing require that appropriate
chain of custody be maintained. Off-site testing, of course,
will also include intermediate transportation information. Log
books must contain all pertinent information.
The "written accountability", as it relates to the actual
biomonitoring, consists of detailed notes of all the inspector's
actions and observations related to the biomonitoring testing.
Furthermore, all notes taken during the course of biomonitoring
tests must reflect total compliance with EPA official protocols
and procedures, which include acclimation of organisms, quality
assurance and chain of custody. Notes must also show unusual
occurrences which may bear on the propriety or impropriety of the
results. All notes must be made as close in time as possible to
the event that they describe. Notes must be complete and coherent
if they are to show that the inspector's opinions are worthy of
consideration. Materials and observations which in fact are
considered relevant, even if they do not fit into a "normal"
pattern, must not be skipped or deleted. Only if this is done can
there be documented assurance of the inspector's objectivity.
All chain of custody documentation, including log books, etc.
should be retained for no less than three years after testing has
been completed. Any unused portions of samples should also be
retained until the enforcement office authorizes disposal. The
transfer of test data and/or results to permanent non-bulky files
such as microfiche could be of great use in the study of environ-
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mental quality trends and in testing protocol evolution and
improvement. When results and samples are needed, as in cases of
active litigation, they shall be retained until case resolution.
Although documentation is performed by the analyst, the documents
themselves are the property of the employee's agency. The inspector
should turn over all such documentation, in accordance with agency
procedures, to the person or persons who are the permanent
custodians of such documents. When these documents are held in
custody in this manner, they are retrievable when needed. As an
example, Appendix G contains the document handling procedures
utilized by EPA's National Enforcement Investigations Center
(NEIC) .*
E. Relations with the Public
1. Financial Activities and Conflicts of Interest
Inspectors must conduct their business affairs in a way that
there can be no suspicion that their activities are motivated by
expectations of personal gain. They generally should not make
investments in or accept outside employment or gratuities from
persons whose activities they regulate. Aside from exercising
personal discretion in these matters, inspectors routinely should
review their agency's related instructions. EPA's instructions are
found in Title 40, Chapter 3 of the Code of Federal Regulations,
and published as "Responsibilities and Conduct of EPA Employees".
*NEIC Policies and Procedure Manual, 1978. National Enforcement
Investigations Center, Denver, Colorado.
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2. Appearance and Dress
Inspectors recognize that to the public they are the embodi-
ment of their agency and that they must represent it in the best
possible light. Inspectors must therefore appear neat and clean
while performing their duties. This does not mean, of course,that
inspectors should not dress in a manner appropriate to protect
themselves from climatic conditions or from job-related hazards
(see Appendix F).
3. Communications with Permittees and other Members of the
Public
It is important that cooperation be obtained and good re-
lations established when working with the permittees and the
public. This can best be accomplished by politeness, diplomacy
and tact. Everyone, even hostile persons, should be treated with
courtesy and respect.
During conversations with permittees and/or their repre-
sentatives, the inspectors should refrain from speculating over
inspection findings or asking any questions beyond those actually
needed for inspection purposes. Inspector behavior and conver-
sation should reveal know-how and objectivity.
4. Disclosure of Official Information
Inspection personnel must always refrain from answering any
questions or making any statements which might endanger any of the
permittee's proprietary materials, right of privacy, or which may
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in an/ way interfere with a monitoring or enforcement activity.
Thus, an inspector should not discuss any matters related to
a permittee whose effluent is being monitored without having first
obtained approval from the appropriate EPA or State enforcement
official.
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SECTION III
PLANNING BIOMONITORING INSPECTIONS
A. Pre-inspection Planning Activities
Pre-inspection planning will insure the best and most
efficient use of resources. Pre-inspection planning activities
can be divided into the following categories:
1. Selection Coordination
Coordination in the selection of candidates for inspections
should involve the exchange of information with other related
programs. Offices with enforcement authority, however, will make
the final selection of candidates for inspection. Factors useful
in selecting candidates for inspections are contained in Appendix
I. These can be used by inspectors and other personnel involved
in NPDES inspections to assist them in their advisory role to
enforcement efforts. Written notification to the permittee is
usually conveyed by means of a 308 letter. Letters of the 308
type perform three basic functions: 1) they inform the permittee
of the upcoming inspection usually without specifying a date, 2)
they detail statutory authority to inspect, and 3) they address
safety and information matters associated with actual inspection.
Each EPA Regional office can continue to use its present 308
letter or other method of notification or it may want to introduce
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alterations that would specifically accommodate biomonitoring
inspections.
2. Selecting the Type of Biomonitoring Inspection to be
Conducted
If not specified by the enforcement program, the inspection
team may also have to decide on the type of biomonitoring inspection
it will conduct on a particular permittee (see flowchart on page
1-6). Flow-through bioassay inspections are very resource intensive
and their use as a routine inspection tool is not advisable. The
office with enforcement authority may find it advantageous to
develop and use specific guidelines for determining the type of
biomonitoring inspection to be employed. Various types of biomoni-
toring inspections are described later in this section.
3. Some Basic Information, Personnel and Material Needs
All biomonitoring teams must include, as one of its members,
at least one inspector experienced in biomonitoring techniques. The
only exception to this rule is the collection of a composite or grab
sample for off-site static bioassay testing. In this case, any
member of the inspecting team may be supplied with sample tags,
chain of custody records, adequate sample containers and careful
instructions on sampling procedures. These instructions may be
found in the NPDES Compliance Sampling Inspection Manual. The type
of sample to be collected (grab, composite) will be specified by
either the permit conditions, if appropriate, or an experienced
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biologist. The experienced biologist will also determine the volume
and preservation techniques to be employed.
Minor equipment items and supplies should be duplicated to
avoid unnecessary delays in case of failure or breakage. The EPA
bioassay technical manual (see Appendix A) is the source from which
to prepare a materials and supplies checklist. The field personnel
should make it a habit to go over a checklist of supplies and
equipment prior to each departure. The checklist should include,
among other items, the following:
a. A copy of the latest NPDES and applicable State
permits.
b. A sketch or copy of a U.S. Geological Survey map
showing the facility location and/or its effluent
discharge points (Overflow and bypass discharge points
should be identified).
c. A summary of names, titles, locations and phone
numbers of the responsible persons (operators, municipal
or industrial officials) involved with the permittee's
waste water control program.
d. A flow chart or summary of the present and/or
planned treatment facilities. (If appropriate, include
industrial production processes.)
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e. Information from or copies of previous inspections
and self-biomonitoring reports (including quality assur-
ance data).
f. Information from the permittee's most recent
Compliance Schedule Report and/or Discharge Monitoring
Report (DMR).
g. Letter of notification of inspection to the
permittee and the response (if applicable).
h. Information from any other recent correspondence
and/or regulatory action, noting the status of requested
actions and/or compliance with enforcement actions.
i. Previous EPA biomonitoring studies, consultant's
reports and laboratory test procedures.
j. Evaluation form for rating self-monitoring practices
associated with biomonitoring.
k. The NPDES Biomonitoring Inspection Manual.
Reports and data from the above checklist are used in eval-
uating the permittee's compliance record with NPDES permit require-
ments.
Knowledge of the distance and location of the sampling point,
electrical power supply and dilution water sources should be used
when positioning the mobile laboratory.
If the inspector foresees the need to transport or ship sam-
ples of any sort, he should obtain in advance the necessary
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containers and agency labels. Proper chain of custody procedures
should be followed (see this manual, page II-5). For the proper
transportation procedures, see the U.S. Department of Transportation
(DOT) regulations and EPA/DOT agreements.
An important part of the pre-inspection activities includes
obtaining information on plant regulations such as gatehouse
check-in and security procedures, in-plant travel restrictions and
safety regulations. Good planning includes providing the inspection
team with proper equipment and attire to meet all safety regulations.
B. Coordination of Inspection Activities
1. Coordination with the Permittee
Prior to contacting the selected permittees, members of the
the inspection team have already conducted a comprehensive review of
the permittee's compliance record.
Once the nature of the permittee's effluent has been studied,
and their compliance history and present compliance status deter-
mined, the inspection team leader may want to establish direct
contact with the permittees to give them an approximate inspection
date and check on the availability of special equipment such as
necessary power supply. If a 308 letter has been sent and the
permittee has responded, this call may not be necessary. If there
is direct verbal communication with permittees, such details as
plant operations, production levels, and related information can be
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obtained. Further, direct exchange of information between the
permittee's representatives and the inspecting team may facilitate
the actual inspection process and prevent misunderstandings from
occurring. The following are some important items of information
that can be obtained from direct verbal contact with permittees:
a. Name, telephone number, mailing address and work
schedule (for immediate period when inspection might
occur) of the person that the permittee has designated
as the contact for inspection purposes.
b. Plant schedule and operational levels.
c. Permittee's in-plant safety requirements and
availability of necessary safety equipment.
d. Accessibility to adequate source of dilution
water.
e. Location and ease of acccess to sampling points.
f. Availability of electrical power supply.
The inspection team leader may want to inform the permittee
of some or all of the following information:
a. Inspection Time Frame - (Approximate Inspection
date). For example, inspection will take place with-
in the next sixty days.
b. Purpose of Inspection - Is the purpose to check
NPDES permit conditions to assess quality assurance of
self-monitoring data, or to establish effluent limits?
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c. Nature of the Inspection - What kind of biomoni-
toring inspection will be conducted? Will there be any
other type of analyses associated with the survey? (i.e.
effluent chemical samples, check samples, laboratory
evaluation).
d. Scope of the Inspection - Number and location of sites
to be sampled, amount of samples to be taken, etc.
e. Who is Going to Participate? - How many people are
going to be involved in the survey and, if possible, who
are these persons? Is any other agency going to be
involved? What part, if any, will the permittee be
expected to carry out? Will the company want to duplicate
any portion of the survey or receive split samples for
chemical analyses?
2. Coordination with Other EPA Programs
There are cases in which the biomonitoring inspection of a
permittee will be conducted simultaneously with a normal Compliance
Sampling Inspection (CSI). This type of combined inspection must be
carefully coordinated to maximize utilization of traveling and man-
power resources. In this case the biomonitoring inspection team
should collect and prepare samples for chemical analysis of the
effluent on which the bioassay is being performed. In this case
advance scheduling of analytical services should be coordinated with
the receiving laboratory.
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3. Coordination with State and Other Agencies
Valuable information may be obtained from State and other
government agencies. This coordination should take place as far
in advance as possible. Information obtained from these agencies
should corroborate information obtained from the permittee.
The following is some of the information that may be ob-
tained from other government agencies.
a. Copy of the latest NPDES permit.
b. Permittee's self-monitoring data.
c. Ambient stream data upstream and downstream
from facility.
d. Results and reports of recent compliance inspections.
e. Reports of fish kills or spills in the stream in
question attributed to the permittee.
f. Any knowledge of operational problems or
changes in plant processes.
g. Recent correspondence and telephone communications
with the permittee.
h. Is the State or other agencies interested in parti-
cipating in the survey?
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SECTION IV
INSPECTION TYPES
A. Announced and Unannounced Inspections
Both announced and unannounced inspections require
essentially the same planning and preparation except for the
pre-inspection facility reconnaissance. Unannounced inspections
will probably require schedule adjustments for this purpose.
For example, delays may occur at the start of unannounced
inspections as permittee personnel are diverted from their normal
duties to accommodate the unexpected inspection activities.
However, if a 308 letter has been sent previously, this delay should
be minimized. The same information that is supplied to permittees
in advance for announced inspections will have to be supplied
quickly and concisely at the beginning of unannounced inspections.
Although it is reasonable for the permittee to cause delays
during unannounced inspections, unreasonably lengthy ones should
not be permitted without adequate justification.
B. Sampling Type Inspections
A biomonitoring sampling inspection is an evaluation of an
effluent based on actual sampling and testing. This is referred to
as a sampling-type inspection so as not to be confused with a
Compliance Sampling Inspection (CSI ) which involves the taking of
samples for subsequent chemical analysis. The inspection consists
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of collecting an effluent sample and determining its biological
impact by exposing test organisms to the sample. Biomonitoring
tests are performed according to specifications in the NPDES permit
and the published EPA test protocol (see Appendix A).
1. On-site Biomonitoring
On-site biomonitoring inspections of a permittee's effluents
includes a combination of the following depending on the specific
needs of the inspection:
- An 8 to 24 hour range finding static bioassay (usually
allowed to run for 24 hours).
- A 24 hour static bioassay.
- A 96 hour flow-through bioassay.
- A 24 hour QA bioassay with reconstituted water and a
standard toxicant.
- Careful examination of the permittee's records and laboratory
facilities (see flowchart page 1-6). If a permittee has a
biomonitoring requirement, the type of biomonitoring test performed
during the inspection should be the same as the test required by the
permittee's NPDES permit.
a) 96-Hour Flow-Through Bioassay
This is one of the most complete of all on-site sampling type
biomonitoring inspections. The methods and materials to be utilized
in conducting this type of biomonitoring inspection are fully
described in Appendix A and Section V (Quality Assurance) of this
manual.
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b) 24-Hour Static Bioassay
The 24-hour static bioassay testing of an effluent grab sample
may be enough in those cases where permittees can prove that proper
mixing and/or long retention time produces a homogenous effluent.
In other cases, a static bioassay using a 24-hour composite sample
or a 96-hour flow-through bioassay must be conducted. For further
details on sampling and holding time see pages 17 and 18 of
Appendix A,
2. Off-site Testing
This type of sampling inspection consists of collecting a
sample of the permittee's effluent and transporting it to EPA or
State laboratories to be tested.. The sample may be a grab sample
or a 24-hour composite sample.
A 24-hour static bioassay would usually be the preferred
off-site test.
C. Evaluation Inspections
1. The Compliance Evaluation Inspection (CEI)
This is the simplest and less resource intensive type of the
evaluation inspections. It consists of an examination of the
permittee's records, laboratory and production facilities. As with
all other types of inspections, the inspector, prior to the visit,
must become acquainted with the permittee's compliance track record
and all relevant aspects of the production and waste treatment
operations.
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No inspection team is necessary for this type of inspection.
An experienced inspector with knowledge in biomonitoring suffices.
Because of its simplicity and few pre-inspection needs, this is
the type of evaluation inspection most likely to be conducted
effectively on an unannounced basis. Refer to the NPDES Compliance
Evaluation Inspection (CEI ) Manual (EPA, July 1976) and to Appendix
C-l of this document for additional guidance.
For purposes of satisfying EPA inspection program resource
commitments, a CEI shall have the same classification or resource
accounting code irrespective of whether it relates to biological,
chemical or combined evaluation of permittee records and facilities.
2. Performance Audit Inspections (PAI)
A biomonitoring performance audit inspection is an announced,
non-sampling audit of the permittee's records, monitoring and
analysis procedures, which assesses the accuracy and completeness of
all aspects of biological monitoring and reporting requirements of
NPDES permits. The performance audit inspection (hereafter referred
to as an audit) serves as a compromise between a simple compliance
evaluation inspection and a comprehensive sampling inspection. It
eliminates the short-comings of a CEI which does not include actual
observation of the permittee's self-biomonitoring procedures.
Furthermore, audits are not as resource intensive as sampling
inspections which can continue for several days or weeks with
round-the-clock sampling of numerous wastewater outfalls.
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Because of its manpower efficiency, the audit is a very useful
tool in any compliance monitoring program. Not only does it require
a minimum number of personnel, but it eliminates extensive on-site
sampling and analyses while permitting in-depth observation of the
permittee's biomonitoring procedures. It functions as an on-site
overall quality control examination. An audit is a highly efficient
mechanism for detecting self-monitoring or permit limitation
deficiencies. Therefore, results of an audit can be the basis for
planning corrective measures and/or initiating comprehensive sampling
inspections by specialists to obtain details of identified problems.
For purposes of satisfying EPA inspection program resource
commitments, an audit (PAI) shall have the same classification or
resource accounting code irrespective of whether it relates to a
biological, chemical or combined audit of the permittee's self-
monitoring program. The following are some of the most important
components of an audit type inspection:
a. Direct observation and audit of the permittee's
bioassay procedures during the course of a complete
self-biomonitoring test.
b. Examination and audit of the permittees:
1) Records (includes DMRs and self-monitoring)
2) Chain of custody procedures
3) Data collection and evaluation methodology
4) All laboratory facilities used in performing
biomonitoring tests.
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SECTION V
QUALITY ASSURANCE
Quality Assurance (QA) practices for effluent bioassays include
all areas that affect the accuracy and precision of the data, such
as: effluent sampling and handling; the test organisms; equipment;
test conditions; instrument calibration; replication; reference
toxicants; record keeping; data evaluation; and chain of custody.
Some of these areas are addressed in the following discussion.
A. Effluent Sampling and Handling
Continuous effluent sampling and flow-through bioassays are
the most sensitive and reliable testing methods. If continuous
effluent sampling is not possible, flow-proportional composite
samples, composite samples or grab samples may be taken. If samples
are collected for offsite testing, the sample preservation proce-
dures given in Appendix A, page 18 should be observed. Any samples
that are to be tested two or more hours after collection should be
kept chilled at 4°C (use only frozen water ice). No air space
should be left between the sample's surface and the container's cap.
Even when sample preservation procedures are properly observed,
testing should always begin within 24 hours after collection.
Toxicity data from effluent samples that have been held more than 24
hours prior to testing may not be used unless it can be proved that
the toxicity of the sample has not changed because of excessive
holding time.
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In running a static test, the volume of sample needed will depend on
the number of dilutions to be tested, test-temperature and the test
organisms loading factor. A five (5) gallon sample may be enough
for Daphnia or other test organism of similar size. For larger test
organisms such as the fathead minnow, volumes up to 15 gallons may
be needed.
B. Test Organisms
For the 24-hour bioassay screening test, with receiving waters
having a salinity less than five 0/00 (parts per thousand), it is
recommended that the fathead minnow (Pimepnales promelas) be used.
Test results from 24-hour static bioassays using juvenile instead of
adult fathead minnows have been found to correlate more closely to
test results from 96-hour flow-through bioassays. For this reason
it is recommended that the fathead minnows used should not be more
than 3 months old nor less than 2 weeks.
With receiving waters having a salinity of greater than 5 0/00,
the mysid shrimp (Mysidopsis bania) is recommended.
Permittees or NPDES inspectors may elect to run additional
tests with other species at any time. These may include species
indigenous to the particular receiving waters.
The fathead minnow and mysid shrimp are recommended rather than
a sensitive indigenous species for the following reasons:
V-2
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1. This gives a simplified national approach where toxicity
data from any NPDES permittee can readily be correlated with all
others.
2. Data does not always exist as to which species is the most
sensitive indigenous species.
3. Many times, because of an effluent's impact on a receiving
water, all sensitive resident species have previously been elimin-
ated and only extremely pollution tolerant species remain. Using
these would give an unrealistic estimate of the effluent's effect on
the receiving waters.
4. Often, the sensitive indigenous species are not commer-
cially available in all areas and would represent significant
costs to the permittee.
5. Both the fathead minnow and mysid shrimp are easily raised
under laboratory conditions and permittees and commercial labs
would be able to maintain their own stocks.
Test organisms should not have been exposed to pollutants or
other stress prior to biomonitoring, and should be free of disease.
Holding conditions should conform to Agency recommended procedures
(see Appendix A).
C. Facilities and Equipment
Laboratory and bioassay temperature control equipment must be
adequate to maintain recommended test water temperatures. To
ensure electrical power, an independent, portable generating unit
is recommended. Thermometers, pH meters, dilutors, and other
measuring devices must be calibrated by the manufacturer's
V-3
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recommended methods before and at appropriate intervals during use.
Glass, stainless steel and perfluorocarbons are to be used for test
chambers, tubing, etc. (see Appendix A). All equipment must be
carefully cleaned.
D. Dilution Water
Receiving water should be used as dilution water wherever
possible. However, if the receiving water has been influenced by
other point-source discharges, contains toxic substances or is
otherwise unsuitable as dilution water, a chemically equivalent or
"reconstituted" water may be employed, (see Appendix A, p. 14.)
This water must have a total hardness, total alkilinity and specific
conductance within 25 percent, and pH within 0.2 units of the
receiving water at the time of testing.
E. Test Conditions
Water temperature ranges for the test organisms must be
maintained within the specified limits. Dissolved oxygen concen-
trations should not fall below 60 percent saturation for cold-water
species and 40 percent saturation for warm-water species. Loading
limits of organisms in test chambers must not be exceeded.
Duplicate test chambers should be provided at each concentration of
effluent (for further details see Appendix A).
F. Reference Toxicants
Reference toxicants are used to establish the validity of
effluent toxicity data generated by bioassay laboratories. Factors
affecting the accuracy of the data include test organism condition
V-4
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and sensitivity, water temperature, and dilutor calibration. The
condition and sensitivity of each batch of test organisms to the
reference toxicant should be determined at least once each month.
No batch of test organisms should be used in an effluent bioassay
unless its condition has been checked against the reference toxicant
within the preceding 7 day period. If preferred, this sensitivity
test may be run on-site in the mobile laboratory concurrently with
the effluent bioassay.
In programs conducting more than one sampling inspection per
month, it may be convenient to run all organism sensitivity tests
on-site, concurrently with the effluent bioassays. The reference
toxicant, sodium dodecyl sulfate (SDS), should be used in a 24-hour
static bioassay, with moderately-hard (Hardness = 160-180 mg/1)
reconstituted water. A minimum of three reference toxicant
concentrations should be employed: one above, one equal to, and one
below the LC50.
The 24-hr LC50 of SDS for fathead minnows (Pimephales prpmelas)
of 2 weeks to 3 months of age should fall in the range of 2-7 ppm.
The 24-hr LC50 of SDS for Mysidopsis bahia should fall in the range
of 2-8 ppm.
If the LC50 of SDS does not fall in the recommended range for
the test organisms, the sensitivity of the organisms and/or the
overall credibility of the test system are suspect. In this case
V-5
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the test system should be examined for defects. A different batch
of test organisms should be employed in the effluent toxicity test.
G. Record Keeping
Proper record keeping is very important. Bound notebooks
should be used to maintain detailed records of test organisms, and
equipment such as: supply source, date of receipt, culture main-
tenance, disease treatment, calibration of the dilutor and other
equipment, and test conditions.
Annotations should be made as close to real-time as possible to
prevent the loss of information.
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SECTION VI
HEALTH & SAFETY
A. General
The same safety rules that are in effect in a fixed laboratory
will apply to all mobile laboratory operations. Evaluation and
sampling inspections at wastewater treatment facilities may involve
significant risks to personal safety and health. Personnel should
protect themselves from injury which may result from unsafe
conditions or practices at the inspection site by taking all safety
precautions necessary for the:
1. Prevention of bodily injury, and contact with corrosive
and/or toxic substances.
