ENVIRONMENTAL PROTECTION AGENCY/
. w \ 1 CORPS OF ENGINEERS
TECHNICAL COMMITTEE ON CRITERIA FOR
DREDGED AND FILL MATERIAL
ADD
ODD
00
PUBLISHED BY
ENVIRONMENTAL EFFECTS LABORATORY
ARMY ENGINEER WATERWAYS EXPERIMENT STATION
VICKSBURG, MISSISSIPPI
-------
m
ENVIRONMENTAL PROTECTION AGENCY/
CORPS OF ENGINEERS
fECHNICAL COMMITTEE ON CRITERIi
DREDGED AND FILL MATERIAL
E
W
? a
ID
JS?1
lojo
FOR
30
Sill
)d|d
TRANSMITTAL OF
"ECOLOGICAL EVALUATION OF PROPOSED DISCHARGE
OF DREDGED MATERIAL INTO OCEAN WATERS"
1. In accordance with Section 227.27(b) of the Federal Register,
Vol. 42, No. 7, Tuesday, 11 January 1977, (referred to hereafter in
this letter as the Register), an implementation manual has been
developed jointly by the Environmental Protection Agency (EPA) and
the Corps of Engineers (CE). This manual will be used in the imple-
mentation of Section 103 of Public Law 92-532, the Marine Protection,
Research, and Sanctuaries Act of 1972. Procedures are presented for
evaluation of potential environmental impacts of the discharge of
dredged material into ocean waters, an evaluation that is required in
considering permit applications for the transportation of dredged
material for ocean dumping.
2.. The manual transmitted herewith represents a multidisciplinary
effort of both agencies to develop procedurally sound, routinely
implementable guidance for complying with the technical requirements
of the Register. The procedures given in the manual are applicable
to evaluation of the potential ecological effects of dumping from
hopper dredges, barges, and scows. The requirements of the
Register are discussed, and detailed guidance is provided on sediment
and water sample collection, preparation, and preservation; chemical
analysis of the liquid phase; bioassays of liquid, suspended particu-
late, and solid phases; estimation of bioaccumulation potential; and
the estimation of initial mixing.
3. The manual Is not intended to establish standards or rigid
criteria and should not be interpreted in such a manner. The document
attempts to provide a balance between the technical state-of-the-art
and routinely implementable guidance for using the procedures
specified in the Register and is intended to encourage continuity and
cooperation between CE Districts and EPA Regions in evaluative programs
-------
for Section 103 permit activities. The manual is particularly im-
portant in forming a foundation to be augmented by more meaningful
and comprehensive evaluation procedures and guidelines as these evolve
from current and future environmental research. It is anticipated
that the manual will be updated as new and more implementable evalua-
tion procedures are developed and verified.
Co-Chairman
U. S. Army Corps of Engineers
FRANK G. WILKES
Co-Chairman
U. S. Environmental Protection Agency
-------
PREFACE
According to Section 103 of Public Law 92-532 (Marine Protection,
Research, and Sanctuaries Act of 1972), any proposed dumping of
dredged material into ocean waters must be evaluated through the use
of criteria published by the Environmental Protection Agency (EPA) in
the Federal Register, Vol. 42, No. 7, Tuesday, 11 January 1977. The
Federal Register states that an implementation manual describing the
applicability of specific evaluative approaches and procedures will
be developed jointly by EPA and the Corps of Engineers (CE). This
manual contains those procedures considered applicable to evaluation
of potential environmental impacts of the ocean disposal of dredged
material, and it will be periodically revised and updated as advances
in the technical state-of-the-art warrant.
By agreement of both agencies, this implementation manual was
developed by the EPA/CE Technical Committee on Criteria for Dredged
and Fill Material, co-chaired by Dr. Frank G. Wilkes of the EPA and
Dr. Robert M. Engler of the CE. Due to the emphasis on bioassay
in the Federal Register, much of the developmental input to the manual
was from the Bioassay/Bioevaluation Subcommittee, co-chaired by
Dr. Jack H. Gentile of the EPA and Dr. Richard K. Peddicord of the
CE. Many individuals within both agencies contributed to the manual,
with major input in various areas from those identified as follows:
Compiler and Technical Editor:
Dr. Richard K. Peddicord, University of California,
Bodega Marine Laboratory, and Environmental Effects
Laboratory (EEL), Waterways Experiment Station (WES), CE
Editor:
Ms. Dorothy P. Booth, EEL, WES, CE
Part I - Introduction and Part II - General Approaches:
Dr. Peddicord
Appendix B - Dredged Material Sample Collection and Preparation:
Mr. James M. Brannon and Dr. Robert M. Engler, EEL, WES, CE
i
-------
Appendix C - Liquid Phase Chemical Analyses:
Mr. Brannon and Dr. Engler
Appendix D - Guidance for Performing Liquid Phase and Suspended
Particulate Phase Animal Bioassays:
Dr. Peter J. Shuba and Dr. Henry E. Tatem, EEL, WES, CE
Appendix E - Guidance for Performing Liquid Phase and Suspended
Particulate Phase Phytoplankton Bioassays:
Drs. Shuba and Tatem and Dr. Jack H. Gentile, EPA,
Narragansett Environmental Research Laboratory,
Narragansett, Rhode Island
Appendix F - Guidance for Performing Solid Phase Bioassays:
Dr. Richard C. Swartz, Mr. Waldemar A. DeBen, and
Ms. Faith A. Cole, EPA, Corvallis Environmental Research
Laboratory, Newport Field Station, Newport, Oregon, and
Drs. Shuba and Tatem.