2. Prevention of infections.
3. Prevention of asphyxiation due to lack of oxygen or
presence of noxious gases.
Prior to site visit, inspection personnel should make sure
that all necessary safety equipment and materials have been ob-
tained and are in good condition.
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B. Personal Conduct
Every work assignment must include review and evaluation of all
potential hazards involved. No job shall be started until the
worker is convinced that all safety factors have been considered.
Care should be taken not to impede or disrupt normal performance of
industrial employees at work site.
C. Safety Equipment
1. Personal Safety Gear
While on duty, inspection personnel should use the safety
equipment as required:
2. Laboratory Safety Equipment
Each lab (including mobile labs) should have safety equipment
such as first aid kit, fire extinguisher, fire blanket, etc.
Inspectors should contact the permittee and inquire as to any
special safety measures and equipment requirements that must be
observed at the permittee's installation. This precaution may save
the inspectors from unnecessary problems and delays and provide
protection from injury.
D< General Laboratory Operation
1. Work with hazardous materials must be performed in
compliance with special rules pertaining to such materials.
Hazardous work will not be performed alone in a mobile laboratory or
anywhere else.
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2. All containers will be adequately labeled to indicate
their contents.
3. Good housekeeping contributes to safety and credibility.
4. The use of electrical equipment or cords not having
Underwriters Laboratories approval is forbidden. Ground-fault
interrupters are required for "wet" lab operators. Mobile
laboratories must be properly grounded to protect against electrical
shock.
E. Transportation
The operation of vehicles and boats shall be performed in
accordance with established rules and regulations in the employees
Field Safety Manual.
F. Emergency Health and F ire Protection
Upon arriving at the plant, inspectors should inquire about the
availability of emergency ambulance service in case of personal
injury or illness and fire protection for mobile labs and vehicles.
G. Accident Reports
EPA personnel shall report all job-connected accidents in
accordance with procedures outlined in the EPA Occupational Safety
and Health Manual.* Employees must furnish complete accident
information to assure accurate reporting.
*USEPA. 1977. Occupational Safety and Health Manual. Office of
Planning and Management, U.S. Environmental Protection Agency,
Washington, D. C.
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Supervisors shall investigate and report within two working
days all job-related accidents on EPA Form 144-9, Supervisor's
Report of accident.
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SECTION VII
CONDUCTING BIOMONITORING INSPECTIONS
A. Facilities Access
In obtaining consent to enter into a particular private
facility for inspection and sampling purposes, the principles or
rules an inspector should follow are:
1. Enter the permittee's premises through the main gate or
through the entrance designated by the permittee in response to the
inspection notification letter.
2. Advise the party at the gate or entrance that you wish
to speak with the owner, lessee, person in charge or other person
you have been instructed to contact.
3. Introduce yourself as an inspector to the owner,
operator or agent in charge and advise that person that you wish
to enter and perform the inspection and/or sampling you have been
instructed to perform.
4. Present your official EPA or State credentials whether or
not identification is requested.
5. Do not sign any "waivers" or "visitor releases" (U.S.
EPA inspectors only) that purport to absolve the permittee of
responsibilty for injury while on the premises.
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6. Limit your conversations with the permittee and their
employees or agents to obtaining directions to reaching sampling
and inspection points and to making arrangements to split samples
or making utility connections to the mobile lab.
7. If you are refused entrance, or are not allowed to
complete your inspection or sampling tasks, immediately notify the
person you have been instructed to notify in such circumstances.
Follow instructions, such as to meet with the U.S. Attorney in the
event that a decision is made to seek a warrant (see Appendix J,
pag e 6).
B. Conducting Sampling-Type Inspections
1. On-site Testing
On-site testing may be performed using either a static or
flow-through bioassay depending on the specific needs of the
inspection. Appendix A of this manual, entitled "Methods for
Measuring the Acute Toxicity of Effluents to Aquatic Organisms"
(EPA-600/14-78-012: Revised July 1978), contains methods and
materials for bioassay testing which are referenced by EPA for
compliance inspection purposes. Appendix A will be used to
fulfill regulatory needs under section 304(h) of the Clean Water
Act. Personnel performing compliance biomonitoring sampling
inspections are referred to Appendix A for step-by-step
procedures. An example of daily routine operations of an on-site
biomonitoring sampling inspection is presented in Appendix D.
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The inspector should avoid any alterations of EPA-approved
bioassay methods and materials. Any such alteration could
invalidate the results obtained from a bioassay. However, should
such alteration be unavoidable, a corresponding and properly
signed and dated logbook entry should be made at the time by the
person{s) who introduced such alteration. Further, this logbook
entry should also include detailed notes of the reasons for having
had to introduce such alteration of methods and/or materials.
The NPDES Compliance Inspection Report (EPA Form 3560-3)
should be used for all on-site inspections.
2. Off-site Testing
Off-site testing is most often performed using a 24-hour
static bioassay. Any experienced member of an EPA inspection team
can collect the effluent sample needed to conduct an off-site 24-
hour static bioassay. Usually, the type of sample collected for
this purpose is a grab sample, but occasionally a 24-hour
composite sample may be collected depending on the nature of the
effluent and the needs of the inspection.
The person collecting the sample should be supplied with the
necessary tags and chain of custody forms. Appendix F supplies
some examples of these types of tags and forms.
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3. Sample Collection
Methods for collecting representative samples are covered in
the NPDES Compliance Sampling Inspection Manual. Some important
considerations for obtaining a representative sample are as
follows:
(a) The sample should be collected where the wastewater is
well mixed. The sample should be collected near the
center of the flow channel, at 40-60% depth, where the
turbulence is at a maximum and the possibility of solids
settling is minimized. Skimming of the water surface or
dragging the channel bottom should be avoided.
(b) in sampling from wide conduits, cross sectional sampling
should be considered. Dye may be used as an aid in
determining the most representative sampling point(s).
(c) The sampling of wastewater which contains immiscible
liquids, such as oil and grease, requires special
attention. Oil and grease may be present in wastewater
as a surface film, an emulsion, or as a combination of
these forms. As it is very difficult to collect a
representative oil and grease sample, the inspector must
carefully evaluate the location of the sampling point.
The most desirable sampling location is the point where
greatest mixing is occurring. Quiescent areas should be
avoided, if possible. Because losses of oil and grease
will occur on sampling equipment, the collection of a
composite sample is impractical.
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(d) If manual compositing is employed, the individual sample
bottles must be thoroughly mixed before pouring the
individual aliquots into the composite container.
C. Conducting Evaluation Inspections
1. Performance Audit Inspection (PAI)
This type of inspection involves observing, interviewing and
recording pertinent information. An inspector with experience is
a major ingredient in conducting a high-quality audit. An equally
important ingredient is a properly completed NPDES Compliance
Inspection Report (EPA Form 3560-3) and all other supporting
inspection documentation.
a. On-site Inspection Responsibilities
Previously, in Section III, the inspector's responsi-
bilities for planning and preparing a biomonitoring audit
were discussed. The following information deals with on-site
responsibilities of the inspector while conducting a
biomonitoring audit.
It is the responsibility of the inspector to determine
the conformance of the permittee's biomonitoring program with
the corresponding permit and regulations. To accomplish
this, the inspector should become thoroughly familiar with
the permit's bioraonitoring requirements and with any
pertinent correspondence which may have modified permit
conditions or procedures. Using a Biomonitoring Audit Form
or a special notebook, the inspector should review all
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necessary documents and conduct a visual inspection of the
permittee's sampling and testing procedures, biomonitoring
equipment and facilities.
b. Effluent Sampling
The inspector is responsible for observing and auditing
sampling practices as performed by the discharger. The
following list contains important items which must be verified
during a biomonitoring audit.
The inspector shall verify:
1) That samples are taken at the locations
prescribed in the NPDES permit;
2) That the sampling location specified in the
permit is adequate to provide a well mixed and
representative sample;
3) That the frequency of sampling (grab samples
and sampling interval for composites) is done in
accordance with the NPDES permit;
4) That grab sample devices, if used, are clean
and properly operated;
5) That containers are clean and appropriate for
collecting samples;
6) That automatic sample collectors, if used,
operate properly;
7) That chemical preservatives are not used in
samples which are to be bioassayed;
8) That samples shipped to a contract laboratory
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are properly preserved (refrigerated) and shipped in
appropriate containers and under approved chain of
custody procedures.
9) That samples are received and bioassay testing
is initiated as soon as possible, but not later than 24
hours after sample collection.
10) That if there is more than one sampling point,
a determination is made so appropriate measures are taken
to prevent cross contamination between samples and that
sample containers are properly identified;
11) That all testing equipment is routinely
calibrated.
c. Laboratory Audit
On-site observation and audit of permittees' or their
contractor's laboratory procedures, equipment and facilities
should be performed by a well-trained inspector.
The following five major areas require detailed obser-
vations and evaluation. Under each listed item is a summary
of "conditions" recommended by the EPA (see Appendix A).
1) Facilities
Effluent toxicity may be performed in a stationary
or mobile laboratory. Depending upon the scope of the
bioassay program, facilities may include equipment for
rearing, holding and acclimating test organisms.
Temperature control is achieved using circulating water
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baths, or environmental chambers. Holding, acclimation,
and dilution water should be temperature-controlled and
aerated whenever possible. Air used for aeration must
be free of oil and fumes; filters to remove oil in water
are desirable. Test facilities must be well ventilated
and free of fumes. During holding, acclimating, and
bioassay, test organisms should be shielded from outside
disturbances.
Materials used °in the construction of the test
equipment which come in contact with the effluent should
be carefully chosen. This is a must if one wishes to
minimize leaching, dissolution, and adsorption (see
Appendix A, pages 4-6).
2) Test Organisms
Inspectors should determine the source of the test
organism. They must be familiar with the requirements of
test organisms and review the handling and holding
procedures used by the permittee or his agent.
3) Dilution Water
Dilution water may be ground water, surface water,
reconstituted water, or dechlorinated tap water.
Criteria for selecting and/or preparing dilution water
are given in section 4, pages 14 to 16 of Appendix A.
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The inspector should determine if the permittee's
dilution meets these criteria.
4) Test Procedure
Perhaps the most important aspect of the biomonitoring
audit consists of observing and carefully scrutinizing
toxicity testing and related laboratory analyses as
performed by the discharger or by a commercial
laboratory under contract to the permittee.
5) Test Results
The inspector should have an in-depth under-
standing of chemical, physical and biological data
required and the calculation methods used for inter-
preting biomonitoring test results. Data needs and
methods for calculating LC50 and EC50 are presented in
pages 24, 25 and 29 to 38 of Appendix A. Methods used
for chemical analysis must be those specified in the
final section 304(h) published as 40 CFR 136 regulations
and Wastes, USEPA, EPA 600/4-79-020 March 1979, unless
the specific NPDES permit has other requirements in
which case they must be followed.
6) NPDES Compliance Inspection Report
Biomonitoring is part of the NPDES program. As
such it shall use for reporting purposes the NPDES
VIl-9
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Compliance Inspection Report (EPA form 3560-3). Addi-
tional information may be submitted using an evaluation
form similar to that included in this manual as Appendix
C - Acute Toxicity Laboratory Evaluation Form.
2. Compliance Evaluation Inspection
Prior to inspection, the inspector has become familiar with
the permittee's operations, compliance track and self-biomoni-
toring activities.
In the compliance evaluation type of inspection, the inspec-
tor examines the production, waste treatment and laboratory
facilities of the permittee. Files and records of the permittee's
self-biomonitoring program are inspected. Additional guidance for
these inspections may be found in the NPDES Compliance Evaluation
Inspection Manual, July 1976.
3. Performance Audit Inspections
Depending on the needs of the enforcement agency, a perfor-
mance audit inspection (PAI) of the permittee's records and
procedures may be required. Guidance of performing PAIs is
currently in draft form, but will be available from EPA HQ Office
of Water Enforcement, Enforcement Division in the fall 1979.
VTI-10
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Section VIII
Post Inspection Activities
A. Data Evaluation
The primary purpose of a Compliance Biomonitoring Inspection
is to establish compliance status with bioassay requirements in an
NPDES permit and/or to evaluate the effluents potential for
toxicity to aquatic life in the receiving waters. This is
accomplished by a thorough evaluation of compliance biomonitoring
data, and is determined through a comparison of the following
parameters:
1. Toxicity of the waste (LC50 or EC50)
2. Instream waste concentration (IWC) of the effluent.
3. Potential for chronic and acute toxicity of waste in the
receiving water, including persistence, carcinogenicity,
mutagenicity, and teratogenicity.
4. Permit limits, if contained in the permit.
5. Chemical parameters of effluent measured in conjunction with
the bioassay such as D. O. , temperature, pH, conductivity,
metals, and organics.
The bioassay serves as a method for measuring the acute
toxicity of an effluent. Because death of the test organisms is
more easily observed and is less ambiguous than other physio-
logical or behavioral effects used in determining toxicity,
VIII-1
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lethality is usually the endpoint in acute toxicity tests.
Several procedures for evaluating lethality, expressed as LC50
(% effluent) are provided in the EPA manual on toxicity testing
(Appendix A, pages 22-38).
The toxic effects that an effluent will have on aquatic
organisms is greatly influenced by the degree of dilution that
occurs in the receiving water. A commonly used parameter to
describe this dilution is the instream waste concentration. The
IWC is expressed as the volume, percent of effluent in the combined
effluent and receiving water and is defined by:
IWC = / Qw
iQr + Qw/ x 100
Where: IWC = Instream waste concentration (%)
Qw = Effluent flow (MGD)
Qr = 7Q10 (7 day, 10 year low
flow of receiving waters
in MGD)
The IWC is a theoretical value which assumes rapid and
complete mixing of the effluent with the receiving water.
However, the validity of this assumption depends on the specific
hydrologic, geologic and morphologic aspects of the receiving
water and basin. For example, in a small, turbulent stream,
mixing may occur very rapidly, and the IWC could give a good
indication of the effluent dilution.
VIII-2
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However, in a larger stream or river, several distinct cur-
rent systems may exist. In this case, the waste discharge will
not mix evenly throughout the entire receiving stream, and will
result in a plume of poorly diluted effluent. Similarly, in lakes
and estuaries, mixing will occur fairly slowly, forming a mixing
zone that contains effluent at a higher concentration than pre-
dicted by the IWC. It has been suggested, however, that the IWC
can be applied to the boundary of the mixing zones in the lake.
Yet even here the applicability is influenced by lake currents,
the retention time of the lake waters, and the persistence of
toxic pollutants from the effluent.
Although the IWC will not, in many cases, describe the
dilution process precisely, it does provide a useful tool for
approximating the dilution that will occur. Consequently, the IWC
is frequently used in conjunction with LC50 or EC50 data, and an
appropriate application factor to predict if a waste discharge may
produce toxic effects on aquatic life in the receiving water. The
application factors create a safety margin for setting water
quality criteria to protect aquatic life and human health.
As supported by Water Quality Standards program guidance(l)
and the recommendations of the 1972 NAS Report(2), an application
1. Quality Criteria for Water, 1976. US EPA, Washington, D.C.
2. Water Quality Criteria, 1972. A report of the Committee on
Water Quality Criteria. National Academy of Sciences - National
Academy of Engineering, Washington, D.C.
VIII-3
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factor of 0.01 for effluents containing persisitent or extremely
toxic pollutants and an application factor of 0.5 for all other
pollutants is recommended. The permittee is required to provide
proof that his effluent does not contain known persistent, carcin-
ogenic, mutagenic, or teratogenic substances in order to use the
0.5 application factor.
If the LC50, multiplied by the appropriate application factor
is equal to or less than the calculated IWC, then the waste is
considered to have the potential for being toxic to aquatic life
in the receiving water. This comparison will serve as the basis
for determining compliance with permit requirements.
pass fail
persistent 0.01 x LC50MWC 0.01 x LC50
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bioassay data. From this information, clues as to the cause of
the toxicity can be obtained; and will aid in choosing corrective
action.
Information on changes in waste characteristics should also
be considered when evaluating toxicity problems.
B. Toxicity Laboratory Evaluation F orm
A suggested format for recording pertinent information is
presented in Appendix C. However, until such time as the current
NPDES inspection report form is amended, the toxicity evaluation
form should be included as an attachment to the regular inspection
report form.
C. Distribution of Inspection Report
The NPDES Compliance Inspection Report (EPA form 3560-3) and
any additional documentation obtained during inspection will be
sent to enforcement personnel who are involved in permit writing
and/or those who have compliance responsibilities. Further dis-
tribution of the report should be made by enforcement personnel
only. Data and/or LC50, EC50, should be made available to
personnel in charge of storing these data in the corresponding ADP
systems (PCS, STORET).
D. Follow-up Activities
It is advisable that informational feedback mechanisms
between enforcement personnel and compliance field inspection
VIII-5
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teams working on the same cases be instituted. This method of
operation will ensure adequate case follow-up and maximum
utilization of information and resources.
VIII-6
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APPENDIX A
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EPA-600/4-78-012
Revised July 1978
METHODS FOR MEASURING THE ACUTE TOXICITY OF EFFLUENTS
TO AQUATIC ORGANISMS
by
William Peltier, Chrm
Bioassay Subcommittee
EPA Biological Advisory Committee
Ecology Branch
Surveillance and Analysis Division
U.S. Environmental Protection Agency
Athens, Georgia 30605
Project Officer
Cornelius I. Weber, Ph.D.
Aquatic Biology Section
Environmental Monitoring & Support Laboratory
Cincinnati, Ohio 45268
ENVIRONMENTAL MONITORING & SUPPORT LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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DISCLAIMER
This report has been reviewed by the Environmental Monitoring and
Support Laboratory, U.S. Environmental Protection Agency, and approved
for publication. The mention of trade names or commercial products does
not constitute endorsement or recommendation for use.
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FOREWORD
Environmental measurements are required to determine the quality of
ambient water, the character of effluents, and the effects of pollutants
on aquatic life. The Environmental Monitoring and Support Laboratory-
Cincinnati conducts research to develop, evaluate, and promulgate methods
to:
Measure the presence and concentration of physical, chemical and
radiological pollutants in water, wastewater, bottom sediments,
and solid waste.
Concentrate, recover, and identify enteric viruses, bacteria, and
other microorganisms in water.
Measure the effects of pollution on freshwater, estuarine, and
marine organisms, including the phytoplankton, zooplankton, peri-
phyton, macrophyton, macroinvertebrates, and fish.
Automate the measurement of the physical, chemical, and biological
quality of water.
Conduct an Agency-wide quality assurance program to assure stand-
ardization and quality control of systems for monitoring water and
wastewater.
The Federal Water Pollution Control Act Amendments (Clean Water Act)
of 1977 (PL 95-217) explicitly state that it is the national policy that
the discharge of toxic substances in toxic amounts be prohibited. Deter-.
mination of the toxicity of effluents, therefore, has high priority in the
EPA water pollution control program. However, suitable, standardized
methodology for effluent bioassays has not been available to EPA regional
and state programs. This report fills an urgent current need for standard-
ized methods to measure the toxicity of effluents to aquatic life.
Dwight G. Ballinger
Director
Environmental Monitoring and
Support Laboratory - Cincinnati
iii
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PREFACE
The effluent toxicity tests described in this document were prepared
by the Bioassay Subcommittee of the EPA Biological Advisory Committee
to provide methods needed for effluent monitoring by EPA Regional and
State NPDES programs and for self-monitoring by NPDES permit holders.
Two types of toxicity tests are described:
1. A preliminary, short-term, static, range-finding (screening) test
for use in determining the concentrations of effluent to be used
in a "definitive" test.
2. A long-term (generally 96-hr), flow-through, definitive test for
use in determining the acute toxicity of the effluent, expressed
as a LC50 or EC50.
These methods will be included in the second edition of the EPA manual,
"Biological Field and Laboratory Methods for Measuring the Quality of
Surface Waters and Effluents," but were printed separately in limited
quantity to make them available prior to the publication of the manual.
Bioassay Subcommittee Members
William Peltier, Chairman, S&A Division, Region IV
John Arthur, Environmental Research Laboratory, Duluth
Bruce Binkley, National Enforcement Investigation Center, Denver
Stanley Hegre, Environmental Research Laboratory, Narragansett
William Horning, Newtown Fish Toxicology Laboratory
Philip Lewis, Environmental Monitoring & Support Lab, Cincinnati
Royal Nadeau, S&A Division, Region II
Ronald Preston, S&A Division, Region III
Steven Schimmel, Environmental Research Laboratory, Gulf Breeze
Milton Tunzi, S&A Division, Region IX
Contributors
Ronald Eisler/Environmental Research Lab, Narragansett
Charles Stephan, " ' " " , Duluth
Lee Tebo, S&A Division, Region IV
Cornelius I. Weber, Environmental Monitoring & Support Lab., Cincinnati
Cornelius I. Weber, Ph.D.
Chairman, Biological Advisory Committee
Chief, Aquatic Biology Section
Biological Methods Branch
Environmental Monitoring & Support Laboratory
iv
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ABSTRACT
This report describes methods for the measurement of the acute
toxicity of effluents to macroinvertebrates and fish. The methods include
a preliminary short-term (8-24 hr), range-finding (screening) test and a
long term (96 hr.)> flow-through (or alternate static) definitive test for
use in determining the LC50 or EC50 of the effluent. The report includes
guidelines on effluent sampling and holding, facilities and equipment,
dilution water, test species selection and handling, and data interpretation.
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CONTENTS
Foreword : ill
Preface iv
Abstract v
Figures viii
Tables ix
1. Introduction 1
2. Facilities and Equipment ..... 4
General Requirements ..... 4
Construction Materials 4
Effluent (Toxicant) Delivery System 5
Test Chambers . . . . . „ 6
Type 6
Cleaning 6
3. Test Organisms 7
Species 7
Source 7
Size 7
Holding and Handling Care 10
Disease Treatment . 10
Transportation and Acclimation 13
4. Dilution Water 14
5. Effluent Sampling and Holding 17
Sampling 17
Holding 18
6, Test Procedure 19
Range-Finding (Screening) Test 19
Definitive Test 19
Test Conditions 19
Number of Test Organisms 20
Loading of Test Organisms 20
Test temperature 20
Oxygen requirements and aeration 21
Beginning the test 21
Feeding 21
Duration 22
7. Test Results 22
Biological data 22
Chemical and physical data 22
Calculation of LC50 and EC50 24
Reports 24
References ', .' 26
Appendices
A. Litchfield and Wilcoxon abbreviated method 29
B. Log-concentration versus percent survival method .... 37
C. Dilutor systems, control panel and equipment lists ... 39
vii
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FIGURES
Number Page
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Data Sheet for Effluent Toxicity Test
Line fitted to data, and LC16, LC50 and LC84 as read
2
Nomograph for obtaining Chi from expected-percent-
affected and observed-percent-affected minus the
expected— percent— affected .....