Appendix G - Guidance for Assessing Bioaccumulation Potential:
Dr. Peddicord
Appendix H - Estimation of Initial Mixing:
Mr. Barry W. Holliday, EEL, WES, CE
Review of the manual was conducted by EPA through the Marine
Protection Branch, Oil and Special Materials Control Division;
Office of Deputy Assistant Administrator for Health and Ecological
Effects; and the Ocean Dumping Bioassay Committee; and by the
Corps of Engineers through the Office, Chief of Engineers, and the
Environmental Effects Laboratory of the Waterways Experiment Station.
ii
-------
CONTENTS
Page
PREFACE i
PART I: INTRODUCTION 1
Background 1
Purpose and Scope 2
Applicability 3
Definitions A
PART II: GENERAL APPROACHES FOR EVALUATION OF
PERMIT APPLICATIONS 6
Applicability (Subpart A) 6
Technical Evaluation (Subpart B) ...... 6
Liquid phase 8
Suspended particulate phase 10
Solid phase 11
Bioaccumulation 12
Initial mixing 14
Trace contaminants 15
General compatibility with the disposal site .... 17
Evaluation of Subpart B results 18
Need for Ocean Dumping (Subpart C) 18
Inpacts on Esthetic, Recreation, and Economics
(Subpart D) 18
Impacts on Other Uses of the Ocean (Subpart E) 18
Site Management Considerations 19
Decision on Permit Application 19
APPENDIX A: Reorganization of Section 227.13 from "Ocean
Dumping-Final Revisions of Regulations and
Criteria" to Incorporate Cross-References
APPENDIX B: Dredged Material Sample Collection and
Preservation
Introduction Bl
Sample Collection and Preservation Bl
Number of samples Bl
Apparatus B2
Water sampling B2
Sediment sampling B3
Liquid Phase Sample Preparation ... B3
Apparatus B4
Sample preparation B5
Disposal Site Water Sample Preparation B7
ili
-------
Page
APPENDIX B (concluded)
Suspended Particulate Phase Sample Preparation B7
Solid Phase Sample Preparation B8
APPENDIX C: Liquid Phase Chemical Analyses
Apparatus CI
Procedures CI
Interpretation of Results CI
APPENDIX D: Guidance for Performing Liquid Phase and
Suspended Particulate Phase Animal Bioassay
Introduction D1
Apparatus Dl
Species Selection ......... D2
Sample Collection and Preservation . .... D5
Experimental Conditions D5
Experimental Procedures D6
Data Analysis and Interpretation D8
Data presentation D8
Statistical analysis ........ D10
Limiting permissible concentration D14
APPENDIX E: Guidance for Performing Liquid Phase and Suspended
Particulate Phase Phytoplankton Bioassays
Introduction . El
Apparatus * E2
Species Selection .... E2
Sample Collection and Preservation E3
Experimental Conditions . . E3
Experimental Procedure . ..... E4
Data Analysis and Interpretation E6
APPENDIX F: Guidance for Performing Solid Phase Bioassays
Introduction F1
Aquarium System F2
Collection of Sediment and Test Organisms F2
Species Selection F3
Experimental Conditions F6
Bioassay Procedure ..... .......... F7
Analysis and Interpretation of Results . F10
Data presentation ... ........ FIX
iv
-------
Page
APPENDIX F (concluded)
Statistical analysis F12
Limiting permissible concentration . . F17
APPENDIX G: Guidance for Assessing Bioaccumulation Potential
Introduction Gl
Field Assessment of Bioaccumulation Potential G2
Apparatus G2
Species selection G2
Sampling design and conduct G3
Analysis and interpretation G5
Laboratory Assessment of Bioaccumulation Potential .... G6
Sampling design and conduct G6
Analysis and interpretation G7
Chemical Analysis G7
Constituents to be assessed G7
Procedures G7
Data Analysis and Interpretation G7
APPENDIX H: Estimation of Initial Mixing
Introduction HI
Initial Mixing Calculations .... Hi
Mathematical models using specific field data
(227.29(a)(1)) HI
WES mathematical models H2
WES model input requirements H3
Similar field data and modeling (227.29(a)(2) .... H4
Theoretical relationship (227.29(a)(3) H5
Release zone method H5
Application to Limiting Permissible Concentration .... H6
Liquid phase - water-quality criteria
(227.27(a)(1) H6
Liquid phase - no water-quality criteria
(227.27(a)(2)) H8
Suspended particulate phase (227.27(b)) H9
v
-------
PART I: INTRODUCTION
Background
1. Section 103 of the Marine Protection, Research, and Sanctuaries
Act of 1972, Public Law (PL) 92-532, specifies that all proposed oper-
ations involving the transportation and dumping of dredged material into
ocean waters must be evaluated to determine the potential environmental
impact of such activities. This must be done by the Secretary of the
Army and the Administrator of the Environmental Protection Agency (EPA)
acting cooperatively through the District Engineer and Regional Adminis-
trator. Environmental evaluations must be in accordance with criteria
published by EPA in the Federal Register, Vol. 42, No. 7, Tuesday,
11 January 1977 (hereafter referred to as the Register), placing
special emphasis on Parts 227 and 228 insofar as potential ecological
effects are concerned.
2. The primary intent of Section 103 of PL 92-532 is to regulate
and limit adverse ecological effects of ocean dumping. Consequently,
the Register emphasizes evaluative techniques such as bioassays and
bioassessments, which provide relatively direct estimations of the
potential for environmental impact. To conduct the required procedures
properly requires considerable expertise in conducting biological evalu-
ations. In addition, significant continuing effort and expense are
required to collect and culture sufficient stocks of all the necessary
species of organisms and maintain them in good condition in the labora-
tory to use whenever an evaluation must be conducted. These considera-
tions argue against obtaining the services of a different group to con-
duct each evaluation. It is highly recommended that a few groups of
demonstrated bioassay capability be selected, with each group con-
ducting evaluations for a number of permit applications. This will
enable these groups to develop adequate culturing and maintenance
capabilities and the expertise and familiarity with the procedures re-
quired to consistently conduct them properly and to provide the most
1
-------
reliable results at the least cost per evaluation.
Purpose and Scope
3. The Register specifies that this technical implementation
manual for the criteria applicable to dredged material be developed
jointly by EPA and the Corps of Engineers (CE). The manual was
developed jointly by the EPA/CE Technical Committee on Criteria for
Dredged and Fill Material, with the major contribution from the
Bioassay/Bioevaluation Subcommittee. The manual is an attempt to
provide a balance between technical state-of-the-art and routinely
implementable guidance for using the evaluative procedures specified
in the Register. Guidance is included on the appropriate uses and
limitations of the various procedures and on sound interpretation of
the results. Its structure follows the general order of test appli-
cation and general priority of importance of testing and evaluation
procedures presented in the Register.
4. This manual contains summaries and discussions of the pro-
cedures for ecological evaluation of dredged material required by the
Register, tests to implement them, definitions, sample collection and
preservation procedures, evaluative procedures, calculations, inter-
pretative guidance, and supporting references required for the
evaluation of permit applications in accordance with the Register.
Even so, this manual cannot stand alone. It is imperative that the
supporting references cited in each appendix be consulted for detailed
or more comprehensive guidance whenever indicated. Before any evalu-
ations are begun, the Register and this manual should be read in their
entirety, and citations and references listed with the appendices
should be consulted to obtain an understanding of the guidance the
manual provides. The technical procedures in this manual were designed
only for dredged material and should not be utilized for any other
materials unless definitive research demonstrates their applicability.
5. This issue of the implementation manual contains evaluative
procedures considered to be acceptable regulatory tools for most
situations. In some instances more sophisticated and complex
2
-------
biological evaluations may be warranted by special circumstances.
However, variations of these procedures should be allowed only when
the District Engineer and the Regional Administrator are able to
justify and defend the technical validity of such variations. The
field of ecological evaluation is a dynamic one, and new and better
regulatory procedures are under development. As warranted by
experience with this manual and the development of new procedures, the
manual will be revised periodically. These revisions will be announced.
6. It should be emphasized that implementation of the criteria is
the joint responsibility of the District Engineer and the Regional
Administrator. This manual was developed by research personnel of both
agencies to contain the best technical guidance available for imple-
mentation. However, it is inevitable that situations will arise that
are not specifically addressed in the manual, as well as occasions when
a choice of the appropriate course of action must be made. Such situ-
ations must be cooperatively worked out by the District Engineer and
Regional Administrator to their mutual satisfaction as they occur.