Nomograph for raising base S to a fractional exponent .
Plotted data and fitted line for log-concentration
versus percent survival method .....
Photographs of dilutor systems. A. Solenoid valve
dilutor system. B. Vacuum siphon dilutor system ....
Solenoid valve dilutor system; general diagram ....
Solenoid valve dilutor system; detailed diagram ....
Vacuum siphon dilutor system; detailed diagram ....
Dilution water chambers
33
34
35
36
38
40
41
42
44
45
47
48
49
50
Vlll
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TABLES
Number Page
1 Recommended species and test temperatures 8
2 Recommended prophylatic and therapeutic treatments
for freshwater fish __.__. 11
3 Preparation of reconstituted fresh waters • 15
4 Preparation of reconstituted sea water 16
5 Percentage of un-ionized ammonia in distilled water
at various temperatures and pH's 23
6 Salinity correction, factor 23
7 Corrected values for 0% or 100% effect 32
8 Values of Chi2 -. 32
IX
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SECTION 1
INTRODUCTION
The Declaration of Goals and Policy of the Federal Water Pollution
Control Act Amendments (Clean Water Act) of 1977 (PL 95-217), Section
101(a)(3), states that "it is the national goal that the discharge of toxic
pollutants in toxic amounts be prohibited"; Current Agency programs for the
protection of aquatic life in receiving waters are based in part on effluent
limitations for individual chemicals. However, toxicity data are available
for only a limited number of compounds. Effluent limitations, therefore, may
not provide adequate protection where the toxicity of the components in the
effluent is not known, where there are synergistic effects between toxic
substances in complex effluents, and/or where a complete chemical charac-
terization of the effluent has not been carried out. Since it is not econom-
ically feasible to determine the toxicity of each of_the thousands of poten-
tially toxic substances in complex effluents or to conduct exhaustive chemical
analyses of effluents, the most direct and cost-effective approach to the
measurement of the toxicity of effluents is to conduct a bloassay with
aquatic organisms representative of indigenous organisms in receiving waters.
For this reason, the frequency of use of effluent bioassays to identify and
control toxic discharges is rapidly increasing within the Agency and state
NPDES programs, and by permittees as a self-monitoring requirement.
The lack of standardized bioassay methodology developed explicitly
for effluents has delayed the implementation of EPA regional and state
effluent toxicity testing programs and has resulted in a lack of uniformity
in test procedures. In response to this problem, a subcommittee was
organized within the EPA Aquatic Biology Methods Advisory Committee,
sponsored by the Environmental Monitoring and Support Laboratory, Cincinnati,
to prepare effluent bioassay methods for the NPDES program. To provide
valid methods, it was essential to include EPA Regional and Enforcement
programs personnel with extensive experience in conducting effluent toxi-
city tests. In completing their task, the subcommittee members drew from
their own experience and borrowed heavily from the report, "Methods for
Acute Toxicity Tests with Fish, Macroinvertebrates and Amphibians,"
(EPA 660/3-75-009), previously prepared primarily to standardize basic
laboratory methods for determining the toxicity of pure compounds.
The acute toxicity tests for effluents described in this report are
used to determine the effluent concentration, expressed as a percent volume,
that is lethal to, or has some other "adverse effect" on, 50 percent of the
organisms within 96 hrs or some other prescribed period of time. If
mortality (death) is the effect measured, the toxicity is expressed as the
median lethal concentration or'LCSO. Where death is not easily detected,
for example, with some invertebrates, other indicators such as immobilization
must be used as the adverse effect. Blood chemistry, biochemical measure-
ments or histological examinations can also be employed to determine the
toxicity of an effluent. The concentration of effluent, expressed as a
percent volume, that causes a defined adverse effect other than death in
50 percent of the test organisms within the prescribed exposure period is
termed the median effective concentration or EC50.
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A two-test sequence is generally required to estimate the acute
toxicity of an effluent: (1) a preliminary, short-term (8-24 hr),
range-finding test is conducted to define the range of effluent dilutions
to be used in the definitive test, and (2) a more rigorous long-term
definitive test is conducted (using the range of effluent dilutions
determined by the range-finding test) over a 96-hour time period to arrive
at .the acute toxicity of the effluent, which is expressed as a LC50 or EC50.
One of the following procedures shall be used for the range-finding
test:
A. Test organisms are placed in suitable containers and exposed
under static conditions to 3-5 widely-spaced dilutions' of the
effluent for a period of 8-24 hours.
B. If the effluent has a high dissolved oxygen demand, or the
organisms require flowing water, a flow-through test is used
to define the range of toxicity of the effluent.
One of the following procedures shall be used for the definitive test:
A. Preferred procedures (flow-through tests), in order of preference
1. Test organisms are exposed to effluent solutions flowing
into and out of test chambers on a once-through basis for
the duration of the test. The effluent is conveyed directly
and continuously from the source to the dilutor system.
2. Test organisms are exposed to effluent solutions flowing into
and out of test chambers on a once-through basis for the
duration of the test as in A.I above. However, the effluent
is supplied to the dilutor system from discrete effluent samples
collected periodically. The interval at which samples are
collected is based on the variability of the effluent charac-
teristics, production schedule, batch processes, retention
time, etc. (see p. 17 - Effluent Sampling and Holding).
The continuous'effluent sampling technique (A.I) is the best of two
methods described.
B. Alternative procedures (static tests), in order of preference
1. Test organisms are exposed to a fresh solution of the same
concentration of effluent every 24 hours, either by trans-
ferring the test organisms from one test chamber to another,
or by replacing the effluent solution in the test chambers.
2. Test organisms are exposed to the same effluent solution for
the duration of the test. However, the effluent solution in
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each test chamber may be filtered, aerated, or sterlized by
continuous circulation through an appropriate apparatus.
3. Test organisms are exposed to the original effluent solution
for the duration of the test without being continuously
circulated through an apparatus as in B.2.
The alternative procedures may be used in emergency situations or
where adequate facilities are not available to the investigator. However,
it must be understood that a test using the alternative procedures is not
valid unless it can be conclusively demonstrated that the chemical charac-
teristics and toxicity of the effluent do not change over time. Because of
toxicant adsorption on the test chambers, uptake by test organisms and the
effect of metabolites on toxicity, it is preferred that the effluent
solution be renewed at least once every 24 hours as described in B.I above.
The special environmental requirements of some organisms, such as
flowing water, fluctuating water levels, or substrate may preclude the use
of static testing.
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SECTION 2
FACILITIES AND EQUIPMENT
GENERAL REQUIREMENTS
Effluent toxicity tests may be carried out in a fixed or mobile lab.
Depending upon the scope of the bioassay program, facilities may include
equipment for rearing, holding and acclimating organisms. Temperature
control is achieved using circulating water baths, or environmental
chambers. Dilution water may be ground water, surface water, reconstituted
water, or dechlorinated tap water. Holding, acclimation, and dilution
water should be temperature-controlled and aerated whenever possible.
Air used for aeration must be free of oil and fumes; filters to remove
oil in water are desirable. Test facilities must be well ventilated and
free of fumes. During holding, acclimating, and testing, test organisms
should be shielded from disturbances.
Some organisms may have special environmental requirements such as
flowing water, fluctuating water levels, or substrate which must be
provided. During holding, acclimating, and testing, immature stream
insects should always be in flowing water; as described by Nebeker and
Lemke (1968); penaeid shrimp and bottom-dwelling fish should be provided
a silica sand substrate. Since cannibalism can occur among many species
of arthropods, they should be isolated by some means (e.g., with screened
compartments), or the claws of crabs and crayfish should be bound.
CONSTRUCTION MATERIALS
Materials used in the construction of the test equipment which
come in contact with the effluent should be carefully chosen. Glass,
No. 316 stainless steel, and perfluorocarbon plastics (TEF-LONR) should
be used whenever possible to minimize leaching, dissolution, and sorption.
Linear polyethylene may also be used with some types of industrial
and municipal effluents, but should be avoided with those containing
synthetic organic compounds or pesticides. Unplasticized plastics such as
polyethylene, polypropylene, TYGON^ and fiberglass can be used for holding,
acclimating, and dilution-water storage tanks, and in the water delivery
system. Copper, galvanized material, rubber, brass, and lead must riot come
in contact with holding, acclimation, or dilution water, or with effluent
samples and test solutions.
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EFFLUENT (TOXICANT) DELIVERY SYSTEM (Flow-through test only)
Although many types of toxicant delivery systems have been designed*,
the flow-through, proportional-dilutor delivery system has proven to be
the best and the preferred system for routine effluent toxicity tests
conducted in, both fixed and mobile laboratories.
The following factors should be considered in designing the system:
(1) whether the apparatus will be installed and used in a fixed or mobile
laboratory; (2) the existence of adequate space and/or structural require-
ments for the delivery system, test chambers, effluent and dilution-water
storage; (3) the applicability of the delivery system to specific effluent
characteristics (high suspended solids, volatiles, etc.); (4) the system's
dependability, durability, flexibility, and ease of maintenance and replace-
ment; (5) the ability to perform within the flow rate and concentration
accuracy limitations; and (6) the cost of the system.
Two types of proportional dilutors are described in the appendix - the
solenoid valve system and the vacuum siphon system. The solenoid valve
'system is preferred, but the vacuum siphon system is acceptable, if funds
are limited.
The flow rate through the proportional dilutor must provide for
at least five complete water volume changes in 24 hours in each test
chamber, plus sufficient flow to maintain an adequate concentration of
dissolved oxygen. It is often desirable to construct the dilutor with
an additional reserve flow capacity, depending on its application and/or
special effluent characteristics. The flow rates through the test
chambers should not vary by more than 10 percent among test chambers at
any time during any test. The dilutor should also be capable of maintain-
ing the test concentration in each test chamber within 5 percent of the
starting concentration for the duration of the test.
The calibration of the dilutor should be checked carefully before
and after each test. This check' should determine the volume of effluent
and dilution water used in each portion of the effluent delivery system
and the flow rate through each test chamber. The general operation of
the dilutor should be checked at least at the beginning and end of each day
during the test.
*(Lowe, 1964; Mount and Brungs, 1967; Sprague, 1969; Freeman, 1971; Cline
and Post, 1972; Granmo and Kollberg, 1972; Bengtsson, 1972; Lichatowich,
S^. JL-L- ' 1973; Schumway and Palensky, 1973; Abram, 1973; Schimmel, Hansen,
and Forester, 1974; DeFoe, 1975; Riley, 1975).
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TEST CHAMBERS
Type
Test chambers used in flow-through tests are constructed of 1/4 inch
plate glass held together with (GE) clear silicone adhesive. Silicone
adhesive absorbs some organochlorine and organophosphorus pesticides, which
are then difficult to remove. Therefore, as little of the adhesive as
possible should be in contact with water; extra beads of adhesive inside
the containers should be removed. Stainless steel (#316) can be used
in the construction of test chambers, but must be of welded, not soldered,
construction.
The size of the chambers may vary according to the size of the test
organism and/or the facilities, but the test solution should have a mini-
mum depth of 5 cm. All chambers should have either a glass or screen
cover to prevent organisms from jumping out.
The test chambers most'commonly used in static tests are wide-mouth,
3.9 liter (1-gallon) or 19.6 liter (5-gallon) soft-glass bottles. Con-
tainers such as 1-liter battery jars or 250-ml beakers may be more
suitable as.test chambers for fish eggs and/or larvae and small Crustacea.
Special glass or stainless steel test chambers can be constructed to
accommodate test organisms requiring special physical conditions.
Cleaning
All test chambers, whether new or used, must be washed in the follow-
ing manner to remove surface contaminants:
A. Soak and wash in suitable detergent in water, preferably heated to
a. temperature of 50° C or higher. The detergent (powder or liquid)
should be entirely synthetic (SPARKLEEN or ALCONOX ).
B. Rinse with water (preferably heated to 50°C or higher).
C. Rinse with a fresh, dilute (5 percent) hydrochloric acid for
removing metals and bases.
D. Rinse with water (preferably heated to 50°C or higher).
E. Rinse with acetone to remove organic compounds. When contaminated
with a pesticide, test chambers must be rinsed with acetone
before they are placed in the hot detergent soak (Item A. above).
F. Rinse twice with water.
When feasible, the outlined cleaning procedure must be used for other
equipment that comes in contact with the dilutor system, pumps, tanks, etc.
All test chambers and equipment must be thoroughly rinsed with the dilution
water prior to each test.
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SECTION 3
TEST ORGANISMS
SPECIES
Whenever possible, effluent toxicity-tests should be conducted with a
sensitive species that is indigenous to the receiving water, readily
available, and either commercially or recreationally important. Acceptable
species include those listed in Table 1, which are sensitive to most
toxicants. The test organisms must be identified to species.
SOURCE
Although effluent tests should be conducted with species that are
indigenous to, or stocked into, the receiving water, the test organisms
do not have to be taken from the receiving water. It is often difficult
to obtain fish of the desired size and condition from the receiving
water. Collection permits are often difficult to obtain, and the
organisms in the receiving water may have been previously exposed to the
effluent. Fish captured by electroshocking must not be used in testing.
The usual sources of freshwater fish used for toxicity tests are private,
state, and Federal hatcheries. If trout are used as test organisms,
they should be obtained from stock that has been certified as disease-
free.
Some fish, such as Fathead and Sheepshead minnows, are easily reared
under laboratory conditions (USEPA, 1972; Schimmel and Hansen, 1974).
However, it is usually more practical to collect marine species from
indigenous populations.
Some invertebrates such as daphnids, midges, and shrimp may be
reared in laboratory cultures. Care must be taken to insure that only
young age groups and early instars are used in testing. Daphnids from
cultures in which ephippia are being produced should not be used in the
tests. Invertebrates not amenable to laboratory rearing are usually
obtained directly from wild populations.
SIZE
Very immature fish (not actively feeding on exogenous food) , spawning
fish, or recently spent fish should not be used. Fish weighing between
0.5 and 5.0 grams each are preferred. In any single test, all fish
should be taken from the same year class, and the total length (tip of
snout to end of tail) of the longest fish should be no more than 1-1/2
times that of the shortest one. A.S stated above, immature invertebrates
should be used whenever possible.
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TABLE 1. RECOMMENDED SPECIES AND TEST TEMPERATURES
Species Test Temperature (°C)
Freshwater
Vertebrates
Coho salmon, Oncorhynchus kisutch 12
Rainbow-trout, Salmo gairdneri 12
Brook trout, Salvelinus fontinalis 12
Goldfish, Carassius auratus 22
5 Fathead minnow, Pimephales promelas 22
Channel catfish, Ictalurus punctatus 22
' Bluegill, Lepomis macrochirus 22
Invertebrates
, Daphnids, Daphnia magna or I), pulex 17
Amphipods, Gammarus lacustris, G_. fasciatus, or 17
G_. pseudolimnaeus 17
Crayfish, Orconectes sp., Cambarus sp., Procambarus 22
sp., or Pacifastacus leniusculus 22
Stoneflies, Pteronarcys sp. 12
Mayflies, Baetis sp. or Ephemerella sp. 17
Hexagenia limbata or E_. bilineata 22
Midges, Chironornus sp. -22
Marine and estuarine
Vertebrates
«Sheepshead minnow, Cyprinodon yariegatus 22
Mummichog, Fundulus heteroc1itus 22
Longnose killifish,. Fundulus similis 22
Silverside, Henidia sp. 22
Threespine stickleback, Casterosteus aculeatus 22
Pinfish, Lagodon rhomboides 22
Spot, Leiostomus xanthurus 22
Shiner perch, Cymatogaster a_ggregata 12
Pacific staghorn sculpin, Leptocottus armatus 12
Sanddab, Citharichthys stigmaeus_ 12
Flounder, Paralichthys dentatus, P.'lethostigma 22
English sole, Parophrys vetulus 12
-------
TABLE 1. (Cont'd)
Species Test Temperature (°C)a
Marine and estuarine
a
Invertebrates
Shrimp, Penaeus setiferus, P_. duorarum, or 22
P_. aztecus
Grass shrimp, Palaemonetes sp. 22
Shrimp, Crangon sp. 22
Oceanic shrimp, Pandalus jordani L2
Blue crab, Callinectes sapidus 22
Dungeness crab, Cancer magister 12
jMysid shrimp, Mysidopsis sp., Neomysis sp. 22
Atlantic oyster, Crassostrea virginica 22
Pacific oyster, Crassostrea gigas 20
freshwater amphipods, daphtvids, and midge larvae and shrimp should be
cultured and tested at the recommended test temperature. Other in-
vertebrates should be held and tested within 5°C of the temperature of
the water from which they were obtained. If the recommended test tempera-
ture is not within this range, they should be tested at the temperature
from the series 7, 12, 17, 22, and 27°C that is closest to the recommended
test temperature and is within the allowed range.
-------
HOLDING AND HANDLING
Disinfect holding tanks or chambers with 0.5 percent commercial
bleach for one hour. Brush thoroughly with the disinfectant. Rinse well
between groups of organisms. Other equipment used to handle organisms
must be disinfected with 0.5 percent commercial bleach (5 ml of bleach
added to 1 liter of water), or 30-percent formalin.
When feasible, holding tanks must receive an uncontaminated, constant-
quality water in a flow-through system with a flow rate of at least 2 tank-
volumes per day. Otherwise, a recirculation system where the water flows
through a charcoal filter to remove dissolved metabolites or passes by an
ultra-violet light for disinfection is necessary. Only as a last resort
should a dechlorinated tap water be used for freshwater organisms or a
synthetic salt water for marine organisms.
When organisms are first brought into a facility, they must be
quarantined for a minimum of 10 days. To avoid unnecessary stress after
collection and transportation, organisms should not be subjected to a change
of more than a 3°C in water temperature or 3-ppt in salinity in any 12-hour
period, or a total change of not more than 6°C or 6 ppt salinity. Inverte-
brates should be held within 5°C of the temperature of the water from which
they were obtained. To maintain organisms in good condition during holding,
crowding should be avoided. The dissolved oxygen concentration must be
greater than 40 percent of saturation for warm water species and greater
than 60 percent of saturation for cold water species. Aeration may be used
if necessary.
Organisms should be fed at least once a day, and excess food and fecal
material should be removed from the bottom of the tanks"at least twice a
week by siphoning. Organisms should be observed carefully for -signs of
disease, stress, physical damage, and mortality. Dead and abnormal speci-
mens should be removed as soon as observed. A daily log of feeding,
behavioral observations, and mortality must be maintained.
Organisms should be handled as little as possible. When handling is
necessary, it should be done as gently, carefully, and quickly as possible
to minimize stress. Organisms that touch dry surfaces or are dropped or
injured during handling must be discarded. Dipnets are best for handling
larger organisms. Such nets are commercially available or can be
made from small-mesh nylon netting, nylon or silk bolting cloth, plankton
netting, or similar material. Nets coated with urethane resin are best
for handling catfish. Smooth glass tubes with rubber bulbs should be used
for transferring smaller organisms such as daphnids and midge larvae.
DISEASE TREATMENT
During holding, fresh and salt water fish should be chemically treated
to cure or prevent disease as recommended in Table 2. However, if the fish
are severely diseased, it is advisable to discard the entire lot. When
invertebrates become diseased, they should be discarded. Tanks which are
contaminated with disease-causing microorganisms must be disinfected with
0.5 percent commercial bleach.
10
-------
TABLE 2. RECOMMENDED PROPHYLACTIC AND THERAPEUTIC TREATMENTS FOR
FRESHWATER FISH*
Disease
Chemical
Concentration Duration of
(mg/1) Treatment
External
Bacteria
Oxytetracycline hydrochloride
(water soluble)
Procaine Penicillin G in
Dihydrostreptomycin sulfate
solution (Franklin Lab, Denver,CO)
Benzalkonium chloride
(HYAMINE 1622 )
Nitrofurazone (water mix)
Neomycin sulfate
25
(3ml/100 gal)
Monogenetic Formalin plus zinc-free
trematodes
fungi, and
external,
d
protozoa
malachite green oxalate
Formalin
Potassium permanganate
Sodium chloride
DEXONR (35% Active Ingredient)
1-2
3-5b
25
25
0.1
150-250
2-6
15000-30000
2000-4000
20
30-60 min
48-72 hrs
30-60 min
30-60 min
30-60 min
1-2 hrs
30-60 min
30-60 min
5-10 min dip
(e)
30-60 min
Parasitic
copepods
Trichlorf on
(MASOTEN )
0 . 25 Continuous
This table indicates the order of preference of treatments that have been
reported to be effective,but their efficacy against diseases and toxicity
to fish may be altered by temperature or water quality. Caution; test
treatments on small lots of fish before making large-scale applications.
Fish should not be treated the first day they are in the facility.
11
-------
TABLE 2 (continued).
Before using a treatment other than those listed in this table, addi-
tional information should be obtained from sources such as Davis (1953),
Hoffman and Meyer (1974), Reichenbach-Klinke and Elkan (1965), Snieszko
(1970), and van Duijn (1973).
b. Active ingredient.
c. Treatment may be accomplished by (1) transferring the fish to a static
treatment tank and back to a holding tank; (2) temporarily stopping
the flow in a flow-through system, treating the fish in a static
manner, and then resuming the flow to flush out the chemical; or (3)
continuously.adding a stock solution of the chemical to a flow-through
system by means of a metered flow or the technique of Brungs and Mount
(1967).
d. One treatment is usually sufficient except for Ichthyophthirius ("Ich"),
which must be treated daily or every other day until no sign of the
protozoan remains. This may take 4-5 weeks at 5-10°C and 11-13 days
at 15-21°C. A temperature of 32°C is lethal to "Ich" in one week.
e. Minimum of 24 hours, but may be continued indefinitely.
f. Continuous treatment should be employed in static or flow-through
systems until no copepods remain, except that treatment should not be
continued for more than 4 weeks and should not be used above 27°C.