7. This manual provides technical guidance to the fullest extent
practical on implementation of the criteria. Yet technical evaluations
can provide only part of the input to the decisionmaking process. Many
of the criteria do not concern subjects amenable to quantitative evalu-
ation. In such cases objective, qualitative decisions must be made.
Indeed, the decision on granting of a permit is ultimately subjective.
The criteria do not prohibit environmental change, but rather "unaccept-
able environmental impact." Consequently, for each permit application,
the Regional Administrator and the District Engineer must decide how
much potential impact is acceptable under the environmental, economic,
social, and political conditions related to the operation in question.
Technical and scientific evaluations provide an important but incom-
plete input to such decisions.
Applicability
8. This implementation manual is applicable to all activities
involving the transportation of dredged material for the purpose of
3
-------
dumping it in ocean waters, These procedures do not apply to
activities excluded in Section 220.1 of the Register. These criteria
pertain to the transportation for ocean dumping of dredged material
outside the baseline from which the territorial sea is measured.
Definitions
9. The following terms are briefly defined as used in this report
and its appendices. See Section 220.2 and Part 227 Subpart G of the
Register for complete definitions of terms used in the criteria.
Constituents. Chemical substances, solids, organic matter, and
organisms associated with or contained in or on dredged material.
Criteria. Procedures and concepts published in the Register for
the evaluation of dredged material ocean-dumping permit applications.
Disposal site. A precise geographical area within which ocean
dumping of wastes may be permitted. Includes both the bottom substrate
and the water column within the specified boundaries.
Dredged material. Bottom sediment or material and the water
associated with such sediment or material that have been dredged or
excavated from the navigable waters of the United States.
Dumping. The disposition of material subject to the exclusions of
paragraph 220.2(e) of the Register.
Initial mixing. Dispersion or diffusion of liquid, suspended
particulate, and solid phases of dredged material that occurs within
4 hr after dumping.
Limiting permissible concentration (LPC) of;
a. Liquid phase; the concentration of dredged material that,
after allowance for initial mixing, does not exceed appli-
cable marine water-quality criteria or a toxicity threshold
of 0.01 of the acutely toxic concentration.
b. Suspended particulate and solid phase: a concentration that
will not cause unreasonable acute or chronic toxicity or sub-
lethal adverse effects including bioaccumulation of toxic
materials in the human food chain.
Liquid phase. The centrifuged and 0.45-p-filtered supernatant re-
maining after 1 hr undisturbed settling of the mixture resulting from
the vigorous 30-mln agitation of a 1:4 ratio of dredged material and
4
-------
disposal site water.
Ocean. Those waters of the open seas lying seaward of the baseline
from which the territorial sea is measured (see paragraph 220.2(c) of
the Register).
Solid phase. All material settling to the bottom within 1 hr in
the liquid-phase procedure. (In practice, bottom sediments of in situ
density may be considered to represent the solid phase.)
Suspended particulate phase. The supernatant, prior to centri-
fugation and filtration, obtained by the liquid-phase procedure.
Water-quality criteria. The criteria given for marine waters in
the EPA publication "Quality Criteria for Water" as published in 1976
or in subsequent editions.
5
-------
PART II: GENERAL APPROACHES FOR EVALUATION OF PERMIT APPLICATIONS
10. The potential effect of the ocean disposal of dredged material
on marine organisms and human uses of the ocean may range from unmeasur-
able to important. These effects may differ at each disposal site and
must be evaluated on a case-by-case basis. The Register provides
criteria for such an evaluation, with an emphasis placed on direct
assessment of biological impacts. The appropriate technical procedures
are found in Parts 227 and 228 of the Register. These procedures are
illustrated diagrammatically in the proper evaluative sequence in
Figure 1.
Applicability (Subpart A)
11. The Register recognizes that dredged material may behave
differently from other materials that may be ocean dumped, but does not
place all dredged material criteria in a separate section. Therefore,
it is necessary to read Part 227, Subpart A, paragraph 227.1(b) care-
fully to determine those sections that are applicable to dredged materi-
al. It is these sections that are discussed in this manual.
Technical Evaluation (Subpart B)
12. The first evaluative consideration shown in Figure 1 involves
the presence of certain substances that may not be ocean dumped under
any circumstances. If any of these are present, the permit application
must be denied without further consideration. Dredged materials, how-
ever, are highly unlikely to contain these substances and must usually
receive the full technical evaluation required by the criteria.
13. There are obvious cases where dredged material is not con-
sidered chemically contaminated and would, therefore, cause negligible
pollutional impact when discharged at an appropriate disposal site.
Thus material that meets the requirements of paragraph 227.13(b) (see
Appendix A, page A2) may be excluded from the technical evaluations
required by Section 227.13 and need be evaluated only in terms of its
compatibility with the disposal site and the considerations of Subparts
C, D, and E and the appropriate sections of Part 228, as illustrated in
Figure 1.