12
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TRANSPORTATION AND ACCLIMATION
Organisms reared and held at a fixed facility are transported to the
test site in the water in which they were reared and held. If the labora-
tory is mobile, the acclimation tank can be used in transporting organisms
from the rearing and holding facilities to the test site. At the test site,
dilution water (receiving water) is pumped to the laboratory for use in the
acclimation of the organisms. If dilution--water is not readily accessible,
it can be transported to the laboratory and stored in a tank for use in
the acclimation procedure and toxicity test. During transport and acclimation
the organisms should not be subjected to a change of more than 3°C in water
temperature or 3-ppt in salinity in any 12-hour period, or a total change of
not more than 6°C or 6-ppt salinity, and the concentration of dissolved
oxygen must not fall below 40 percent of saturation for warm water species
and 60 percent of saturation for cold water species.
Upon arriving at the test site, the organisms are acclimated to the test
dilution water and temperature by gradually changing from 100-percent holding
water to 100-percent dilution water« over a period of 24 hours. All organisms
must be exposed to 100-percent dilution water for at least 24 hours before
they are used for the tests, and must be held at the test temperature (+2°C)
for at least 24 hours before tests are begun.
A group of organisms must not be used for a. test if they appear to be
diseased or otherwise stressed, or if more than 5 percent die during the 48
hours immediately preceding the test. If the organisms fail to meet these
criteria, the entire group must be discarded and a new group obtained. The
same acclimation procedure must be followed for the new group or ogranisms.
Mortality of the test organisms during the acclimation period may be due
to the presence of toxicants in the dilution water (receiving water). If
excessive mortality occurs during the acclimation of the second group of
test organisms, an alternate source of dilution water must be used.
The acclimation of marine organism for effluent toxicity tests poses
special problems because most effluents discharged into the marine environ-
ment consist of adulterated freshwater. Therefore, when the effluent is
diluted with the receiving water (salt water), the higher percent effluent
volumes will have a low salinity (the salinity will be inversely proportional
to the percent volume of effluent). If the effluent is essentially fresh-
water, it is obvic \s that 100% effluent cannot be used with the marine test
organisms. The highest effluent concentration (lowest salinity) tested will
depend upon the salinity of the receiving water and the tolerance of the test
organism. (Sheepshead minnows and mysid shrimp are known to be tolerant to a
salinity range of 5-35 ppt, but the tolerance of other marine species in
Table 1 must be established by the investigator). Under the circumstances
described above, it will be necessary to acclimate marine organisms to a
series of salinities, ranging from 5-35 ppt. It would also be advisable
to culture the test organisms at a series of salinity levels, including
at least 10, 20, and 30 ppt, so that changes in salinity upon acclimation
do not exceed 6 ppt.
13
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SECTION 4
DILUTION WATER
A dilution water is acceptable if healthy test organisms survive
in it through the acclimation period and tpxicity test without showing signs
of stress, such as discoloration or unusual behavior. For effluent toxi-
city testing, the dilution water should be a representative sample of the
receiving water, and should be obtained from a point as close as possible
to, but upstream of or outside of, the zone influenced by the effluent.
It is preferable to pump the dilution water continuously to the acclima-
tion tank and dilutor. However, it may be more practical to transport
batches of water in tanks to the testing site as the need arises, and
then continuously pump water to these systems.
In an estuarine environment, the investigator should collect uncon-
tarainated water having a salinity as near as possible to the salinity of
the water at the receiving site.
Pretreatment of the dilution water should be limited to filtration
through a nylon sieve having 2-mm or larger holes to remove debris
and/or break up large floating or suspended solids. The water should be
obtained from the receiving water as close as possible to the time the
test begins. It should not be obtained more than 96 hours prior to
testing. If acceptable dilution water cannot be obtained from the
receiving water, some other uncontaminated, well-aerated surface or
ground water, or commercially available media, can be used. This water
must have a. total hardness, total alkalinity, and specific conductance
within 25 percent, and pH within 0.2 units, of the receiving water at
the time of testing.
, If the substitute dilution water must be modified, reconstituted
water must be prepared for use as the diluent. Recommended procedures
are given in Tables 3 and 4. There are also commercially available salt
water media such as INSTANT OCEANR and RILA SALTSR.
With highly toxic effluents requiring very large volumes of dilu-
tion water, it may be convenient to locate the testing facility near the
source of the dilution water, and transport the effluent.
14
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TABLE 3. PREPARATION OF RECONSTITUTED FRESH WATERS
3a. Quantities (mg/1) of reagent grade chemicals required to prepare
recommended reconstituted fresh waters and the resulting water
qualities.
Water
Type
Reagent Added
NaHCO, CaSO -2H00 MgSO. KCL
3 42 4
Final Water Quality
, Alka- .
b C C
pH Hardness linity
Very
Soft
Hard
Very
soft
hard
12
48
192
384
7.
30.
120.
240.
5
0
0
0
7.
36!
120.
240.
5
0
0
0
0.5
2.0
8.0
16.0
6.
7.
7.
8.
4-6.8
2-7.6
6-8.0
0-8.4
10-13
40-48
160-180
280-320
10-13
30-35
110-120
225-245
3b. Quantities of reagent-grade chemicals to be added to aerated, soft
reconstituted freshwater for buffering pH. The solutions should not
be aerated after addition of these chemicals.
Volume (ml)'of solution added to 15 liters of water
PH
6.0
6.5
7.0
7 c
/ . J
8.0
8.5
9.0
9.5
10.0
l.ON NaOH
1.3
,5.0
19.0
19.0
6.5
8.8
11.0
16.0
1.0 m KH0PO.
2 4
80.0
30.0
30.0
20.0
•———• —
0.5 m H3B03
„-___
40.0
30.0
20.0
18.0
a. From Marking and Dawson (1973).
b. Approximate equilibrium pH after aeration'and with fish in water.
c. Expressed in mg/1 as CaCO .
d. Approximate equilibrium pfi with fish in water.
15
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TABLE 4. PREPARATION OF RECONSTITUTED SEA WATERa'b
Add the following reagent-grade chemicals in the amounts and order listed
to 890 ml distilled water. Each chemical must be dissolved before another
is added.
Chemical Amount
NaF 3 mg
SrCl2'6H20 20 mg
H3B03 30 mg
KBr 100 mg
KC1 700 mg
i20 1.47 g
4.00 g
i20 10.78 g
NaCl "23.50 g
'9H 0 20 mg
Na4EDTA(c) 1 mg
NaHCO 200 mg
a. If the resulting solution is diluted to 1 liter, the salinity should be
34*0.5 g/kg, and the pH 8.0*0.2. The desired salinity is attained by
dilution at time of use.
b. From Kester e_t^ a_L^. (1967), Zaroogian _et_ al_. (1969), and Zillioux
et al. (1973).
c- Tetrasodium ethylenediaminetetraacetate. This coumpound should be
omitted when toxicity tests are conducted with metals. When tests are
conducted with plankton or larvae, the EDTA should be omitted and the
medium should be stripped of trace metals (Davey et al., 1970).
16
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SECTION 5
EFFLUENT SAMPLING AND HOLDING
SAMPLING
The effluent sampling point must be the same as that specified in the
NPDES discharge permit. Conditions for exception would be: (1) better
access to a sampling point between the final treatment and the discharge
outfall, or (2) if the processed waste is chlorinated prior to discharge
to the receiving waters, the sampling point may be located prior to con-
tact with the chlorine if the purpose of the test is to determine toxicity
levels of the unchlorinated effluent. Sampling should be based on an under-
standing of the short and long-term operations and schedules of the dis-
charger. It is desirable to evaluate an effluent sample that most closely
represents the "normal" or "typical" discharge and operating conditions
of the plant in question. The retention time of the effluent in the waste
water treatment facility, as indicated in Par. A.l.a-c and B.l-3 below,
must be measured using dye studies. The only way in which the sample may
be altered prior to testing is by filtering through a TEFLON or stainless
steel screen with 2-mm or larger holes.
A. Flow-through test
1. If the industrial or municipal facility discharges continu-
ously, the effluent should be pumped directly and continuously
from the discharge line to the dilutor system for the
duration of the test. The use of the effluent grab samples
should be avoided. However, if the effluent cannot be pumped
directly and continuously to the dilutor system, the fol-
lowing' alternative methods may be employed for collection
of the effluent:
a. When the measured minimum retention time of the effluent
is less than 96 hours, as determined from dye studies, a
6-hour composite sample, consisting of equal volumes taken
every 30 minutes, must be collected and transported to the
dilutor every 6 hours for the duration of the test.
b. When the measured minimum retention time of the effluent
is between 4 days (96 hours) and 14 days, as determined
from dye studies, then a 24-hour composite sample, consist-
ing of equal volumes taken every hour, may be collected
daily for the duration of the test.
17
-------
c. When the measured minimum retention time of the effluent
is greater than 14 days, as determined from dye studies,
a single grab sample may be collected daily for the
duration of the test.
2. If the industrial or municipal facility discharges inter-
mittently (i.e. where the-waste is discharged over a single
8-hr work shift, or is accumulated and discharged at the end
of the shift, or end of the week), a composite sample, consist-
ing of equal volumes collected every 30 minutes, may be taken
for an 8-hr operating shift or for the duration of the plant
operating schedule, or a single grab sample may be taken in
the case of a batch discharge.
B. Static test
If a flow-through test cannot be used, a static test may be
conducted with effluent collected by one of the following methods:
1. When the measured minimum retention time of the effluent is
less than 96 hours, as determined with dye studies, four
consecutive 6-hr composite samples, each consisting of equal
volumes taken every 30 minutes, are collected and used in
setting up 4 separate static tests.
2. When the measured minimum retention time of the effluent is
between 4 days (96 hours) and 14 days, as determined with dye
studies, a 24-hour composite sample, consisting of equal volumes
taken every hour, is collected daily and used in the test.
3. When the measured minimum retention time of the effluent is
greater than 14 days, as determined with dye'studies, a
single grab sample may be collected and used in the test.
HOLDING
Effluent grab samples must be stored in covered, unsealed containers.
Violent agitation must be avoided. However, undissolved materials must be
uniformly dispersed by gentle agitation. This agitation must immediately
precede adjustment of any aliquot of the effluent to test temperatures
before adding it to the dilution water. Although it is desirable to
refrigerate samples prior to the test, it is often convenient to store
samples in a constant-temperature water bath or controlled-environment
room at the temperature at which the test is conducted. The test should
be initiated as soon as possible, but no later than 24 hours after collection
of the effluent.
The persistence of the toxicity of an effluent may be a factor in deter-
mining specific toxicity limits in an NPDES permit, and is determined by
measuring its toxicity upon collection and again after holding 96 hours.
If after holding the effluent 96 hours, its toxicity has not decreased 50%
or more, it is classified as persistent. (When special tests, such as
persistence are conducted, the exact methodology must be detailed in the
report.)
18
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SECTION 6
TEST PROCEDURE
RANGE-FINDING(SCREENING) TEST
Unless the approximate toxicity of the effluent is already' known, it
is necessary to conduct an abbreviated, preliminary, range-finding or
screening test to determine the concentrations that should be used in the
definitive tests. This test can be either a static or flow-through test.
However, the test most often used is an abbreviated static test in which
groups of 5 organisms are exposed to three to five widely-spaced effluent
dilutions, and a control, for 8 to 24 hours.
Because the characteristics of the effluent and the receiving water
may vary significantly within short periods of time, the toxicity observed
in a range-finding test may not be representative of the toxicity of the
effluent. If the range-finding test is to be conducted with the same
sample of the effluent with which the definitive test is to be conducted,
the duration of the range-finding test cannot exceed 24 hours (see limits
in holding time for effluents, p. 18).
DEFINITIVE TEST
Test Conditions
The determination of a LC50 or EC50 must employ a control and at
least five concentrations of effluent in an exponential series. To
calculate the LC50 or EC50 with reasonable accuracy, a definitive test
must meet both of the following criteria:
A. Each concentration of the effluent must be at least 50
percent of the preceding concentration.
B. One concentration must have killed (or affected) more than 65
percent of the organisms exposed to it, and one concentration other
than the control must have killed (or affected) less than 35
percent of the organisms.
If 100-percent effluent does not kill (or affect) more than 65 percent
of the organisms exposed to it, the percentage of organisms killed (or
affected) by various levels of the effluent in the receiving water must be
reported.
The control shall consist of the same dilution water, conditions,
procedures, and organisms used in testing the effluent. A test is not
acceptable if more than ten percent of the organisms die in the control.
Number of Test Organisms
At least 20 organisms of a given species must be exposed to each treat-
ment. More than one species may be used In the same test chamber in a
19
-------
given test, if segregated. One-half of the organisms of each species
exposed to each treatment should be placed in separate test chambers to
serve as replicates. To qualify as true replicates, no water connections
can exist between replicate test chambers. Randomization of treatments is
desirable.
Test animals are normally captured for' transfer from acclimation tanks
to test chambers by dip netting. No more~~than 20 percent of the total
number of organisms transferred to each chamber should be added from a
given net capture.
Loading of Test Organisms
For all tests, a limit must be placed on the weight of organisms
per liter of test solution. This practice will minimize the depletion of dis-
solved oxygen, the metabolic conversion of effluent constituents, the accumu-
lation of metabolic waste products, and/or stress induced by crowding, any
of which could significantly affect the test results.
For flow-through tests, loading in the test chambers must not exceed
5 grams per liter at temperatures of 20°C or less, or 2.5 grams per liter
at temperatures above 20°C.
For static tests, loading in the test chambers must not exceed 0.8
grams per liter at temperatures of 20°C or less and 0.4 grams per liter at
temperatures above 20°C.
Test Temperature
For flow-through tests, it is desirable to hold the temperature
within +2.0°C of the acclimation temperature throughout the test. This
can be accomplished by passing the effluent and/or dilution water through
separate stainless steel coils immersed in a heating or cooling water bath
prior to entering the dilutor system.
For static tests, the temperature may be that at which the test
organisms were held, prior to transportation or acclimation at the site.
The instantaneous ambient temperature should not vary more than +2°C at any
time during the test.
Dissolved Oxygen
Aeration may alter the results of toxicity tests and, as a general
rule, should not be employed. It can reduce the apparent toxicity of an
effluent by stripping it of highly volatile toxic substances, or increase
its toxicity by altering the pH. However, the dissolved oxygen concentration
(DO) in the test solution should not be permitted to fall below 40 percent
saturation for warm water species and 60 percent saturation for cold water
species. In most flow-through tests, DO depletion is not a problem in the
test chambers because aeration occurs as the liquids pass through the
dilutor system.
20
-------
If the DO concentration decreases to a level that would be a
source of additional stress, the turnover rate of the solutions in the
test chambers must be increased sufficiently to maintain acceptable DO
levels. If the increased turnover rate does not maintain adequate DO
levels, aerate the dilution water prior to the addition of the effluent,
and aerate all test solutions.
Caution must be exercised to avoid excessive aeration, and it
should be. used only as a last resort in maintaining adequate DO levels.
When aeration is used, the exact methodology must be detailed in the report.
Beginning the Test
The test begins when the test organisms are first exposed to the
effluent.
A. Flow-through test
The dilutor system should be in operation 24 hours prior to
the addition of the test organisms and the beginning of the test.
During this period, necessary adjustments can be made in the
percent effluent volumes, temperature, and flow rate through
the test chambers.
B. Static test
The effluent is added to the dilution water and mixed well
by stirring with a glass rod. The test organisms are placed
in the chambers within 30 minutes. This procedure conserves DO
and is sufficient for the effluent to become evenly dispersed
in the dilution water.
Feeding
Organisms should not be. fed during the test unless they are newly
hatched or very young. In the case of fish, feeding should be terminated
48 hrs before the beginning of the test. Problems caused by feeding, such
as the possible alteration of toxicant concentration, the build-up of
food and metabolic wastes and resulting oxygen demand, are common in
static test systems, but are minimal in flow-through systems.
Duration
The test duration may range from a minimum of 8 hours to 96 hours,
depending on the test organism used, the purpose of the test and whether
it is a. range-finding test or a definitive test.
21
-------
SECTION 7
TEST RESULTS
BIOLOGICAL DATA
The lengths and weights of the test organisms should be determined by
sacrificing and measuring representative organisms before the test or
by obtaining the lengths and weights of all surviving organisms at the
end of the test. The number of dead (or affected) organisms in each
test container should be counted 24, 48, 72, and 96 hours after the
beginning of the test. (See data sheet in Appendix, Fig. 1, p. 33).
Dead organisms must be removed at least once every 24 hours.
Death is the "effect" most frequently used for determining toxicity
to aquatic organisms. The criteria usually employed in establishing
death are: (1) no movement (especially no gill movement in fish), and
(2) no reaction to gentle prodding. Death is not easily determined for
some invertebrates, and some other effect often must be used. The
effect generally used for determining toxicity to daphnids and midge
larvae is immobilization, which is defined as the inability to move
except for minor activity of appendages. With crabs, crayfish, and
shrimp, the effects used are immobilization and loss of equilibrium.
Other effects can be used for determining an EC50, but the effect and
its definition must always be reported. General observations on such
things as erratic swimming, loss of reflex, discoloration, changes in
behavior, excessive mucus production, hyper-ventilation, opaque eyes,
curved spine, hemorrhaging, molting, and cannibalism should be reported.
CHEMICAL AND PHYSICAL DATA
The dissolved oxygen concentration and pH must be measured at the
beginning of the test, and every 24 hours thereafter, in the control
and in the high, medium, and low effluent concentrations, for the
duration of the test. The specific conductance, total alkalinity,
total hardness, and salinity, where applicable, should be measured at
the beginning of the test in the control and the high, medium and low
effluent concentrations. There may be a build-up of ammonia in the static
toxicity tests. It is advisable, therefore, to measure the concentration
of total ammonia nitrogen in the control, high, medium, and low effluent
concentrations at the beginning and end of each static test. The percentage
of un-ionized ammonia in the test containers can be determined from
Tables 5 and 6. Temperature should be recorded at least hourly in at least
one container during the acclimation period and test.
22
-------
TABLE 5. PERCENTAGE OF AMMONIA THAT IS UN-IONIZED IN DISTILLED
WATER AT VARIOUS TEMPERATURES AND pH's*
PERCENT UN-IONIZED AMMONIA
Temperature
-------
Water samples collected for chemical analysis should be taken
midway between the top, bottom, and sides of the test containers
and should not include any surface scum or material stirred up from
the bottom or sides.
Methods used for chemical analysis must be those specified in
Section 304(g) of the Federal Water Pollution Control Act
Amendments of 1972 (Chemical Methods Manual-, USEPA, 1977).
CALCULATION OF LC50 AND EC50
For each set of data, the 96-hr LC50 or EC50 and its 95-percent
confidence limits must be calculated on the basis of the initial volume
percent of the effluent in the test solutions. The "volume percent"
equals "(100 x volume of effluent)/(volume of effluent + volume of
dilution water)." If other (24-,48-,72-hr) LC and EC values are
calculated, their 95 percent confidence limits must also be determined.
A variety of methods are available to calculate a LC and EC (Finney,1
1964, 1971). The most widely used are the log-concentration versus-
percent-survival, probit, logit, moving-average, and Litchfield-Wilcoxon
(1949) methods.
Two examples of calculating a LC50 using a hypothetical set of
data have been provided in the Appendix. The two methods used in the
calculations are: (1) the Litchfield-Wilcoxon and, (2) the log-
concentration-versus-percent-survival. If more than 10 percent of the
control organisms die, none of the previously mentioned methods may
be used in calculating LC and EC values, and the remaining test
results must be used with caution in evaluating the toxic effect.
REPORTS
A report of the results of a test must include the following:
A. The name of the test method, investigator and laboratory, and
the date the test was conducted.
B. A detailed description of the effluent, including its source,
date and time of collection, composition, known physical and
chemical properties, and variability.
C. The source of the dilution water, the date and time of its
collection, its chemical characteristics, and a description of
any pretreatment.
D. Detailed information about the test organisms, including scientific
name, length and weight, age, life stage, source, history, observed
diseases, treatments, and acclimation procedure used.
24
-------
E. A description of the test procedure: the test chambers, including
the depth and volume of solution; the way the test was begun; the
number of organisms per treatment; and the loading. For the flow-
through system, the water volume changes per 24 hours in each test
chamber must be calculated and reported.
F. The definition of the adverse effect (death, immobility, etc.) used
in the test, and a summary of general observations on other effects or
symptoms.
G. The number and percentage of organisms in each test chamber (including
the control chambers) that died or showed the "effect" used to measure
the toxicity of the effluent.
H. A 24-, 48-, 72-, and 96-hr LC50 or EC5Q value for the test
organisms, depending on the duration of exposure. If 100 percent
effluent did not kill or affect more than 65 percent of the test
organisms, report the percentage of the test organisms killed or
affected by various concentrations of the effluent.
I. The 95-percent confidence limits for the LC50 and EC50 values and the
method used to calculate them.
J. The methods used for and the results of all chemical analyses.
K. The average and range of the acclimation temperature and the test
temperature.
L. Any deviation from this method.
M. Any other relevant information.
25
-------
REFERENCES
1. Abram, F.S.H. 1973. Apparatus for control of poison concentration
in toxicity studies with fish. Water Res. 7:1875-1879.
2. Bengtsson, B.E. 1972. A simple principle for dosing apparatus in
aquatic systems. Arch. Hydrobiol. _70:413-415.
3. Brungs, W. A., and D. I. Mount. 1967. A device for continuous treatment
of fish in holding chambers. Trans. Amer. Fish. Soc. 96:55-57.
4. Cline, T.F. and G. Post. 1972. Therapy for trout eggs infected with
Saprolegnia. Prog. Fish-Cult. 34:148-151.
5. Davey, E.W., J.H. Gentile, S.J. Erickson, and P. Betzer. 1970.
Removal of trace metals from marine culture media. Limnol. Oceanogr.
15:486-488.
6. Davis, H.S. 1953. Culture and diseases of game fishes. Univ. Calif.
Press, Berkeley. 332 pp.
7. DeFoe, D.L. 1975. Multichannel toxicant injection system for flow-
through bioassays. J. Fish. Res. Bd. Can. 32:544-546.
8. Finney, D.J. 1964. Statistical Method in Biological Assay, 2nd ed.
Hafner Publ. Company, New York. 668 pp.
9. Finney, D.J. 1971. Probit Analysis. Cambridge Univ. Press,
London. 333 pp.
10. Freeman, R. A. 1971. A constant flow delivery device for chronic
bioassay. Trans. Amer. Fish. Soc. 100:135-136.
11. Granmo, A. and S. 0. Kollberg. 1972. A new simple water flow system
for accurate continuous flow tests. Water Res. 6:1597-1599.
12. Hoffman, G.L.}Jand F. P. Meyer. 1974. Parasites of freshwater fishes.
THF Publ., Inc., Neptune City, N.J. 224 pp.
13. Kester, D.R., I.W. Dredall, D.N. Connors, and R.M. Pytokowicz. 1967.
Preparation of artificial seawater. Limnol. Oceanogr. 12:176-179.