6
-------
ECOLOGICAL EVALUATION OF PROPOSED DISCHARGE
c=o
Apply Evaluations in 33 CFR 209.120
or 33 CFft 209.145
TP J I
Applicability of Criteria
Part 227
Subpart A
TP 12-36
Environmental Impact
Subpart B
IP !2
Prohibited Materials
Sec 227,5
TP 13
Exclusion from Technical Evaluation
Sec 227.13(b)
Exclusion
Disallowed
Sec 227.13(c)
Exclusion
Allowed
(Benthlc Impacts)
(Water Column Impacts)
W> 1», AI-J2
W 10-19
Solid Phase
Suspended Particulate
Bioassay
Phase Bioassay
Sec 227.13(c)(3)
Sec 227.13(cK3)
Appendix F
Appendices D&E
1
1
5PI6-IJ
Liquid Phase
Bioassay
Sec 227 13 (c)(2)
Appendices D&£
*7?
Water-Quality
Criteria
Sec 227.13(c)(2)
227.13(d)
Appendix C
JP33
Possible SpecJaJ Studies
Sec 227.6(d)
*>35
General Compatibility of the Material
With the Oisposal Site
Sec 227.9, 227.10
*36
DENy
PERMIT
Sec 227.3
Impacts on Esthetics, Recreation, & Economics
Subpart 0
Impacts on Other Uses of the Oceans
Subpart E
*40
Site Management Considerations
Sec 227.13,
228.4(e), 228.9, 228.12
Note: Numbers within the boxes refer to Sections and
paragraphs in the Register.
Paragraph ( w ) and appendix citations inside the
boxes refer to this manual.
*>4t
Request Additional
Information
Sec 225.2(b)
9*43
*» 42
DENY PERMIT
Waiver of Criteria
Sec 225 2(e)
Sec 225.3
225.4
Figure 1„ Sequence of testing and evaluation procedures
-------
14. Dredged material that does not meet the conditions for exclu-
sion of paragraph 227.13(b) must receive a full technical evaluation of
its potential for environmental impact. The evaluative procedures
emphasize biological effects, rather than simple chemical presence, of
possible contaminants. Dredged material is separated for evaluation
into three phases, as defined in paragraph 227.32(b)(1) of the Register
(see Appendix A, page A3). The liquid phase and the suspended particu-
late phase are considered to have the greatest potential for impact on
the water column and are evaluated with this in mind. The solid phase
has the greatest potential for impact on benthic organisms, and evalu-
ative emphasis is placed there. All three phases must be evaluated, as
indicated in Figure 1.
Liquid phase
15. The liquid phase of dredged material may be evaluated in
either of two ways, as specified in paragraph 227.13(c)(2) of the
Register. Where there is concern about specific contaminants that may
be released in soluble form, the liquid phase may be analyzed chemically
and the results evaluated by comparison to water-quality criteria for
those contaminants after allowance for initial mixing. The period of
initial mixing, discussed in paragraphs 26 and 27, must be allowed be-
fore comparing the predicted concentrations to water-quality criteria.
This provides an indirect evaluation of potential biological impacts of
the liquid phase, since the water-quality criteria were derived from
bioassays of solutions of the various contaminants. Chemical evaluation
of the liquid phase is possible only for those contaminants for which
specific water-quality criteria have been established. The major con-
stituents to be analyzed in the liquid phase are to be selected cooper-
atively by the District Engineer and the Regional Administrator, as
discussed in paragraph 227.13(d) of the Register. Sample collection
and preservation methods are given in Appendix B, and the appropriate
laboratory procedures and supporting references may be found in
Appendix C of this manual.
8
-------
16. If the water-quality criteria approach is not taken, the
liquid phase must be evaluated by bioassays, as indicated in Figure 1.
The direct bioassay approach is to be used when the liquid phase may
contain major constituents not included in the water-quality criteria
or when there is reason to be concerned about possible synergistic
effects of certain contaminants. In these cases liquid phase bioassays
can aid in evaluating the importance and the total net impact of dis-
solved chemical constituents released from the sediment during disposal
operations.
17. Liquid phase bioassays must be conducted with "appropriate
sensitive marine organisms." Paragraph 227.27(c) of the Register (see
Appendix A, page A8) defines this to mean at least three species con-
sisting of one phytoplankton or zooplankton species, one crustacean or
mollusc, and one fish. Phytoplankton bioassays can indicate the
potential for algal stimulation or inhibition by the dredged material
in question. However, it is widely felt that potential effects on
phytoplankton are generally of little environmental concern at ocean
dredged material disposal sites, due to the extremely dynamic and
variable characteristics of natural phytoplankton assemblages and to
the rapid mixing and dilution that occurs in the water column. For
these reasons, unless there is a specific reason to be concerned about
potential effects of the proposed operation on phytoplankton, it is
recommended that a zooplankton species be selected to fulfill that
portion of the bioassay species requirement. Laboratory procedures for
conducting liquid-phase animal bioassays may be found in Appendix D;
guidance on phytoplankton bioassays, if they are felt necessary, is
contained in Appendix E of this manual.
18. It should be recognized that dredged material bioassays cannot
be considered precise predictors of environmental effects. They must
be regarded as quantitative estimators of those effects, making
interpretation somewhat subjective. In order to avoid adding more un-
certainty to their interpretation, the animal bioassays given in this
manual all utilize mortality as an end point. The significance of this
9
-------
response to the individuals involved is clear, but the state of ecologi-
cal understanding is such that it remains impossible to predict the
ecological consequences of the death of a given percent of the local
population of a particular species. For example, there is presently no
basis for estimating whether the loss at the disposal site of 10 percent
of a particular crustacean species would have inconsequential or major
ecological effects. This interpretative uncertainty becomes over-
powering when a parameter whose ecological meaning is not as clear as
mortality is used as the bioassay end point. In view of the inter-
pretative difficulties, the bioassays in this manual specify death as
the response to be measured. Interpretative guidance does not attempt
to consider the ecological meaning of the mortality observed, but takes
the environmentally protective approach prescribed in the Register that
any statistically significant increase in mortality compared to the
controls is potentially undesirable. It is important to realize, how-
ever, that a statistically significant effect in a laboratory bioassay
cannot be taken as a prediction that an ecologically important impact
would occur in the field. Bioassay results must be evaluated in light
of initial mixing (Figure 1) as discussed in paragraphs 26 through 28.
Suspended particulate phase
19. The suspended particulate phase of dredged material may be
evaluated for potential environmental impact only by use of bioassays.
No chemical procedure has yet been devised that will determine the
amount of environmentally active contaminants present in the suspended
particulate phase of dredged material. Therefore, bioassays are used
to evaluate directly the potential for biological impacts due to both
the physical presence of suspended particles and to any biologically
active contaminants associated with the particles and/or the dissolved
fraction. Suspended particulate phase bioassays must also be conducted
with appropriate sensitive marine organisms, as described in paragraph
17 for liquid phase bioassays. In addition to the discussion there
concerning the general advisability of phytoplankton bioassays with
dredged material, it should be noted that suspended particulate phase
10
-------
phytoplankton bioassays are extremely difficult to conduct and interpret
validly. This is due to interferences and predation on the test species
by protozoans contained in the dredged material being tested. Conse-
quently, in most cases the maximum amount of useful information on
potential effects of the proposed disposal operation will be obtained
by bioassaying zooplankton, a crustacean or mollusc, and a fish.
Solid phase
20. It is generally felt that if a dredged material is going to
have an environmental impact, the greatest potential for impact lies in
the solid phase. This is because it is not mixed and dispersed as
rapidly or as greatly as the liquid and suspended particulate phases,
and bottom-dwelling animals live and feed in and on the deposited solid
phase for extended periods. Therefore, unless there is reason to do
otherwise, the major evaluative efforts should be placed on the solid
phase. The Register requires that bioassays be used to evaluate the
potential impact of the solid phase. No chemical procedures exist that
will determine the environmental activity of any contaminants or combi-
nation of contaminants present in the solid phase of dredged material.
Therefore, animals are used in a bioassay to provide a measurement of
environmental activity of the chemicals found in the material.
21. Solid phase bioassays, described in Appendix F, must be con-
ducted with "appropriate sensitive benthic marine organisms." Paragraph
227.27(d) of the Register (Appendix A, page A8) defines this to mean at
least three species, consisting of one filter-feeding, one deposit-
feeding, and one burrowing species. These are broad overlapping general
categories, and it is recommended that the species be selected to in-
clude a crustacean, an infaunal bivalve, and an infaunal polychaete.
22. Paragraph 18 is a key discussion pertinent to all bioassay
procedures in this manual, including solid phase bioassays, which also
measure mortality as the end point because of its clear physiological
significance. However, as pointed out in paragraph 18, the ecological
meaning of the death of a given percent of the animals of one or several
species at the disposal site cannot be estimated at present. Therefore,
11
-------
the interpretative guidance presented for the solid phase bioassay is
environmentally protective in that any statistically significant
increase in mortality compared to the controls is considered potentially
undesirable. This approach is environmentally conservative in that it
does not attempt to consider the ecological meaning of the mortality
observed, but assumes that any mortality might be adverse. Again, it
must be emphasized that a statistically significant effect in a labora-
tory bioassay does not necessarily imply that an ecological important
impact would occur in the field. Solid phase bioassay results must also
be interpreted in light of initial mixing as discussed in paragraph 28.