14. Lichatowich, J.A., P.W. O'Keefe, J.A. Strand, and W. L. Templeton.
1973. Development of methodology and apparatus for the _bioassay of oil.
In: Proceeding of Joint Conference on Prevention and Control of Oil
Spills. American Petroleum Institute, U.S. Environmental Protection
Agency and U.S. Coast Guard, Washington, D.C. pp. 659-666.
15. Litchfield, J.T. Jr. and F. Wilcoxon. 1949. A simplified method of
evaluating dose-effect experiments. J. Pharm. Exp. Ther. 96:99-113.
26
-------
16. Lowe, J.I. 1964. Chronic exposure of spot, Leiostomus xanthurus, to
sublethal concentrations of toxaphene in seawater. Trans. Amer. Fish.
Soc. 93: 396-399.
17. Marking, L. L.., and V. K. Dawson. 1973. Toxicity of quinaldine
sulfate to fish. Invest. Fish Contr. No. 48., U.S. Fish & Wildlife
Service, Washington, D.C. 8 pp.
18. Mount, D. I. and W. A. Brungs. 1967. A simplified dosing apparatus
for fish toxicological studies. Water Res. 1:21-29.
19. Nebeker, A. V. and A. E. Lemke. 1968. Preliminary studies on the
tolerance to aquatic insects to heated waters. J. Kans. Entomol.
Soc. 41:413-418.
20. Reichenbach-Klinke, H., and E. Elkan. 1965. The principal diseases of
lower vertebrates. Academic Press, New York, 600 pp.
21. Riley, C. W. 1975. Proportional diluter for effluent bioassays.
JWPCF. 47:2620-2626.
22. Schimmel, S. C., D. J. Hansen, and J. Forester. 1974. Effects of
Aroclor 1254 on laboratory-reared embryos and fry of sheepshead min-
nows _(Cy_p_rinodon variegaituŁ). Trans. Amer. Fish. Soc. 103:582-586.
23. Schimmel, S. C. and D. J. Hansen. 1974. Sheepshead Minnow (Cyprinodon
variegatus): An estuarine fish suitable for chronic (entire life-cycle)
bioassays. Proceedings of the 28th Annual Conference of the South-
eastern Association of Game and Fish Commissioners, pp. 392-398.
24. Shumway, D. L. and J. R. Palensky. 1973. Impairment of the flavor of
fish by water pollutants. Ecological Research Series No. EPA-R3-73-
010. U. S. Environmental Protection Agency, Washington, D. C. 80 pp.
25. Skarheim H. P. 1973. Tables of the fraction of ammonia in the
undissociated form. SERL Report No. 73.-5. University of California,
Berkeley, 33 pp.
.*
26. Sniewzko, S. F. (ed.). 1970. A symposium on diseases of fishes and
shellfishes. Spec. Publ. No. 5, Amer. Fish. Soc., Washington, D.C.
526 pp.
27. Sprague, J. B. 1969. Measurement of pollutant toxicity to fish. I.
Bioassay methods for acute toxicity. Water Res. 3:793-821.
28. Thurston, R.V., R. C. Russo, and K. Emerson. 1974. Aqueous ammonia
equilibrium calculations. Tech. Rep. No. 74-1. Fisheries Bioassay
Laboratory, Montana State University, Bozeman. 18 pp.
29. U.S. Environmental Protection Agency. 1972. Recommended bioassay
procedure for fathead minnow Pimephales promelas Rafinesque chronic
tests. U.S. Environmental Protection Agency, National Water Quality
Laboratory, Duluth, Mn. 13 pp.
27
-------
30. U.S. Environmental Protection Agency. 1974. Methods for chemical
analysis of water and x^astes. U.S. Environmental Protection Agency,
National Environmental Research Center, Methods Development and
Quality Assurance Research Laboratory, Cincinnati, Oh. 298 pp.
31. U.S. Environmental Protection Agency. 1975. Methods for acute
toxicity tests with fish, macroinvertebrates and amphibians.
U.S. Environmental Protection Agency^ National Environmental
Research Center, National Water Quality Research Laboratory,
Duluth, Mn. 61 pp.
32. van Duijn, C., Jr. 1973. Diseases of fishes. 3rd ed., Charles C.
Thomas Publ., Springfield, II. 309 pp.
33. Zaroogian, G. E., G. Pesch, and G. Morrison. 1969. Formulation of an
artificial sea water media suitable for oyster larvae development.
Amer. Zool. 9:1141.
34. Zillioux, E..J., H.R. Foulk, J.C. Prager, and J. A. Cardin. 1973.
Using Artemia to assay oil dispersant toxicities. JWPCF. 45:2389-
2396.
28
-------
APPENDIX
A. LITCHFIELD AND WILCOXON ABBREVIATED METHOD OF DETEBMINING THE LC50
General Procedure
Step 1: Tabulate the data (see sample data sheet, Fig. 1, p. 33) showing
the percent-effluent volumes used, the total number of organisms exposed to
each percent-effluent volume, the number of affected organisms, and the
observed percent-affected organisms ^(see Example 1 below). Do not list
more than 2 consecutive 100 percent 'affects at the higher percent-effluent
volumes or more than two consecutive^O percent affects at the lower percent-
effluent volumes.
Step 2: Plot the percent-affected organisms against the percent-effluent
volume on 2 cycle, logarithmic probability paper (Fig. 2), except for
0 percent or 100 percent affect values. With a straight edge, fit a
temporary line through the points, particularly those in the region of
40 percent to 60 percent affects.
Step 3: Using the line drawn through the points, read and list an
"expected" percent^affect for each percent-effluent volume tested. Disre-
gard the "expected" 'percent value for any of the percent^volumes less than
0.01 or greater than 99.99. Using the expected-percent-affect, calculate
from Table 7 a "corrected" value for each 0 percent or 100 percent affect
obtained in the test. (Since the expected values in the table are whole
numbers, it will be necessary to obtain intermediate values by interpolation.)
Plot these values on the logarithmic probability paper (Fig. 2) used in
Step 2 and inspect the fit of the line to the completely plotted data. If
after plotting the corrected expected values for 0 percent and—100 percent
affected, the fit is obviously unsatisfactory, redraw the line and obtain
a new set of expected values.
Step 4; List the difference between each observed (or corrected) value and
the corresponding expected value. Using each difference and the corres-
ponding expected value, read and list the contributions to Chi-square (Chi2)
from Fig. 3 ( a straight edge connecting a value on the Expected-Percent
Affected scale with a value on the Observed-Minus-Expected scale, will
indicate at the point of intersection of the Chi^ scale, the contribution to
Chi2. Sum the contributions to Chi and multiply the total by the average
number of organisms per effluent volume, i.e., the number of organisms used
in K concentrations divided by K, where K is the number of percent-affected
organism values plotted. The product is the "calculated" Chi2 of the line.
The degrees of freedom (N) are 2 less than the number of points plotted,'
i.e., N =K-2. If the calculated Chi2 is less than the Chi2 given in Table 8
for N degrees of freedom, the data are non-heterogeneous and the line is
a good fit. However, if the calculated Chi2 is greater than the Chi2 given
in Table 8 for N degrees of freedom, the data are heterogeneous and the line
is not a good fit. In the event a line cannot be fitted (the calculated
Chi2 is greater than the tabular Chi2),, the data can not be used to calcu-
late a LC50 or EC50. Litchfield and Wilcoxon provided an alternate
method for calculating the 95 percent confidence limits under these cir-
cumstances. However, the toxicity test should be repeated.
29
-------
Step 5: Determine the confidence limits of the LC50.
a. Read from the fitted line (Fig. 2), the percent effluent volumes for
the corresponding 16, 50, 84 percent affects (LC16, LC50 and LC84).
b. Calculate^the slop'e function, S, as:
S = LC84/LC50 + LC50/LC16
2
c. From the tabulation of the data determine N', which is defined as the
total number of test organisms used within the percent-affected-
organism interval of 16 percent and 84 percent. Calculate the exponent
(2.77//N7) for the slope function and the factor, f;LC50» used to
establish the confidence limits for the LC50 (or EC50).
LLC50
.(2.77//N7)
LC50 can ^e obtained directly from the nomogram in Fig. 4 by laying
a straight-edge across the appropriate base and exponent values and
reading the resultant "f" value.
e. Calculate the confidence limits of the LC50 as follows:
(1) Upper limit for 95% probability = LC50 X fTP,-n
JL»L»DU
(2) Lower limit for 95% probability = LC50/f7
Example
'LC50
Steps 1-4: The data were tabulated and plotted (Fig.-2) and the
expected values were read from the graph.
STEP ONE
STEP THREE
STEP FOUR
% Number of Observed % Observed
Effluent Number of Affected Affected Expected % Minus „
Volume Organisms Organisms Organisms (Fig. 2) Expected Chi
3.2
5.6
10.0
18.0
32.0
56.0
100.0
20
20
20
20
20
20
20
0
1
11
1
12
18
20
0(.2)b
5
55
35
60
90 b
100 (99.0)
.6
3.5
(14.5)a
38.0
67.0
87.5
97.0
0.4 0.003
1.5 0.006
Aberrant Value
3.0 0.004
7.0. 0.024
2.5 0.006
2.0 0.014
Total
0.057
a. Percent-affected organisms at the 10 percent effluent volume is obviously
an aberrant value and should be omitted when fitting the line in Step 2.
b. Step 3 "Corrected" affected values from Table 7.
30
-------
Step 4 (Cont.) :
Calculation of Chi
a. Mean number of organisms used in 'K' (K=6) concentrations = —7— - 20.
o
(Note that the data for the 10% effluent volume were aberrent and not
used. Therefore, K=6, and the total number of organisms - 120)
b. Calculated Chi2 = 20 x 0.057 = 1.14
c. Degrees of Freedom (N) = K-2 = 6-2 = 4
2
d. From Table 8, the Chi for 4 degrees of freedom =9.49
2 2
e. The calculated Chi is less than the tabular Chi . Therefore, it is
assumed the line is a good fit, and the data are non-heterogeneous.
Step 5:
a. From the fitted line in Fig. 2, determine the (percent) effluent
concentrations corresponding to the 16%, 50% and 84% affected
organism values:
b. LC84 effluent concentrations * 50.0%
LC50 " " - 23.0%
LC16 " " 10.5%
c. Calculate the slope function, *S', as:
S = LC84/LC50 + LC50/LC16 50.0/23.0 + 23.0/10.5
2.17
2
+ 2.19
2 '
4.36 2.18
2 ~
2
d. N' - 40 (From Figure 2)
e. Calculate the exponent (Nr) and factor, fTr>1-n
_ 2.77/-/F_ 2.77//40 2.77/6.32 1R0.438
TC50 ~" ^--Lo • Z.lo = 2.18 a 1.41
f. Calculate the confidence limits of the LC50
(1) Upper limit for 95% probability = LC50 X fTrc;n - 23.0 X 1.4.- 32.2%
(2) Lower limit for 95% probability - LC50/fT_,n = 23.0/1.4 - 16.4%
LC5U
31
-------
TABLE 7. CORRECTED VALUES OF 0% OR 100% EFFECT
Expected
Value
Corrected Value
0
0
10 3.2
20 6.0
30 8.3
40 9.9
50
60 90.1
70 91.7
80 94.0
90 96.8
1
0.3
3.5
6.2
8.4
10.0
89.5
90.2
91.9
94.3
97.1
2
0.7
3.8
6.5
8.6
10.1
89.6
90.4
92.2
94.5
97.4
3
1.
4.
6.
8.
10.
89.
90.
92.
94.
97.
0
1
7
8
2
6
5
4
8
7
4
1.3
4.4
7.0
9.0
10.3
89.6
90.7
92.6
" 95.1
98.0
5
1.6
4.7
7.2
9.2
10.4
89.7
90.8
92.8
95.3
98.4
6
2.0
4.9
7.4
9.3
10.4
89.7
91.0
93.0
95.6
98.7
7
2.3
5.2.
7.0
9.4
10.4
89.8
91.2
93.3
95.9
99.0
8
2.6
5.5
7.8
9.6
10.4
89.9
91.4
93.5
96.2
99.3
9
2.9
5.7
8.1
9.8
10.5
90.0
91.6
93.8
96.5
99.7
TABLE 8. VALUES OF Chi* (p = 0.05)
Degrees of Freedom (N)
1
2
3
4
5
6
7
8
9
10
Chi2
3.84
5.99
7.82
9.49
11.1
12.6
14.1
15.5
16.9
18.8
32
-------
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33
-------
r
LL
Q
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CO
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• o
LC50=23.0:
LC16=10.5 c
EElL
* I • r • •
% EFFLUENT VOLUME
FIG. 2. LINE FITTED TO DATA, AND LCI6,
LC50, AND LC84 AS READ FROM
THE LINE (STEPS 2, 3, AND 5).
-------
EXPECTED
AFFECTED
5of—50
Chi
. 2
7O
80
90
95
96-
97- —3
98-
99-
99.5
99.6
99.7-
99.8
99.9
99.95
99.96-
99.9 {
— 30
— 20
—10
—5
1.0
- .5
-.4
-.3
.2
05
.04
'-+.03
.02
OBSERVED MINUS
EXPECTED
— 50
40
— 30
— 20
— 10
• 5
4
-3
— 2
.5
.4
--.3
- -.2
--.05
— 2.0
— 1.0
.50
— .40
— .30
— .20
— .10
— .05
— .04
— .03
— .02
— .01
— .005
.004
— .003
— .002
—.001
FIG. 3. NOMOGRAPH FOR OBTAINING Chi2 FROM
EXPECTED % AFFECTED AND
OBSERVED-MINUS-EXPECTED (STEP 4).
35
-------
B/
10 —
-
5.0 —
40 —
3.0 —
-
2.0—
-
1.30 —
1.40 —
1.30—
-
™"
1.20 —
_
_
-
1.10—
\SE S .p
L
EXPONE
.C50
- 100
— 50
— 10.0
— 5.0
— 4.0
— 3.0
— 2.0
— 1.5
- 1.4
— 1.3
— 1.2
— 1.10
- 1.05
— 1.04
— 1.03
- 1.02
NT
— 4.0
-
— 3.0
-
— 2.0
— 1.5
-
— 1.0
— .90
— .80
— .70
— .60
— .50
-
— .40
-
— .30
—
.20
FIG. 4. NOMOGRAPH FOR RAISING BASE S TO
A FRACTIONAL EXPONENT
36
-------
B. LOG-CONCENTRATION VERSUS PERCENT-SURVIVAL METHOD OF DETERMINING
THE LC50
General Procedure
Step 1: Plot the percent effluent volumes and the corresponding
percent survival on semi-logarithmic paper (Fig. 5).
Step 2; Locate the 2 highest points on the graph which are separated
by the 50 percent survival line and connect them with a diagonal straight
line. However, if one of the points is an aberrant value, the next
lowest or highest percent-effluent volume is used.
Step 3: Read on the scale for percent-effluent volume, the value of
the point where'the diagonal line and the 50 percent survival line
intersect. This value is the LC50 percent-effluent volume for the
test. If by chance one of the effluent concentrations happens to have
50 percent survival, no graphing is ^necessary.
Example
Step 1: The percent-effluent volumes and the corresponding percent
survival data from the Litchfield and Wilcoxon example are plotted in
Fig. 5.
Step 2: The two highest points which are separated by the 50 percent
survival line (65 percent and 40 percent) are located and connected with a diagonal
diagonal straight line. The percent survival in the 10 percent-effluent
volume was considered an aberrant value and, therefore, was omitted from the
evaluation.
Step 3: An LC50 of 25.4 percent-effluent volume for the test was
derived from the point where the diagonal line and the 50 percent
survival line intersected in Fig,5.
37
-------
0 10 20 30 40 50 60 70 80 90 100
% SURVIVAL
FIG. 5. PLOTTED DATA AND FITTED LINE FOR
LOG-CONCENTRATION VERSUS
% SURVIVAL METHOD
38
-------
C. DILUTOR SYSTEMS, CONTROL PANEL AND EQUIPMENT LISTS
Two proportional dilutor systems are illustrated: (1) The Solenoid
Valve System, and (2) the Vacuum Siphon System. The designs incorporate
features from devices developed by many other Federal and State pro-
grams, and have been shown to be very versatile for on-site bioassays
in mobile labs as well as in fixed (central) labs. The Solenoid Valve
System is fully controlled by solenoids (Figs. 6, 7, & 8), and is the
preferred system. The Vacuum Siphon System (Figs. 6, 9, & 10), how-
ever is acceptable. Both systems employ the same control panel
(Fig. 14).
If in the range-finding test, the LC50 of the effluent falls in the
concentration range, 5.6% to 100%, premixing is not required, and the
mixing chamber is by-passed by running a TEFLONR tube directly from
the effluent in-flow pipe to chamber E-l. Chambers D-l and D-2 and the
mixing chamber are deactivated.
To provide the capability of using the dilutor system to carry out
tests of the toxicity of pure compounds, the control panel is equipped
with an auxiliary power receptacle to operate a metering pump to deliver
an aliquot of the stock solution of the pure compound directly to the
mixing chamber during each cycle. In this case, chamber D-l is
deactivated and chamber D-2 is calibrated to deliver a volume of
2000 ml, which is used to dilute the aliquot to the highest concen-
tration used in the toxicity test.
39
-------
FIG. 6. PHOTOGRAPHS Gi DILUTOR SYSTEMS:
SOLENOID VALVE SYSTEM (LEFT), AND
VACUUM SIPHON =5 /STEM (RIGHT).
40
-------
FLOW CONTROL
VALVES-
NORMALLY OPEN
SOLENOID VALVES
DILUTION WATER
INFLOW
EFFLUENT
INFLOW
CYCLE
COUNTER
LAPSE
TIME
CLOCK
LIQUID LEVEL
SWITCH
DILUTION WATER CHAMBERS
MIXING CHAMBER
NORMALLY OPEN
SOLENOID VALVE
EFFLUENT
CHAMBERS
MIXING CHAMBERS
TEST CHAMBERS 4—20 LITERS CAPACITY
FIG. 7. SOLENOID VALVE DILUTOR SYSTEM,
GENERAL DIAGRAM.
-------
ADJUSTABLE
STANOPIPE
DRAIN
DILUTION WATER CHAMBERS
NORMALLY CLOSED
SOLENOID VALVES
6mm 0 D DELIVERY TUBE.
NORMALLY OPEN SOLENOID VALVE
6 mm 0.0. DELIVERY TUBE
EFFLUENT CHAMBERS
NORMALLY CLOSED
SOLENOID VALVE
6 mm OD DELIVERY TUBE
MIXING CHAMBER
1200 ml CAPACITY
10 mm 00 DELIVERY TUBE
TEST CHAMBER
4-20 LITER CAPACITY
FIG. 8. SOLENOID VALVE DILUTOR SYSTEM,
DETAILED DIAGRAM.
42
-------
Solenoid System Equipment List
1. Diluter Glass.
2. Stainless Steel Solenoid Valves
a. 3 - normally open, two-way, 55 psi, water, 1/4" pipe size,
9/32" orifice size, ASCO 8262152, for incoming effluent and
dilution water pipes and mixing chamber pipe.
b. 1 - normally closed, two-way, 15 psi, water, 3/8" pipe size,
3/8" orifice size, ASCO 8030B65, for D-2 chamber evacuation
pipe.
c. 12 - normally closed, two-way, 36 psi, water, 1/4" pipe
size, 9/32" orifice size. ASCO 8262C38, for remaining dilution
chamber and effluent chamber evacuation pipes.
3. Stainless steel tubing, seamless, 316-grade, austenetic.
a. 10 ft - 3/8" OD, 0.035" wall thickness, for dilution water and
effluent pipes.
b. 60 ft - 1/4" OD, 0.035" wall thickness, for dilution water and
effluent pipes.
c. 1 ft - 3/4" OD, 0.035"'wall thickness, for standpipe in D-l
chamber.
4. Swagelok tube connectors, stainless steel.
a. 4 - male tube connectors, male pipe size 1/4", tube OD 3/8".
b. 2 - male tube connectors, male pipe size 1/2", tube OD 3/8".
c. 26 - male tube connectors, male pipe size 1/4, tube OD 1/4"
d. 2 - male tube connectors, male pipe size 3/8", tube OD 3/8".
e. 2 - male adapter, tube to pipe, male size 1/2", tube OD 3/8".
5. 7 - 1200 ml stainless steel beakers.
6. Several Ibs each of Neoprene stoppers, size 00, 0, and 1, 1 Ib of siz 5.
7. ]4 - aquarium 2-5 gal.
8. Magnetic stirrer.
9. 2 - PVC ball valves, 1/2" pipe size.
10. Dilutor control panel - see Fig. 14 and equipment list.
11. Plywood sheeting, exterior grade: one - 4' x 8' x 3/4", one -
4' x 8' x 1/2".
12. Pine or redwood board, 1" x 8", 20 ft.
13. Epoxy paint, 1 gal.
14. Assorted wood screws, nails, etc.
15. 25 ft - 1/4" ID,-TEFLONR tubing, to connect the mixing chambers
to the test chambers (see Fig. 10).
43
-------
.NORMALLY OPEN
'SOLENOID VALVES
-FLOW CONTROL VALVES
•i \ V—
'DILUTION INFLOW [f \" \
DILUTION WATER CHAMBERS
LIQUID LEVEL SWITCH
,NORMALLY CLOSED
/ SOLENOID VALUE
MIXING CHAMBERS
TEST CHAMBERS 4-20 LITERS CAPACITY
FIG. 9. VACUUM SIPHON DILUTOR SYSTEM, GENERAL DIAGRAM.
44
-------
D-l
D-3
ADJUSTABLE
STANDPIPE
DRAIN
DILUTION WATER CHAMBERS
VACUUM LINE
6mm O.D. CONNECTING TUBE T FORM
10 mm 0.0. U SHAPE SYPHON TUBt
6mm O.D. VACUUM LINE TUBE
STAINLESS STEEL HOSE CLAMP
10 mm I D. CONNECTING TUBES Y FORM
10 mm O.D. DELIVERY TUBE
120 ml BOTTLE VACUUM BLOCK
10 mm O.D. DELIVERY TUBE
10 mm O.D. DELIVERY TUBE
10 mm O.D. AUTOMATIC SYPHON TUBE
EFFLUENT CHAMBERS
10 mm O.D. U SHAPE SYPHON TUBE
10 mm I.D. CONNECTING TUBE Y FORM
10 mm O.D. DELIVERY TUBE
MIXING CHAMBER 1200 ml. CAPACITY
10 mm O.D. DELIVERY TUBE
TEST CHAMBERS CAPACITY
4- ZQ LITERS
FIG. 10. VACUUM SIPHON DILUTOR SYSTEM, DETAILED DIAGRAM.
45
-------
Vacuum Siphon System Equipment List
1. Diluter Glass.
2. Stainle&s^ steel solenoid valves.
a. 2 - normally open, two-way, 55 psi, water, 1/4" pipe size,
9/32" orifice size, ASCO 8262152, for incoming effluent and
dilution water pipes.
b. 1 - normally closed, two-way, 15 psi, water, 3/8" pipe size,
3/8" orifice size, ASCO 8030B65, for dilution water evacuation
pipe.
3. Stainless steel tubing, seamless, 316-grade, austenetic.
a. 60 ft - 3/8" OD, 0.035" wall thickness, for dilution water-and
effluent pipes.
b. 20 ft - 5/16" OD, 0.035" wall thickness, for standpipes in
mixing chambers.
c. 1 ft - 3/4" OD, 0.035" wall thickness, for standpipe in D-l
chamber.
4. Swagelok tube connectors, stainless steel
a. .4 - male tube connectors, male pipe size 1/4", tube OD 3/8".
b. 2 - male tube connectors, male pipe size 3/8", tube OD 3/8".
c. 2 - male adapter, tube to pipe, male pipe size 1/2", tube OD 3/8".
d. 2 - male tube connectors, male pipe size 1/2", tube OD 3/8".
5. 7 - 1,200 ml stainless steel beakers.
6. Several Ibs each of NEOPRENER stoppers, size 00, 0 and 1; 1 Ib of
size 5.
7. 14 - aquariums, 2-5 gal.
8. Magnetic stirrer.
9. 2 - PVC Ball valves, 1/2" pipe size.
10. Dilutor control panel equipment - see Fig. 14 and equipment list.
11. 7 - 120 ml NALGENER bottles.
12. 3 ft, l-in-2 aluminum bar, for siphon support brackets.
13. Stainless steel set screws, box of 50, for securing SS tubing in
siphon support brackets.
14. Stainless steel hose clamps, box of 10, size #4 or 5,(need 3 boxes).
15. 6 - NALGENER T's, 5/16" OD.
16. 12 - TYGONR Y connectors, 3/8" I.D.
17. TYGONR tubing, 3/8" OD, 10 ft.
18. Plywood sheeting, exterior grade: one - 4' x 8x x 3/4",
one - 4' x 8' x 1/2".
19. Pine or redwood board, 1" x 8", 20 ft.
20. Epoxy paint, 1 gal.
21. Assorted wood screws, nails, etc.
22. 25 ft - 5/16" ID, TEFLONR tubing, to connect the mixing chambers to
the test chambers.
46
-------
231mm
A
J N
x
D-l
i 1
B
(
-»
0-2
1
1
D-3
1 !
B
D-4
I 1
D
1 1
-5
B
0-6
1
0-7
1
L
0-8
i i
20 mm HOLE FOR VACUUM
.. .* SIPHON SYSTEM ONLY.
- - (See Fig. 6)
609 mm
20 mm HOLE CENTERED AT INDICATED DISTANCE
oo o o-o o o o o
I—I—. . , . , , , ,—I
33mm 70mm 165 mm 248mm 297mm 350mm 411 mm 477mm 558mm
DRAIN HOLES IN BOTTOM PLATE (C) SHOWN FOR SOLENOID VALVE DILUTOR SYSTEM. FOR VACUUM
SIPHON DILUTOR SYSTEM. SINGLE DRAIN HOLE IS REQUIRED ONLY FOR CHAMBER D-1.
INDIVIDUAL PART SIZE AND NUMBER OF
PIECES USING 6mm (I/4")PLATE GLASS
A 225 mm X. 95mm - 3
B 200 mm X 95 mm - 6
C €09 mm X 95mm - I (Bottom Plate)
0 609 mm X 231 mm - 2 (Side Panels)
INSIDE CELL MEASUREMENTS AND APPROXIMATE VOLUME
D-l 95 mm X 225 mm X 95 mm -2030ml
D - 2 115 mm X 20O mm X 95 mm -2185ml
0-3 40 mm X 20O mm X 95 mm - 76Oml
0-4 45mm X 20O mm X 95mm- 855ml
0-5 50mm X 200 mm X 95mm -950ml
0-6 60 mm X 20O mm X 95 mm - 114 0 ml
0-7 SO mm X 200 mm X 95mm -1140ml
0-8 90mm X 200mm X 95mm-1710ml
";OTE: 1/8" - 316 GRADE AUSTEIMISTIC STAINLESS STEEL
MAY BE SUBSTITUTED FOR GLASS IN PART C.
FIG. II. DILUTION WATER CHAMBERS
47
-------
ISO mm
"1 E
E-l
J M
J E
E-2
I !
1 E
E-3
! 1
1 E
E-4
.11
a
E-5
E-6
,,,l.l
20 mrn HOLE
FOR VACUUM
SIPHON SYSTEM ONLY.
(See Fig. 6)
367 mm
20 mm HOLE CENTERED AT INDICATED DISTANCE
o
o
0
o
o
o
61 mm
154mm 213mm 254mm 290mm 336mn
DRAIN HOLES IN BOTTOM PLATE (C) SHOWN FOR SOLENOID VALVE DILUTOR SYSTEM. FOR VACUUM
SIPHON DILUTOR SYSTEM. SINGLE DRAIN HOLE IS REQUIRED ONLY FOR CHAMBER D-1.
INDIVIDUAL PART SIZE AND NUMBER OF
PIECES USING 6mm ttM'lPLATE GLASS
A I74 mm X 40 mm - 2
B I55 mm X 40 mm - 5
C 367mm X 40 mm - I (Bottom Plate)
D 367mm X 180mm- 2 (Side Panels)
INSIDE CELL MEASUREMENTS AND APPROXIMATE VOLUME
E-l 110mm X 155mm X40mm~682ml
E-2 65mmXI55mmX40mm-403ml
E-3 40mm X l55mmX40mm-H48ml
E'4 30mm X !55mmX 40mm- 186ml
E-5 30 mm X 155 mmX 40mm- 186ml
E-S 50mm X 155 mm X 40 mm - 3 18 ml
NOTE: 1/8" - 316 GRADE AUSTENISTIC STAINLESS STEEL
MAY BE SUBSTITUTED FOR GLASS IN PART C.
FIG. 12. EFFLUENT CHAMBERS
48
-------
M2
I86mm
SIDE VIEW
M-l
f 20 mm
DIAMETER
HOLE
\ 90 mm
-M-3
180 m m
PART M-l
END VIEW
. 20 mm DIAMETER HOLE
20 mm
I O 5 mm
INDIVIDUAL PART SIZE AND NUMBER OF
PIECES USING 6mm (I/4")PLATE GLASS
M-l 180mm X 105 mm-l
M-2 180mm X 105 mm-l
M-3 190mm X 105 mm-l (BOTTOM PLATE)
M- 4 190mm X !86mm-2 (SIDE PANELS)
(APPROXIMATE CAPACITY 3365ml)
FIG. 13. MIXING CHAMBER
49
-------
50
-------
Dilutor Control Panel Equipment List*
Designation
Al
CTR-1
ET
F,
2
L.S.
SJ,
SJ,
CKT Description
Encapsulated amplifier
Cycle counter
Elapsed time indicator
Input power fuse
Recepticle
Aux A.C. output jack
Main input power cord
Fill indicator light
Emptying indicator light
Level sensor (Dual Sensing Probe)
Plug
On-off main power switch (spst)
On-off aux power switch (spst)
Solenoid
Manufacturer
Cutler Hammer 1353H98C
Rodington #P2-1006
Courac #636W-AA H&T
Little fuse 342038
Amphenol 91PC4F
Stand. 3-prong AC Recpt.
Stand. 3-prong AC maleplug
Dialco 95-0408-09-241
Dialco 95-0408-09-241
Cutler Hammer 13653H2
Amphenol 91MC4M
Cutler Hammer 7580 K7
Cutler Hammer 7580 K7
(See Solenoid and Vacuum
System equipment lists)
it ii ii
SJ. - SJ-,
4 16
TDR-1
TDR-2
Additional Solenoids for
Solenoid Valve System
Time delay relay
Aux time delay relay
ii it ii
Dayton 5x829
Dayton 5x829
*Consult local electric supply house.
51
-------
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1 . REPORT NO.
EPA-600/4-78-012
3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
METHODS FOR MEASURING THE ACUTE TOXICITY OF
EFFLUENTS TO AQUATIC ORGANISMS
5. REPORT DATElssued January, 197Ł
Revised July,..!978
i. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
William Peltier
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Bioassay Subcomm., EPA Biological Advisory Committee
Ecology Branch, Surveillance & Analysis Division
U.S. Environmental Protection Agency
Athens, Georgia 30605
10. PROGRAM ELEMENT NO.
1BD612
11. CONTRACT/GRANT NO.
12 SPONSORING AGENCY NAME AND ADDRESS
Environmental Monitoring & Support Laboratory
Office of Research and Development
U. S. Environmental Protection Agency
Cincinnati, Ohio 45268
13. TYPE OF REPORT AND PERIOD COVERED
In-House
14. SPONSORING AGENCY CODE
EPA/600/06
15. SUPPLEMENTARY NOTES
Supplement to "Biological Field and Laboratory Methods for Measuring the
Quality of Surface Waters & Effluent"
16. ABSTRACT
This report describes methods for the measurement of the acute
toxicity of effluents to macroinvertebrates and fish. The methods include a
preliminary short-term (8-24 hr), range-finding (screening) test and a long-
term (96 hr) flow-through, or alternate static, definitive test for use in
determining the LC50 or EC50 of the waste. The report includes guidelines
for effluent sampling and holding, facilities and equipment, dilution water,
test .species selection and handling, and data interpretation.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
Effluents
Bioassay
Toxicity
Industrial Wastes
Sewage
Water Pollution
Fishes
Invertebrates
Fresh Water Biology
Marine Biology
13. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (This ReportI
UNCLASSIFIED
21. NO. Of PAGES
62
20. SECURITY CLASS (This page)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
52
* n-t soYBumar rnfont wnct an— 7 5 7 - n o /13 5 a
-------
Appendix B
TOXICITY TEST REPORT
I. Sumnary and Recommendations
A. State lethality of effluent in terms of LC50 (or EC50).
B. Review toxicity in receiving waters - IWC.
C. Recommend appropriate action, e.g. change in permit
requirements, self-monitoring, use of diffusers, process
alterations, alternate disposal methods, pretreatment, etc.
II. Introduction
A. Dates of study.
B. Dischanrge Serial Number
C. Principal investigators
D. Receiving water - include 7Q10 or, if tidal, with
approximate salinity, amplitude range.
E. Trailer location.
F. Industrial representatives.
G. State, Federal or other observers to the study.
H. S.I.C. Code number
Plant Operation
A. Schedule of operation.
III. B. Major products and raw materials (enter SIC code).
C. Wastewater treatment schedule - periodic or continuous.
D. Daily wastewater flow rates for week prior to study and
during study.
B-l
-------
E. Retention time of effluent - arithmetic or actual.
F. Description of outfall and receiving water - submerged
diffuser, etc.
G. Diagram of wastewater treatment facilities.
H. General maintenance of plant area (as pertains to the
potential for contaminating runoff).
I. Description of industrial process.
J. Estimated effluent composition.
IV. Toxicity Test Methods
A. Citation of methods.
B. Explain LC50 (or EC50) data analyses used.
C. Organisms used in test: source, age and/or length and weight,
health conditions.
D. Brief description of physical test facilities:
1. Test chamber capacity.
2. Aquarium turnover rate.
3. Use areation.
4. Volume of test solution (statics).
E. Chemical analyses run (wet-lab and monitor) on effluent,
dilution water and samples of test solutions.
F. Type of biomonitoring test used (e.g. static, flow-through,
etc.
G. Standard toxicant used.
V. Sampling Procedure
A. Effluent
1. Location and description of sampling point.
B-2
-------
2. Note the date and time of each composite or grab sample
for test or analytical purposes was recorded.
3. Note the physical and chemical parameters for which data
was recorded.
4. Note types of instruments used for specific parameters
and include identification labels or serial numbers.
B. Dilution water
1. Type, origin and composition if known, of dilution water
used.
2. Note if a fresh/salt-water mixture was needed to
achieve test salinity.
3. Note for each sample collected:
a. Location and description of sampling point.
b. Date and time.
c. DO, temperature, alkalinity, hardness
(freshwater),and salinity (salt water). (In marine
work, these parameters should be reported for the
freshwater, saltwater, and resulting mixture if the
dilution water is salinity-adjusted by mixing.)
C. Document control numbers
1. Sample tag numbers
2, Chain of custody record number
Results
VI. A. Report toxicity results of effluent - include LCSO's (UCL and
LCL) and list 24, 48, 72, 96 hour LCSO's where appropriate (or
EC50).
B-3
-------
B. Report characteristics of effluent, dilution and receiving
waters (color, odor, solids, etc.)
C. Report data, in tabular form from physical and chemical
analyses recorded throughout test.
D. Report data or trends observed from continuous monitor
tracings.
VII.Discussion
A. Describe events which may have affected results of toxicity
tests such as aquarium turnover rate, treatment upsets, power
failure, aeration, rainfall, etc.
B. Describe condition of live test organisms during and at the
end of the test.
C. Relate chemical data to toxicity where applicable.
D. Calculate in-stream wastewater concentrations using dilution
ratios, application factors 0.05 and 0.01 LC50 values to determine
if acute or chronic toxicity exist in the receiving waters.
B-4
-------
Appendix C
INSTRUCTIONS FOR COMPLETING THE ACUTE
TOXICITY LABORATORY EVALUATION FORM
1. Laboratory or Industry - Enter the complete name of the
laboratory or industry conducting the acute toxicity test.
l.a. Industry SIC Code - Enter this number and briefly describe
the type of industry, raw materials used,and estimated effluent
composition if available.
2. Location - Enter the address of the laboratory or industry
conducting the acute toxicity test.
2.a. NPDES Permit No. - Enter the corresponding number and other
necessary permit identification such as date of issuance and
expiration.
3. Date - Enter date of evaluation.
4. Investigator - Enter name and title of person conducting
evaluation.
5. Company Representative - Enter name of person(s) interviewed
and telephone number (if available).
6. Test Method - Enter brief narrative of the test being
conducted and the reference where written instructions on the
methodology appears (i.e. 96-hour static bioassay; or reference: EPA
660/3-75-009 April, 1975).
7.a. Dilution Water - Source - Enter the source of the dilution
water; the date and time of its collection.
7.b. Dilution Water; Chemical Analyses Performed - Enter specific
C-l
-------
chemical tests performed on dilution water if any. Also enter
chemical characteristics recorded by the analyst (average and/or range
values).
7.c. Dilution Water: Pretreatment - Enter a description of any
pretreatment of dilution water.
8.a. Effluent Water: Source - Enter the source of the effluent to
be tested, the date and time of its collection.
8.b. Effluent Water: Variability - Enter a description of the
physical or chemical variability of the effluent (i.e. constant flow of
effluent from a lagoon with 14-days detention time or batch process
releasing effluent having variable flow and chemistry directly into the
receiving water).
8.c. Effluent Water: Sampling Technique - Enter a brief
description of the method used to collect the sample(s) of effluent.
8.d. Effluent Water: Holding time and Conditions - Enter the
amount of time and conditions under which the test effluent is held
before being used in the toxicity study.
8.e. Effluent Water: Pretreatment - Enter a description of any
pretreatment of the effluent.
8.f. Effluent Water: Chemical Analyses Performed - Enter specific
chemical tests performed on effluent. Also enter chemical
characteristics recorded by the analyst (average and/or range values).
9.a. Test Organism: Species - Enter the common and scientific
name of the test organism.
9.b. Test Organism: Life State - Enter the age, life stage, as
well as length and weight (if apporpriate) of the test organism.
C-2
-------
9.c. Test Organism: Source - Enter the specific source of the test
organism; the date and time of the collection (i.e. Brown Fish
Hatchery, Central City, Iowa; collected 0800 hours on January 10,
1978).
9.d. Test Organism: Holding Facilities - Enter a brief description
of the facility used to hold test organisms prior to the biomonitoring
study (i.e. 500-gallon Minnow-Kool tank with flow-through dechlorinated
tap water).
t~
9.e. Test Organism: Aclimation Procedure - Enter a brief
A,
description of the procedure used to acclimate the test organism to
laboratory conditions prior to biomonitoring tests.
9.f. Test Organism: Treatment - Enter any observed diseases and
specific treatment rendered if any. State the number of treatments and
dates.
10.a. Experimental Design: Equipment Cleaning Procedure - Enter a
brief description of step-by-step pre-cleaning procedure for equipment
(tanks, etc.) used in biomonitoring tests. List trade name and
scientific name of cleaning compounds (if available).
lO.b.(l) Experimental Design: Test Chambers: Construction Material -
Enter the type of material used in constructing the test chambers.
10.b.(2) Experimental Design: Test Chambers: Dimensions - Enter the
specific size of the test chambers (length, width, height).
10.b.(3) Experimental Design: Test Chambers: Volume - Enter the
designated volume of the test chambers as well as the specific depth
and volume of solution used during the biomonitoring test.
10.b.(4) Experimental Design: Test Chambers: Volumetric Exchange Rate
- Enter the rate of exchange of test solution in flow
through/continuous-flow test chambers.
C-3
-------
10.c. Experimental Design: Test concentrations - Enter a list of
solution concentrations in which test organisms were exposed.
10.d. Experimental Design: Number of organisms per concentration -
Enter the number of test organisms exposed to each concentration of
test solution.
lO.e. Experimental Design: Loading Rate - Enter the weight of test
organisms per liter of test solution (i.e. 5 grams/liter).
19.f. Experimental Design: Test Temperature - Average and Range -
Enter the temperature (average and range) of the solution in which test
organisms are exposed during the biomonitoring study.
lO.g Experimental Design: Chemical Parameters Monitored and
Frequency - Enter the type of chemical tests performed and the
^.H
frequency which each chemical test is performed during the
biomonitoring study.
lO.h. Experimental Design: Duration and Frequency of Test - Enter
the time period of the biomonitoring test and the number of times the
biomonitoring test is performed each year. (Record both as
"performed" and "as required in NPDES permit").
10.i. Experimental Design: Definition of adverse effect - Define
the endpoint of the biomonitoring test (i.e. death).
10.j. Experimental Design: Frequency of Observations - Enter the
time intervals when test organisms were observed during the
biomonitoring study (i.e. observed each 12-hour period).
10.k. Experimental Design: Method of calculating EC50 or LC50 -
Enter the name of the calculation procedure used and the reference
citation.
C-4
-------
10.1. Experimental Design: Special Conditions - Briefly describe
test conditions not addressed elsewhere in this questionnaire (i.e.
dead organisms not removed during the test; or test chambers aerated
continuously with pure 02 during test). Attach a supplement sheet if
needed.
11. Methods Used for All Chemical Analyses - Enter the reference
cited for the chemical analyses performed during the biomonitoring
study.
12. Other Relevant Information - Enter explanations of infor-
mation provided elsewhere in the Acute Toxicity Laboratory Evaluation
questionnaire or other pertinent information not presented in the audit
questionnaire (i.e., quality assurance program, training and experience
of analyst, adequacy of laboratory equipment and facilities, etc.).
C-5
-------
Appendix C
ACUTE TOXICITY LABORATORY EVALUATION FORM
1. Laboratory or industry
a. Industry or SIC Code_
2. Location
a. NPDES Permit No.
3. Date
4. Investigator
5. Company Representative^
6. Test Method
7. Dilution Water
a. Source
Date: Time
b. Chemical Analyses Performed
C-6
-------
c. Pretreatment
8. Effluent Water
a. Source
b. Variability
c. Sampling Technique_
d. Holding time and conditons_
e. Pretreatment
f. Chemical analyses performed
9. Test Organism
«
a. Species
b. Life stage_
c. Source
d. Holdinq facilities
C-7
-------
e. Acclimation Procedure
f. Treatment (schematic or flow chart, if available)
10. Experimental Design
a. Equipment Cleaning Procedure
b. Test Chambers
(1) Construction material^
(2) Dimensions
(3) Volume
(4) Volumetric exchange rate_
c. Test concentrations
d. Number of organisms per concentration
e. Loading rate_
f. Test temperature - average and range_
g. Chemical parameters monitored and frequency
h. Duration and frequency of test
C-8
-------
i. Definition of adverse effect
j. Frequency of observations_
k. Method of calculating EC50 or LC50_
1. Special conditions_
11. Methods used for all chemical analyses
12. Record Keeping_
13. Other relevant information
C-9
-------
Appendix D
I. General Compliance Sampling Inspection Daily Activities
The following is a suggested day-to-day inspection activities check
list:
A. Day 1
1. Get power connected to mobile laboratory.
2. Level and stabilize laboratory.
3. Collect dilution water.
4. Begin acclimation (approximately 2 tank volumes in 24
hours) (Appendix A, page 13).
5. Set up static range finding test (Appendix A, page 19).
6. Make necessary entries in logbooks and fill in necessary
forms.
B. Day 2
1. Check results of range finding test and make necessary
logbook entries.
2. Assemble dilution board and delivery system.
3. Calibrate dilution board.
4. Activate diluter and begin filling test tanks.
5. Cease flow to acclimation tank.
6. If a composite sample is to be used for the static test;
the compositer should be set up this day.
7. Collect dilution water.
8. Make all necessary logbook entries.
D-l
-------
C. Day 3
1. Check all systems to ascertain that all have worked
overnight.
2. Check temperatures to see if the acclimation temperature
and test temperature are approximately the same.
3. Start the pump in the circulating water bath and turn
the thermal equilizing unit on.
4. Collect the sample for the static test whether it be
grab or composite.
5. Set up static test tanks.
6. Perform temperature, D.O., pH, and conductivity readings
in all test containers.
7. Introduce the test organisms to both the static and
flow-through test containers.
8. Collect additional dilution water.
D. Days 4, 5, and 6
1. Check all systems to ascertain that all have worked
overnight.
2. Record test organism mortality in all test containers
and remove dead organisms where appropriate.
3. Perform length and weight measurements on dead fish
(make necessary logbook entries).
4. Calibrate the appropriate meters and take meter
readings.
5. Collect dilution water.
6. When scheduled, conduct a compliance biomonitoring
evaluation inspection (see section IV C.)
D-2
-------
7. Make all necessary logbook entries.
E. Day 7
1. Check all systems to ascertain that all have worked
overnight.