Bioaccumulation
23. The criteria require that all biological evaluations of the
suspended particulate and solid phases include an assessment of the
potential for contaminants from the dredged material to be bioaccumu-
lated in the tissues of marine organisms (Figure 1 and paragraphs 227.6
(c)(2) and (3) of the Register). This is intended to assess the po-
tential for the long-term accumulation of toxins in the food web to
levels that might be harmful to the ultimate consumer, which is often
man, without killing the intermediate organisms. In order to use bio-
accumulation data in a permitting decision, it is necessary to predict
whether there will be a cause-and-effeet relationship between the
animals' presence in the dredged material and a meaningful elevation of
body burdens of contaminants above those of similar animals not in the
dredged material.
24. Since concern about bioaccumulation is focused on the possi-
bility of gradual uptake over long exposure times, primary attention is
usually given to the solid phase that is deposited on the bottom. Bio-
accumulation from the suspended particulate phase is considered to be
of secondary concern except in special cases, due to the short exposure
time resulting from rapid dispersion of the suspended particulates by
mixing. Should this be a major consideration for the operation in ques-
tion, the laboratory bioaccumulation procedures given in Appendix G may
be used to assess the suspended particulate phase. Because of the long-
12
-------
term nature of the concerns, bioaccumulation from the solid phase is at
present best evaluated in the field where possible. This can be done
only when an historical precedent exists for the proposed operation, as
discussed in paragraphs 2 and 3 of Appendix G. Under these conditions,
a field assessment provides the most useful information because the
animals have been exposed to the sediment under natural conditions for
longer periods than are now generally practical in the laboratory. To
the extent that source control has prevented increased input of contami-
nants, it will generally be true that sediment quality at dredging sites
will not be lower than at the time of previous dredging and disposal
operations. Therefore, since the same disposal site is traditionally
used repeatedly for each dredging site, a valid historical precedent
probably exists at present for most disposal operations utilizing sites
designated in Section 228.12 of the Register.
25. The environmental interpretation of bioaccumulation data is
even more difficult than for bioassays because in most cases it is
impossible to quantify either the ecological consequences of a given
tissue concentration of a constituent that is bioaccumulated or even
the consequences of that body burden to the animal whose tissues con-
tain it. Almost without exception in the marine environment, there is
no technical basis for establishing, for example, the tissue concen-
tration of copper in a species of polychaete that would be detrimental
to that organism, not to mention the impossibility of estimating the
effect of that organism's body burden on a predator. Therefore, in
order to ensure environmental safety, the interpretative guidance
assumes that any statistically significant bioaccumulation relative to
animals not in dredged material, but living in material of similar sedi-
mentological character, is potentially undesirable. The evaluation of
experimental results using this approach requires the user to recognize
the fact that a statistically significant difference cannot be pre-
sumed to predict the occurence of an ecologically important impact.
Bioaccumulation results must also be evaluated in light of initial mix-
ing as discussed in the next section.
13
-------
Initial mixing
26. All data from chemical analysis of the liquid phase, bioassays,
and bioaccumulation studies must be interpreted in light of initial
mixing, as illustrated in Figure 1. This is necessary since biological
effects (which are the basis for water-quality criteria) are a function
of biologically available contaminant concentration and exposure time
of the organism. Laboratory bioassays expose organisms to relatively
constant concentrations for fixed periods of time, while in the field
both concentration and exposure time to a particular concentration change
continuously. Since both factors will influence the degree of biological
impact, it is necessary to incorporate the mixing expected at the site
in the interpretation of biological data.
27. Initial mixing is defined in Section 227.29 of the Register
(Appendix A, page A6) and guidance on estimation of initial mixing may
be found in Appendix H of this manual. Methods for incorporation of
mixing estimations into the interpretation of water-quality results are
included in Appendix C, and these methods for liquid and suspended
particulate phase bioassay data are included in Appendix D.
28. Although the Register allows the consideration of initial
mixing and dispersion of the sediment after it reaches the bottom in
interpreting solid phase bioassay data, no objective method of doing so
has been devised. Rather, there has been an attempt to incorporate the
phenomenon of solid phase sediment dispersion into the bioassay design
to some extent. The concept is expressed in the environmental impact
statement on the ocean dumping criteria* that "EPA has chosen to allow
some change in sediment characteristics or water chemistry as being
reasonable, but no damage to the biota out-side the region of initial
mixing is allowed under these criteria." The solid phase bioassay
technique, therefore, does not evaluate the physical effects of massive
sediment deposition immediately under the discharging vessel, since the
* U. S. Environmental Protection Agency, "Proposed Revisions to Ocean
Dumping Criteria: Final Environmental Impact Statement," 1977, Office
of Water Program Operations, Washington, D. C., p 128.