2. Record test organism mortality in all test containers
and remove the dead organisms where appropriate.
3. Calibrate the appropriate meters and take meter
readings.
4. Recalibrate diluter board.
5. Make all necessary entries in logbooks.
6. Dismantle laboratory and secure equipment.
7. Inform permittee of your departure and sign out with
gate security guard.
D-3
-------
Appendix E
Sample Tags and Chain of
Custody Form
-------
Appendix E
Page 1 Qf 2
SAMPLE TAG*
0
Proj. Coda
Station No.
Sequence No,
Mo./Day/Yr.
Station Location
ENVIRONMENTAL
OFFICE OF
PROTECTION AGENCY
ENFORCEMENT
Com p.
Time
Grab
H*
cn
o
M
Samplers: (Signature)
obverse
Sample Type. Preservative^)
1. General Inorganics/Ice
2. Metals/HNO,
3. Nutrients/H.,S04 & Ice
4. Oil & Grease/H,S04 & Ice
5. Phenclics/H.PO, & CuS04 & Ice
6. Cyanide/NaOH & Ice
7. Organic Characterization/Ice
8. Volatile Organics/lce
9. General Organics/lce
10. Tracer/None
11. Solids-Inorganics/Ice or Freeze
12. Solids-Organics/lce or Freeze
13. Biol. - Inorganics/Ice or Freeze
14. Biol.-Organics-Ice or Freeze
15. Source Filter/None
16. Frcbe Wash, None
17. Irupinger Catch/ None
IS. Ambient Filter/None
19. Solid Adsorbant/lce or Freeze
20. Ambient Impinger/Amb. or Ice
21. Benthos/ Ethanol or Formal
22. Bacteriology/Ice
23. Plankton/Formal; HgCI,; Lugol's
24. Chlorophyll/Ice or Freeze
25. Pathogenic Bacteria/Ice
26. Bioassay
Remarks:
reverse
•Modified and adopted from: NEIC Policies and Procedures Manual, May 1978. - Denver, CO
-------
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6. Cyanide
7. Organic Characterization
S. Volatile Organics
9. General Organics
10. Tracer
11. Solids - Inorganics
12. Solids - Organics
13. Biol - Inorganics
14. Biol •> Organics
15. Source Filter
16. Probe Wash
17. Impinger Catch
18. Ambient Filler
19. Solid Adsorbanl
20. Ambient Impinger
21. Benthos
22. Bacteriology
23. Plankton
24. Chlorophyll
25. Pathogenic Bacteria
26. Blo»a««y
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-------
APPENDIX F
DOCUMENT CONTROL
The goal of the NEIC Document Control Program is to assure that
all documents for a specific project issued to. or generated by NEIC
personnel will be accountable when the project is completed. This
program includes a serialized document number system, a document
inventory procedure, and a central filing system, all under the
supervision of a Document Control Officer (DCO).
Accountable documents used or generated by NEIC employees include
items such as logbooks, field data records, correspondence, sample
tags, graphs, chain of custody records, bench cards and photos (see
page 11-27 for a more complete list). Each document bears a serialized
number and is listed, with the number, in a project document inventory
assembled by each Branch at the project's completion.
Unless prohibited by weather, waterproof ink is used in recording
all data on serialized accountable documents.
SERIALIZED DOCUMENTS
The DCQ is responsible for assigning the necessary serialized
NEIC documents to project personnel for field activities. Once a
Project Coordinator is appointed, all field logbooks, field data
records, field laboratory logbooks, sample tags and chain-of-custody
records are assigned to this person. The Coordinator is responsible
for ensuring that a sufficient supply of documents is obtained for an
investigation and that these documents are properly distributed to
the appropriate personnel. The DCO provides the Project Coordinator
F-l
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with a list of all serialized project documents that were assigned to
personnel for field activities.
PROJECT LOGBOOKS
The logbook of the Project Coordinator will document the transfer
of logbooks to the individuals who have been designated to perform
specific tasks on the survey. All pertinent information should be
recorded in these logbooks from the time each individual is assigned
to the project until the project is completed.
Logbook entries should be dated, legible and contain accurate
and inclusive documentation of an individual's project activities.
Since the logbook forms the basis for the later written reports, it
must contain only facts and observations. Language should be objec-
tive, factual and free of personal feelings or other terminology
which might prove inappropriate. Entries made by individuals other
f
than the person" to whom the logbook was assigned are dated and signed
by the individual making the entry.
Field analysts who conduct their assigned project analyses in a
mobile laboratory are assigned a Branch logbook bythe Chemistry
Branch. In addition to information documenting the analysis performed,
field analysts document in their logbooks the date and results of any
*
calibration of mobile laboratory equipment. A record is also kept of
any incidents related to the survey; for example, the electricity
going off in the lab, tampering with government vehicles or equipment,
etc. When appropriate, visitors to the mobile lab, such as facility
personnel, are noted in the logbook.
All project logbooks are the property of NEIC and ares to be
turned over to the Project Coordinator when a survey assignment has
been concluded.
F-2
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FIELD DATA RECORDS
Where appropriate, serialized Field Data Records (in the form of
individual sheets or bound logbooks) are maintained for each survey
sampling station or location. The Project Coordinator numbers the
FDR's with the appropriate project code and station number. All
in-situ measurements and field observations are recorded in the FDR's
with all pertinent information necessary to explain and reconstruct
sampling operations. Each page of a Field Data Record is dated and
signed by all individuals making entries on that page. The Coordinator
and the field team on duty are responsible for ensuring that FDR's
are present during all monitoring activities and are stored safely to
avoid possible tampering.
SAMPLE IDENTIFICATION DOCUMENTS
Assignment of all serialized sample tags to field personnel is
recorded in the Project Coordinator's logbook. Individuals are
accountable for each tag assigned to them until it has been filled
out, attached to a sample, and transferred to another individual with
the corresponding Chain-of-Custody Record. At no time are any
sample tags to be discarded; if any of these forms are lost, voided
or damaged, it is noted in the appropriate FDR or logbook immediately
upon discovery. Tags attached to those samples split with the facil-
ity or another government agency will be accounted for as described
below.
At the completion of any reconnaissance or field-sampling investi-
gation, all unused sample tags are returned to the Project Coordinator
by the individual to whom they were originally assigned. This individual
lists the serial numbers of the returned items in the Coordinator's
logbook and signs and dates the transfer.
F-3
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CHAIM-OF-CUSTODY RECORDS
All serialized Chain-of-Custody Records are assigned and accounted
for in a manner similar to that for the sample tags as described
above. When samples are transferred from a field sampler or courier
to field laboratory personnel, the analyst, after signing, retains
the white (original) custody record and files it in a safe place.
The copy of the custody record is returned to the Project Coordinator.
A similar procedure is followed when dispatching samples via common
carrier, mail, etc., except that the original accompanies the shipment
and is signed and retained by the receiving laboratory sample custodian.
When samples are split with the facility or another government
agency, the separate custody record that is prepared (see page 24) is
labeled to indicate this. In addition, the serial numbers from all
the tags are recorded on the custody record. The person relinquishing
the samples to the facility or agency should request the signature of
a representative of the appropriate party, acknowledging receipt of
the' samples. -If a representative is unavailable or refuses to sign,
this is noted in the "received by" space. When appropriate, as in
the case v/here the representative is unavailable, the custody record
should contain a statement that the samples were delivered to the
designated location at the designated time. The copy of the custody
record may be given to the facility or agency upon request; all white
originals are returned to the Project Coordinator.
ANALYST, INSTRUMENT AND SAMPLE ENTRY LOGBOOKS
Logbooks and data sheets that are used for various purposes
(chemical or biological analyses, equipment calibration, etc.) within
the NEIC laboratories are not handled by the DCO, but rather are
accountable by practices instituted by individual Branches.
F-4
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All laboratory observations and calculations not recorded on
serialized'bcnch cards, instrument graph printouts, etc., are entered
in serialized logbooks assigned by a Branch file custodian. The
logbook should contain information sufficient to recall and describe
succinctly each step of the analysis performed should the analyst be
required to testify in subsequent enforcement proceedings. Sufficient
detail should be provided to enable others to reconstruct the analysis
should the analyst not be available to do so. Any irregularities
observed during the testing process should be noted. If, in the
technical judgment of the analyst, it is necessary to deviate from a
particular analytical method, the deviation shall be properly justified
and documented.
When an individual is assigned a logbook for use en a variety of
projects, each page contains information about only one project and
is labeled with the project code, dated, and signed by the individual.
All bench cards, instrument printouts, and other separate documents
are labeled similarly. Notes (taken at meetings, from research articles,
etc'!) which do not relate to a particular NEIC project shall not be
kept in the assigned logbook. When a laboratory logbook is completed,
it is returned to the Branch file custodian and a new logbook is
issued. The custodian or other appropriate staff member maintains an
inventory sheet for the logbook, listing the project code for each
page. These books that have been completed and turned in are used
for reference purposes only.
Where applicable, the Branch file custodian issues a serialized
instrument logbook in which all information relating to calibration
and maintenance of a particular laboratory instrument is recorded. A
serialized sample entry logbook is used in the laboratory to record
the entry of the samples to the laboratory or laboratory instrument
for analysis. Again, each page should contain information about one
project only.
F-5
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PHOTOGRAPHS
When movies, slides or photographs are taken which visually show
the effluent or emission source and/or any monitoring locations, they
are numbered to correspond to logbook entries. The name of the
photographer, date, time, site location, and site description are
entered sequentially in the logbook as photos are taken. Once devel-
oped, the slides or photographs should be serially numbered corre-
sponding to the logbook descriptions.
CORRECTIONS TO DOCUMENTATION
As previously noted, unless prohibited by weather conditions,
all documentation in logbooks, FDR's, sample tags, custody records
and other data sheets are filled out with waterproof ink. None of
the accountable serialized documents listed above are to be destroyed
or thrown away even if they are illegible or contain inaccuracies
which required-a replacement document.
If an error is made in a project logbook assigned to one indivi-
dual, that individual may make corrections simply by crossing a line
through the error and entering the correct information. Changes made
subsequently are dated and initialed. If an error is discovered on a
sample tag, custody record or FDR, when possible the person who made
the er^or should correct it. Corrections or insertions are made by
inserting the word or abbreviation for "corrected," the date, and the
correcting person's initials beside the correction. The procedure
applies to words or figures inserted or added to a prior recorded
statement.
If a sample tag is lost in shipment, or a tag was never prepared
for a sample(s), or a properly tagged sample was not transferred with
F-6
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a formal NEIC Chairrof-Custody Record, the following procedure applies.
A written statement is prepared detailing how the sample was collected,
air-dispatched or hand-transferred to the field or NEIC laboratory.
The statement should include all pertinent information, such as
entries in field logbooks regarding the sample, whether the sample
was in the sample collector's physical possession or in a locked
compartment until hand-transferred to the" laboratory, etc. Copies of
the statement are distributed to the Project Coordinator, the Assistant
Director for Technical Programs and the appropriate Branch project
files.
CONSISTENCY OF DOCUMENTATION
Before releasing any analytical sample results to the Project
Coordinator, the Chemistry and/or Biology Branches assemble and
cross-check information on corresponding sample tags, custody records,
bench cards, analyst logbooks and sample entry logbooks to ensure
that data pertaining to each particular sample is consistent through
out the record. A statement that all project evidentiary data in the
Branch's possession has been accounted for accompanies the transfer
of any analytical data from the NEIC laboratories to the Project
Coordinator.
The Project Coordinator then conducts a cross-check of evidentiary
data in his possession (FDR's, logbooks, custody records, etc.) to
ensure that information recorded corresponds to information from each
of the Branch laboratories and is consistent throughout the project
record.
F-7
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DOCUMENT NUMBERING SYSTEM AND INVENTORY PROCEDURE
In ordar to provide document accountability to the appropriate
individuals, each of the document categories discussed above features
a unique serialized number for each item within the category. Logbooks,
FDR's, sample tags and custody records are serially numbered by the
DCO before assignment to project personnel. The logbooks and FDR's
are usually given a five-digit number, with the project code as the
first three digits followed by a two-digit document number. Sample
tags and custody records are labeled with a four digit document
number and the project code appears elsewhere on the document. All
Branch documentation not covered by the above (logbooks, data sheets,
graphs, etc.) are uniquely and serially numbered using the project
code as part of the number when appropriate.
All other documents (such as recorder graph paper, data calcula-
tion sheets, memos, correspondence, photos, etc.) which are generated
during a project are sequentially numbered with the project code, the
Branch initiate and a serialized number (e.g., 707-CH-01), usually at
the time the Branch file is assembled.
BRANCH FILES
After a Branch has completed its work for a particular investi-
gation, all- documents generated from that project should be assembled
in the Branch file. Individuals may retain clean (no handwritten
comments) copies of documents for their personal files but only after
personally verifying that the original or similar copy is in the
Branch file. The Chief of each Branch in Technical Programs is
responsible for assuring the collection, assembly, and inventory of
all documents relative to a particular project at the time the project
objectives are completed. The file then becomes accountable. Any
records leaving the file must be signed out.
F-8
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CENTRAL FILE
When the NEIC has completed the project objectives, all inven-
toried Branch file documents are reviev/ed and submitted to tha Central
File by each Branch Chief. By this time each document will have been
labeled with a unique serialized number as specified above. The
format of the Central File covers the following document classes:
A. Project Logbooks
B. Field Data Records
C. Sample Identification Documents
D. Chain-of-Custody Records
E. Analytical Logbooks, Lab Data, Calculations, Bench Cards,
Graphs, etc.
F. Correspondence
1. Intra-office
2. EPA
3. Industry
4. Record of Confidential Material
G. Report Notes, Calculations, etc.; Drafts
H. References, Literature
I. Sample (on-hand) Inventory
J. Check-out Logs
K. Litigation Documents
L. Miscellaneous - photos, maps, drawings, etc.
Once deposited in the Central File, documents may only be checked
out through the DCO or designated representative.
REPORTS
All draft reports are numbered and accountable. They are stamped
DRAFT REPORT FOR AGENCY REVIEW ONLY, DO NOT DUPLICATE on the cover
page. .The author is responsible for disseminating draft reports for
internal NEIC review, and preparing a memorandum for the Assistant
Director for Technical Programs to transmit copies to Regional Offices,
F-9
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Headquarters, etc. All draft copies of the report are to be returned
to the author. Once comments have been incorporated and the final
report has been prepared, all draft copies are disposed of. However,
Regional Offices may retain a copy of the draft report with their
comments until they receive the final report at which time the draft
will be returned to the NEIC.
LITIGATION DOCUMENTS
Any court documents, litigation reports, letters, memos, etc.
from the Chief, Enforcement Specialist Office, EPA Regional Office(s)>
State Pollution Control Offices, etc., which discuss legal matters
or strategies, should be placed in a separate file folder (see Central
File format) which is reviewed by the Enforcement Specialist Office
at the appropriate time.
CONFIDENTIAL IRFORMATION
Any information received by NEIC with a request of confidentiality
is handled as "confidential." A separate, locked file is maintained
in the Central File room for the segregation and s'torage of all
confidential and trade-secret information. Upon receipt by NEIC,
this information is directed to and recorded in the Confidential
Inventory Cog by the DCO. The information is then made available to
NEIC personnel, but only after it has been logged out. The informa-
tion should be returned to the locked file at the conclusion of each
working day. Confidential information may not be reproduced except
upon approval by and under the supervision of the DCO. Any reproduction
should be kept to an absolute minimum. The DCO will enter all copies
into the document control system and apply the same requirements as
for the original. In addition, this information may not be entered
F-10
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Appendix G
DEFINITIONS
Acclimation - The process of adjusting to environmental changes.
Acute Toxicity - Short-term effect of a toxicant on test organisms.
Death is the end point in acute toxicity tests.
Announced Inspection - Is the type of inspection in which the permittee
is made aware of the exact dates in which the inspection is
to take place.
Application Factor (AF) - Is an indirect method used in estimating the
maximum allowable toxicant concentration (MATC) or the safe
concentration (SC). Application factors are generally
estimated but their true value is the ratio between MATC and
the incipient LC50 or: AF = MATC
Incipient LC 50
(See pages 686 and 689 of the 14th Edition of Standard
Methods).
Audit (or Performance Audit Inspection-PAI) - Is a non-sampling type
inspection that assesses all the elements of a permittee's
self-monitoring program while they are being performed. This
includes review of quality assurance, sample collection,
files and analytical laboratory data.
Bioassay - Is a test which utilizes any biological system to detect or
measure the presence or effect of one or more substances,
waste, or environmental factors alone or in combination.
Biomonitoring - For this manual's purposes, biomonitoring refers to
acute toxicity bioassays performed for the NPDES program.
Chain of Custody - Includes all the administrative procedures directed
at protecting and certifying the integrity and therefore the
acceptability of evidence in a legal proceeding.
Chronic Toxicity - Long-term effect of a toxicant on test organisms.
Effective concentration (EC) as determined by sublethal
behavioral or physiological response of the test species is
the end point.
Composite Sample - The type of sample made out of several discrete
samples collected either at equal time intervals or
proportional to the flow rate.
G-l
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over the compositing period (see pages 28 to 32 of EPA's
NPDES Compliance Sampling Manual, June 1977).
Definitive Test - Full scale bioassay consisting of at least five
different concentrations of effluent in and exponential
series with each concentration and control being tested
against no less than 20 organisms of a given species.
Dilution Water - Mixing water to be used for preparing the
different test exponential dilution series of the
effluent. This water is usually collected from a point
as close as possible but away from the zone of influence
of the effluent's discharge.
Discharge Monitoring Report (DMR) - Form used by NPDES permittees
to report the results of their self-monitoring which is
required by their NPDES permit, must be submitted to the
respective NPDES permitting authority at least
quarterly. At present EPA Form 3320-1 (Rev. 10-77) is
used for reporting purposes.
Document Control - Administrative procedures used for the purpose
of tracking and maintaining adequate records of all
documents issued by or generated by a particular program
(see Appendix G of this manual).
EC50 - Or median effective concentration is the concentration
producing a specific response, other than death, in 50%
of the test organisms. Responses can be behavioral, a
developmental abnormality, or a deformity.
Effluent - For the purposes of this manual, is an outflow from a
point source with some of its physical, chemical, and
biological parameters being regulated by an NPDES
permit.
Evaluation Inspections - Inspections that involve a review and
evaluation of all self-monitoring and better records
required by an NPDES permit.
Flow-through Bioassay - Or continuous flow bioassay is the type of
test where different concentrations of the effluent are
prepared by mixing it with adequate quality dilution
water and then tested by allowing such effluent
concentrations to flow at predetermined rates into
chambers containing the test organism.
Grab Sample - Individual sample collected over a period of time
not to exceed 15 minutes.
Incipient Lethal Level - The concentration at which acute toxicity
ceases, that is, the concentration at which 50% of the
test organism's population can live for an indefinite
ti me.
G-2
-------
In-stream Waste Concentration (IWC) - The concentration (expressed as
percent) of permittee's waste in the receiving stream at the
7Q10.
Lethal Units (LU) - Normally given in LU per gallon (LU/g) are defined
as LU/g = 100%
LC50%
Maximum Allowable Toxicant Concentration (MATC) - The concentration of
toxic waste that may be present in the receiving water
without causing significant harm to its productivity and
uses. Usually determined by a long-term bioassay (from egg
to egg or beyond).
Monitoring - In this manual, the term refers to overview by actual
sampling and/or evaluation of NPDES permittee's compliance
with permit conditions.
NPDES or National Pollutant Discharge Elimination System - Refers to
permit system developed by EPA under the authority granted by
section 402 of the Clean Water Act.
Quality Assurance (QA) - For this manual's purposes refers to all
scientific tests and administrative procedures used to ensure
the scientific and legal validity of results obtained from
biomonitoring tests.
Range Finding Test - Refers to a short-term (8-24 hours) flow-through
or static bioassay (usually static) used for determining the
approximate concentrations, above and below the LC50, to be
used in the definite test. In this test, groups of five
organisms are exposed from three to five widely spaced
effluent dilutions.
Sampling Point - Particular site whose location may be specified in a
permit and from which effluent samples are to be collected
for testing and evaluation.
Standard Toxicant - Toxic reference material used for QA purposes in
the biomoni toring program. Its main functions are to
determine the reproducibility of test results and differences
in sensitivity among batches of test organisms.
Unannounced Visits - Inspections where the permittee is not given
notice of the exact date on which the inspection will take
place except through a general 308 letter.
7Q10 - The once in 10 year, seven day consecutive low flow of a
stream.
G-3
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Appendix H
FACTORS USEFUL IN SELECTING CANDIDATES FOR INSPECTION
Any consideration of factors useful in prioritizing biomonitoring
resources for enforcement purposes must involve two types of activities:
0 Problem area monitoring - a reaction program with candidates
selected on the basis of known problems.
Several factors may trigger the need for a compliance biomoni-
toring inspection at an NPDES permitted facility including:
0 Fish kills in receiving waters
0 Permit violations associated with discharges containing
potentially toxic substances.
0 Citizen complaints
0 Questionable or inadequate self-monitoring data
0 Biological monitoring studies indicating that a receiving body
of water is not supporting a productive and diverse biota when it
normally should.
The actual use of resources to investigate problem areas is
normally based on the judgment of the enforcing agency on a case by case
basi s.
H-l
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0 Preventive monitoring - a carefully planned long-term program
aimed at detecting potential problems before they occure and/or
detecting problems that are not readily apparent. Problem area
biomonitoring activity has first priority.
A preventive monitoring program should result in a lower incidence
of problems related to the discharge of toxic substances.
The following problems should be considered when selecting sites
for bioassay inspections:
0 Industrial category - this is the first order of importance in
evaluating the potential for discharge of toxic substances by a
permittee. This also includes municipals receiving industrial
wastes.
° Instream waste concentration (IWC) - for a given industrial
type the potential for toxicity of a waste discharge to aquatic
life is a function of concentration. For flowing waters a measure,
or estimate of the 7Q10 (see section VI A.) flow should be used in
calculating the IWC. In lakes and estuaries the permittees should
be ranked on the basis of the volume of the waste discharged. In
most cases, any discharges to lakes or estuaries should be
considered more critical than discharges to rivers.
H-2
-------
0 Quality of receiving water - As a first approximation the
State-Federal water quality standards for the receiving waters
should be determined and the receiving waters ranked in order of
highest use.
0 Status of permit - The permittees should be ranked on the basis
of the time remaining before an existing permit is to expire.
Those permits whose expiration date is earliest would receive
highest priority.
H-3
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APPENDIX I
Conduct of Inspections After the Barlow's Decision;
Development of Neutral Administrative Inspection Schemes
-------
\
^| UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
^ WASHINGTON. D.C. 20460
11 APR 1979
OFFICE OF ENFORCEMENT
MEMORANDUM
TO: Deputy Assistant Administrators for Enforcement
FROM: Assistant Administrator for Enforcement
SUBJECT: Development of Neutral Administrative Inspection Schemes
It is essential that all enforcement programs develop, as soon
as possible, neutral schemes for the conduct of all inspections. This
requirement is mandated by the decision of the Supreme Court in
Marshall v. Barlow's, Inc., U. S. , 98 S. Ct. 1816 (1978). As you
know, the Barlow's decision addressed the issue of the need for and use
of warrants in conducting administrative inspections under various regula-
tory schemes. The Court stated, in general, that a warrant was necessary
when requested by the owner or person-in-charge of the establishment to be
inspected, but that the warrant need not be based on a showing of criminal
probable cause. Father, a warrant would be issued if it could be shown that
the establishment was being inspected pursuant to a neutral administrative
scheme.
The Department of Justice and the Office of General Counsel agree
that there is an urgent need for each compliance nonitoring program to develop
a neutral administrative schene for inspections, and that such schep.es must
be put into writing. If the schemes are not documented until an inspection
warrant is sought, they will appear to be post hoc rationalizations for
ill-conceived or harassing inspectional programs and will not be favored by
the courts.
I believe that most of our compliance monitoring programs have
developed inspection programs which utilize neutral criteria. In some
instances, however, the total neutral scheme for each program has not been
formally docunented. Examples of neutral criteria which could satisfy the
requirements of Barlow's are random selection; inspecting all or a fixed
percentage of certain types of facilities on an annual basis; ranking
inspections by the amount of controlled chemical substances manufactured,
processed or distributed in commerce; re-inspection of establishments which
exhibited prior violations; and other reasonable factors that show that the
establishment being inspected has not been selected for any arbitrary or
invidious reason. The Office of General Counsel will cooperate with the
Office of Enforcement in assuring that the neutral administrative schemes
developed by each prograna are compatible with the Barlow's decision.
-------
-2-
I am sending to the Regions a guidance document, in which you have
already concurred, entitled "Conduct of Inspections after the Barlow*s
Decision." The document informs the Regions that inspections, other
than those based upon probable cause, must be based on a neutral
administrative scheme, and that such schemes are being prepared at
Headquarters for each compliance monitoring program. As new enforce-
ment programs are developed, a neutral administrative inspection scheme
must be developed in writing by the time of program implementation.
tfor programs already in existence, a written neutral administrative
schema should be developed, and submitted to the Office of General
Counsel, by May 31, 1979.
Marvin B. Burning
-------
| UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON. D.C. 20460
11 APR 1979
MEMORANDUM OFF1CE OF ENFORCEMENT
TO: Regional Administrators
Surveillance and Analysis Division Directors
Enforcement Division Directors
FROM: Assistant Administrator
for Enforcement
SUBJECT: Conduct of Inspections After the Barlow's Decision
I. Summary
This document is intended to provide guidance to the Regions in
the conduct of inspections in light of the recent Supreme Court decision
in Marshall v. Barlow's, Inc., U.S. , 98 S. Ct. 1816 (1978).
The decision bears upon the need to obtain warrants or other process for
inspections pursuant to EPA-administered Acts.
In Barlow's, the Supreme Court held that an OSHA inspector was not
entitled to enter the non-public portions of a work site without either
(1) the owner's consent, or (2) a warrant. The decision protects the
owner against any penalty or other punishment for insisting upon a warrant.
In summary, Barlow's should only have a limited effect on EPA
enforcement inspections:
o Inspections will generally continue as usual;
o Vhere an inspector is refused entry, EPA will seek a warrant through
the U.S. Attorney;
*»
o Sanctions will not be imposed upon owners of establishments who insist
on a warrant before allowing inspections of the non-public portions
of an establishment.
The scope of the Barlow's decision is broad. It affects all current
inspection programs of EPA, including inspections conducted by State
personnel and by contractors. The Agency's procedures for inspections,
particularly where entry is denied, vere largely in accord with
the provisions of Barlow's before the Supreme Court issued its ruling.
Nevertheless, a number of changes in Agency procedure are warranted.
Thus, it is important that all personnel involved in the inspection
process be familiar with the procedural guidelines contained in this docu-
ment.
-------
- 2 -
This document focuses on the preparation for and conduct of inspec-
tions, including (1) how to proceed when entry is denied, (2) under what
circumstances a warrant is necessary, and (3) what showing is neces-
sary to obtain a warrant.
II. Conduct of Inspections
The following material examines the procedural aspects of conducting
inspections under EPA-administered Acts. Inspections are considered in
three stages: (1) preparation for inspection of premises, (2) entry onto
premises, and (3) procedures to be followed where entry is refused.
A. Preparation
Adequate preparation should include consideration of the following
factors concerning the general nature of warrants and the role of personnel
conducting inspections.
(1) Seeking a Warrant Before Inspection
The Barlow's decision recognized that, on occasion, the Agency may
wish to obtain a warrant to conduct an inspection even before there has
been any refusal to allow entry. Such a warrant may be necessairy when
surprise is particularly crucial to the inspection, or when a company's
prior bad conduct and prior refusals make it likely that warrantless
entry will be refused. Pre-inspection warrants may also be obtained where
the distance to a U.S. Attorney or a magistrate is considerable so that
excessive travel time would not be wasted if entry were denied.
At present, the seeking of such a warrant prior to an initial inspection
should be an exceptional circumstance, and should be cleared through
Headquarters. If refusals to allow entry without a warrant increase, such
warrants may be sought more frequently. (For specific instructions on
how to obtain a warrant, see Part D.)
(2) Administrative Inspections y. Criminal Investigations
It is particularly important for both inspectors and attorneys to
be aware of the extent to which evidence sought in a civil inspection can
be used in a criminal matter, and to know when it is necessary to secure a
criminal rather than a civil search warrant. There are three basic rules
to remember in this regard: (1) If the purpose of the inspection is to
discover and correct, through civil procedures, noncompliance with regulatory
requirements, an administrative inspection (civil) warrant may be used;
(2) if the inspection is in fact intended, in whole or in part, to gather
evidence for a possible criminal prosecution, a criminal search warrant
must be obtained under Rule 41 of the Federal Rules of Criminal Procedure;
and (3) evidence obtained during a valid civil inspection is generally
admissible in criminal proceedings. These principles arise from the recent
Supreme Court cases of Marshall v. Barlow's, Inc., supra; Michigan v. Tyler,
U.S. , 98 S.Ct. 1942 (1978); and U.S. v. LaSalle National Bank,
U.S. , 57 L. Ed'. 2d 221 (1978). It is not completely clear whether
a combined investigation for civil and criminal violations may be properly
conducted under a civil or "administrative" warrant, but we believe that
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a civil warrant can properly be used unless the intention is clearly to
conduct a criminal investigation.
(3) The Use of Contractors to Conduct Inspections
Several programs utilize private contractors to aid in the conduct
of inspections. Since, for the purpose of inspections, these contractors
are agents of the Federal government, the restrictions of the Barlowjs
decision also apply to them. If contractors are to be conducting
inspections without the presence of actual EPA inspectors, these con-
tractors should be given training in how to conduct themselves when
entry is refused. With respect to obtaining or executing a warrant,
an EPA inspector should always participate in the process, even if
he was not at the inspection where entry was refused.
(4) Inspections Conducted by State Personnel
The Barlow's holding applies to inspections conducted by State
personnel and to joint Federal/State inspections. Because some EPA
programs are largely implemented through the States, it is essential
that the Regions assure that State-conducted inspections are conducted
in compliance with the Bar la-/' s decision, and encourage the State inspec-
tors to consult with their legal advisors when there is a refusal to
allow entry for inspection purposes. State personnel should be encouraged
to contact the EPA Regional Enforcement Office when any questions con-
cerning compliance with Barlow's arise.
With regard to specific procedures for States to follow, the
important points to remember are: (1) The State should not seek for-
cible entry without a warrant or penalize an owner for insisting upon
a warrant, and (2) the State legal system should provide a mechanism for
issuance of civil administrative inspection warrants. If a State is
enforcing an EPA program through a State statute, the warrant process
should be conducted through the State judicial system. Where a State
inspector is acting as a contractor to the Agency, any refusal to allow
entry should be handled as would a refusal to an Agency inspector as
described in section II.B.3. Where a State inspector is acting as a
State employee with both Federal and State credentials, he should utilize
State procredures unless the Federal warrant procedures are more, advantageous,
in which case, the warrant should be sought under the general procedures
described below. The Regions should also assure that all States which
enforce EPA programs report any denials of entry to the appropriate
Headquarters Enforcement Attorney for the reasons discussed in section
II.B.4.
B. Entry
(1) Consensual Entry
One of the assumptions underlying the Court's decision is that
most inspections will be consensual and that the administrative inspec-
tion framework will thus not be severely disrupted. Consequently, inspec-
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tions will normally continue as before the Barlow's decision was issued.
This means that the inspector will not normally secure a warrant before
undertaking an inspection but, in an attempt to gain admittance, will
present his credentials and issue a notice of inspection where required.
The establishment owner may complain about allowing an inspector to enter
or otherwise express his displeasure with EPA or the Federal government.
However, as long as he allows the inspector to enter, the entry is voluntary
and consensual unless the inspector is expressly told to leave the premises.
On the other hand, if the inspector has gained entry in a coercive manner
(either in a verbal or physical sense), the entry would not be consensual.
Consent must be given by the owner of the premises or the person in
charge of the premises at the time of the inspection. In the absence
of the owner, the inspector should make a good faith effort to determine
who is in charge of the establishment and present his credentials to
that person. Consent is generally needed only to inspect the non-public
portions of an establishment - i.e., any evidence that an inspector obtains
while in an area open to the public is admissible in an enforcement
proceeding.
(2) Withdrawal of Consent
The owner may withdraw his consent to the inspection at any time.
The inspection is valid to the extent to which it has progressed before
consent was withdrawn. Thus, observations by the inspector, including
samples and photographs obtained before consent was withdrawn, would be
admissible in any subsequent enforcement action. Withdrawal of consent
is tantamount to a refusal to allow entry and should be treated as
discussed in section II.B.3. below, unless the inspection had progressed
far enough to accomplish its purposes.
(3) When Entry is Refused
Barlow's clearly establishes that the owner does have the right
to ask for a warrant under normal circumstances.1 Therefore, refusal
to allow entry for inspectional purposes will not lead to civil or criminal
penalties if ,the refusal is based on the inspector's lack of a warrant
and one of the exemptions discussed in Part C does not apply. If the
owner were to allow the inspector to enter his establishment only in
response to a threat of enforcement liability, it is quite possible that
any evidence obtained in such an inspection would be inadmissible. An
inspector may, however, inform the owner who refuses entry that he intends
to seek a warrant to compel the inspection. In any event, when entry is
1
FIFRA inspections are arguably not subject to this aspect of Barlow's
See discussion, p. 5 and 6.
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refused, the inspector should leave the premises immediately and telephone
the designated Regional Enforcement Attorney as soon as possible for
further instructions. The Regional Enforcement Attorney should contact
the U.S. Attorney's Office for the district in which the establishment
desired to be inspected is located and explain to the appropriate Assistant
United States Attorney the need for a warrant to conduct the particular
inspection. The Regional Attorney should arrange for the United States
Attorney to meet with the inspector as soon as possible. The inspector
should bring a copy of the appropriate draft warrant and affidavits.
Samples are provided in the appendix to this document.
(4) Headquarters Notification
It is essential that the Regions keep Headquarters informed of
all refusals to allow entry. The Regional Attorney should inform the
appropriate Headquarters Enforcement Attorney of any refusals to enter
and should send a copy of all papers filed to Headquarters. It is
necessary for Headquarters to monitor refusals and Regional success in
obtaining warrants to evaluate the need for improved procedures and to
assess the impact of Barlow's on our compliance monitoring programs.
C. Areas Where a Right of Warrantless Entry Still Exists
1. Emergency Situations.
In an emergency, where there is no time to get a warrant, a warrant-
less inspection is permissible. In Camara v. Municipal Court, 387 U.S. 523
(1967), the Supreme Court states that "nothing we say today is intended
to foreclose prompt inspections, even without a warrant, that the law has
traditionally upheld in emergency situations". Nothing stated in Barlow's
indicates any intention by the court to retreat from this position. The
Regions will always have to exercise considerable judgment concerning
whether to secure a warrant when dealing with an emergency situation.
However, if entry is refused during an emergency, the Agency would need
the assistance of the U.S. Marshal to gain entry, and a warrant could
probably be obtained during the time necessary to secure that Marshal's
assistance.
An emergency situation would include potential imminent hazard
situations, as well as, situations where there is potential for destruction
of evidence or where evidence of a suspected violation may disappear during
the time that a warrant is being obtained.
(2) FIFRA Inspections.
There are some grounds for interpreting Barlow's as not being
applicable to FIFRA inspections. The Barlow's restrictions do not apply
to areas that have been subject to a long standing and pervasive history
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of government regulation. An Agency administrative law judge held recently
that even after the Barlow's decision, refusal to allow a warrantless
inspection of a FIFRA regulated establishment properly subjected the
owner to civil penalty. N. Jonas & Co., Inc., I.P. & R Docket NO. III-121C
(July 21, 1978). For the present, however, FIFRA inspections should be
conducted under the sane requirements applicable to other enforcement
programs.
(3) "Open Fields" and "In Plain View" situations.
Observation by inspectors of things that are in plain view, (^.e_.,
of things that a member of the public could be in a position to observe) does
not require a warrant. Thus, an inspector's observations from the public
area of a plant or even from certain private property not closed to
the public are admissible. Observations made even before presentation of
credentials while on private property which is not normally closed to the
public are admissible.
D. Securing a Warrant
There are several general rules for securing warrants. Three
documents have to be drafted: (a) an application for a warrant, (b) an,
accompanying affidavit, and (c) the warrant itself. Each document should be
captioned with the District Court of jurisdiction, the title of the action,
and the title of the particular document.
The application for a warrant should generally identify the statutes
and regulations under which the Agency is seeking the warrant, and should
clearly identify the site or establishment desired to be inspected
(including, if possible, the owner and/or operator of the site),
The application can be a one or two page document if all of the factual
background for seeking the warrant is stated in the affidavit, and the
application so states. The application should be signed by the U.S.
Attorney or by his Assistant U.S. Attorney.
The affidavits in support of the warrant application are crucial
documents. Each affidavit should consist of consecutively numbered para-
graphs, which describe all of the facts that support warrant issuance. If
the warrant is sought in the absence of probable cause, it should recite
or incorporate the neutral administrative scheme which is the basis for
inspecting the particular establishment. Each affidavit should be signed
by someone with personal knowlege of all the facts stated. In cases where
entry has been denied, this person would most likely be the inspector
who was denied entry. Note that an affidavit is a sworn stataient that
must either by notarized or personally sworn to before the magistrate.
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The warrant is a direction to an appropriate official (an EPA
inspector, U.S. Marshal or other Federal officer) to enter a
specifically described location and perform specifically described
inspection functions. Since the inspection is limited by the terms of
the warrant, it is important to specify to the broadest extent possible
the areas that are intended to be inspected, any records to be inspec-
ted, any samples to be taken, any articles to be seized, etc. While
a broad warrant may be permissible in civil administrative inspections,
a vague or overly broad warrant will probably not be signed by the
magistrate and may prove susceptible to constitutional challenge
The draft warrant should be ready for the magistrate's signature at the
time of submission via a motion to quash and suppress evidence in
Federal District court. Once the magistrate signs the draft warrant, it
is an enforceable document. Either following the magistrate's signature
or on a separate page, the draft warrant should contain a "return of
service" or "certificate of service". This portion of the warrant should
indicate upon whom the warrant was personally served and should be signed
and dated by the inspector. 'As they are developed, more specific warrant-
issuance documents will be drafted and submitted to the Regions.
E. Standards or Bases for the Issuance of Administrative Warrants.
The Barlow's decision establishes three standards or bases for the
issuance of administrative warrants. Accordingly, warrants may be obtained
upon a showing: 1) of traditional criminal probable cause, 2) of civil
probable cause, or 3) that the establishment was selected for inspection
pursuant to a neutral administrative inspection scheme.
1. Civil specific probable cause warrant.
Where there is some specific probable cause for issuance of a warrant
such as an employee complaint or competitor's tip, the inspector should be
prepared to describe to the U.S. Attorney in detail the basis for this
probable cause.
The basis for probable cause will be stated in the affidavit in
support of the warrant. This warrant should be used when the suspected
violation is one that would result in a civil penalty or other civil
action.
2. Civil probable cause based on a neutral administrative
inspection scheme.
Where there is no specific reason to think that a violation has been
committed, a warrant may still be issued if the Agency can show that the
establishment is being inspected pursuant to a neutral administrative
scheme. As the Supreme Court stated in Barlow's:
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"Probable cause in the criminal law sense is not required.
For purposes of an administrative search, such as this, probable
cause justifying the issuance of a warrant may be based not only
on specific evidence of an existing violation, but also on a
showing that "reasonable legislative or administrate standards
for conducting an ... inspection are satisfied with respect
to a particular [establishment]". A warrant showing that a speci-
fic business has been chosen for an OSHA search on the basis of a
general administrative plan for the enforcement of the act derived
from neutral sources such as, for example, dispersion of employees
in various type of industries across a given area, and the desired
frequency of searches in any of the lesser divisions of the area,
would protect an employers Fourth Amendment rights."
Every program enforced by the Agency has such a scheme by which it prioritizes
and schedules its inspections. For example, a scheme under which every permit
holder in a given program is inspected on an annual basis is a satisfactory
neutral administrative scheme. Also, a scheme in which one out of every three
known PCB transformer repair shops is inspected on an annual basis is satis-
factory, as long as, neutral criteria such as random selection are used to
select the individual establishment to be inspected. Headquarters will prepare
and transmit to the Regions the particular neutral administrative scheme under
which each program's inspections are to be conducted. Inspections not based
on specific probable cause must be based on neutral administrative schemes for
a warrant to be issued. Examples of two neutral administrative schemes are
provided in the appendix. (Attachments II and III)
The Assistant U.S. Attorney will request the inspector to prepare and
sign an affidavit that states the facts as he knows them. The statement
should include the sequence of events culminating in the refusal to allow
entry and a recitation of either the specific probable cause or the
neutral administrative scheme which led to the particular establishment's
selection for inspection. The Assistant U.S. Attorney will then present
a request for an inspection warrant, a suggested warrant, and the inspector's
affidavit to a magistrate or Federal district court judge.
3. Criminal Warrants.
Where the purpose of the inspection is to gather evidence for a
criminal prosecution, the inspector and the Regional Attorney should request
that the U.S. Attorney seek a criminal warrant under Rule 41 of the Federal
Rules of Criminal Procedure. This requires a specific showing of probable
cause to believe that evidence of a crime will be discovered. Agency policy
on the seeking of criminal warrants has not been affected by Barlow' s. The
2
The Barlow's decision states that imposing the warrant requirement
on OSHA would not invalidate warrantless search provisions in other
regulatory statutes since many such statutes already "envision resort
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distinction between administrative inspections and criminal vrarrant
situations is discussed in Section II.A.2.
F. Inspecting with a Warrant
Once the warrant has been issued by the magistrate or judge, the
inspector may proceed to the establishment to commence or continue the
inspection. Where there is a high probability that entry will be refused
even with a warrant or vfaere there are threats of violence, the inspector
should be accompanied by a U.S. Marshal when he goes to serve the warrant
on the recalcitrant owner. The inspector should never himself attempt
to make any forceful entry of the establishment. If the owner refuses
entry to an inspector holding a warrant but not accompanied by a U.S.
Marshal, the inspector should leave the establishment and inform the
Assistant U.S. Attorney and the designated Regional Attorney. They will
take appropriate action such as seeking a citation for contempt. Where
the inspector is accompanied by a U.S. Marshal, the Marshal is principally
charged with executing the warrant. Thus, if a refusal or threat to
refuse occurs, the inspector should abide by the U.S. Marshal's decision
whether it is to leave, to seek forcible entry, or otherwise.
The inspector should conduct the inspection strictly in accordance
with the warrant. If sampling is authorized, the inspector must be sure
to carefully follow all procedures, including the presentation of receipts
for all samples taken. If records or other property are authorized to be
taken, the inspector must receipt the property taken and maintain an
inventory of anything taken from the premises. This inventory will be
examined by the magistrate to assure that the warrant's authority has
not been exceeded.
2 continued from page 8.
to Federal court enforcement when entry is refused". There is thus
some question as to whether the existence of a non-warrant Federal
court enforcement mechanism in a statute requires the use of that
mechanism rather than warrant issuance. We believe that the Barlow's
decision gives the agency the choice of whether to proceed through warrant
issuance or through an application for an injunction, since the decision
is largely based on the fact that a warrant procedure imposes virtually
no burden on the inspecting agency. In addition, an agency could attempt
to secure a warrant prior to inspection on an ex parte basis, something
not available under normal injunction proceedings. Several of the acts
enforced by EPA have provisions allowing the Administrator to seek
injunctive relief to assure compliance with the various parts of a
particular statute. There may be instances where it would be more appro-
priate to seek injunctive relief to gain entry to a facility than to
attempt to secure a warrant for inspection, although at this point we
cannot think of any. However, since the warrant process will be far
more expeditious than the seeking of an injunction, any decision to
seek such an injunction for inspection purposes should be cleared through
appropriate Headquarters staff.
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G. Returning the Warrant.
After the inspection has been completed , the warrant must be returned
to the magistrate. Whoever executes the warrant, (i.e., whoever performs
the inspection) , must sign the return of service form indicating to whom
the warrant was served and the date of service. He should then return
the executed warrant to the U.S. Attorney who will formally return it to
the issuing magistrate or judge. If anything has been physically taken
from the premises, such as records or samples, an inventory of such items
must be submitted to the court, and the inspector must be present to certify
that the inventory is accurate and ccmplete.
III. Conclusion
Except for requiring the Agency to formalize its neutral inspection
schemes, and for generally ending the Agency's authority for initiating
civil and/or criminal actions for refusal to allow warrantless inspections,
Barlow's should not interfere with EPA enforcement inspections.
Where there is doubt as to how to proceed in any entry case,
do not hesitate to call the respective Headquarters program contact for
assistance.
Marvin B. Burning
>}• U. S. GOVERNMENT PRINTING OFFICE 1979 - 677-09^/1101 Reg. 8
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