14
-------
primary concern is that damage not extend beyond the region of initial
mixing. Instead, the technique generally approximates conditions near
the disposal site boundary where sediment dispersion has reduced the
depth of deposited dredged material to a few centimetres. The solid
phase bioassay technique measures the effects of chemicals associated
with this deposited sediment, rather than physical effects of the sedi-
ment. It is apparent that there will be a gradient of decreasing effects
with increased distance due to dispersion away from the disposal site,
although the nature of this gradient cannot be determined. Therefore,
the environmentally protective assumption is made that a statistically
significant effect in the solid phase bioassay indicates a real po-
tential for environmental impact from the solid phase.
29. The Register also requires consideration of initial mixing
in interpreting the results of bioaccumulation studies. In contrast to
the situation with liquid and suspended particulate phase bioassays, no
objective semiquantitative method for incorporating mixing and dilution
into the interpretation of results has been developed for bioaccumula-
tion data. If, in light of paragraph 24, bioaccumulation from the sus-
pended particulate phase is of concern, evaluation of the results must
subjectively consider the effects of mixing on exposure time and concen-
tration, and thus on bioaccumulation. In field evaluations of bio-
accumulation potential, mixing is fully Incorporated into the experi-
mental design, since the animals have lived in the sediment under the
natural conditions at the site since the previous disposal operation.
This is a major advantage of field assessment of bioaccumulation
potential from the solid phase over laboratory evaluations.
Trace contaminants
30. As illustrated in Figure 1, the presence or absence of trace
contaminants must be determined for all three phases of the material.
Section 227.6 is perhaps the key section of the criteria, since dredged
material may not be ocean dumped if it contains any of the listed
substances in greater than trace amounts. This is not defined in terms
of numerical chemical limits whose environmental meaning is uncertain,
15
-------
but rather* "...EPA came to the conclusion that the basis for regulation
[of trace contaminants] should be the probable impact of these constitu-
ents on the biota and that the measurement technique used should be
bioassays on the waste itself." Section 227.6(b) of the Register ex-
presses in regulatory language the idea that trace concentrations be
defined as those too low to cause an environmental effect.
31. In other words, marine organisms are regarded, in a sense, as
analytical instruments for determining the environmentally active por-
tions of any contaminants present. Implementation of this approach to
the definition of trace contaminants requires that lack of effect in
bioassays and bioaccumulation studies be taken to mean that contaminants
are absent, or present only in amounts and/or forms which are not
environmentally active, and therefore do not exceed so-called "trace
concentrations." When effects do occur in dredged material bioassays,
it is not possible within the present state-of-knowledge to determine
which constituent(s) caused the observed effects. Therefore, it must be
assumed they are due to Section 227.6 materials because it cannot be
established that this is not the case. This would mean some contami-
nant (s) is present in greater than trace concentrations. In practice,
the exact identity of the contaminant(s) causing the effect is of little
concern from a regulatory viewpoint, since dredged material that might
cause an environmental effect for any reason should not be ocean dumped
except perhaps under special circumstances. Following this reasoning,
bioaccumulation of any potentially harmful constituent, whether listed
in Section 227.6 or not, could make the material undesirable for ocean
dumping.
32. Since the assessment of trace contaminants depends upon the
determination of effects, it cannot be made until the bioassays (and/or
water-quality studies), and bioaccumulation studies are completed and
interpreted with consideration of mixing. Only then can effects, and
thus the presence of contaminants in other than trace concentrations,
be estimated. This sequence is illustrated in Figure 1.
* ibid., p 83.
16
-------
33. Paragraph 227.6(d) allows special studies to estimate the
potential environmental impact of materials believed to contain com-
pounds identified as carcinogens, mutagens, or teratogens for which
there are no water-quality criteria. This paragraph is expected to
apply to relatively few permit applications for the ocean disposal of
dredged material. In cases where it does apply, the required special
studies would have to be specifically designed for the contaminant of
concern under the particular conditions of the operation in question.
Such highly site- and problem-specific studies are beyond the scope of
a manual such as this and must be designed for each situation in which
they are needed.
34. The prohibitions and limitations of Section 227.6 do not apply
when it can be demonstrated that the material in question is environ-
mentally acceptable, as described in paragraphs 227.6(f) or (g) of the
Register (Appendix A). Again, the studies necessary to demonstrate
compliance with these paragraphs would have to be designed specifically
for the environmental conditions and contaminants in question, making
them so site specific as to be beyond the scope of this manual. Both
these studies and those discussed in paragraph 33 would have to be
designed cooperatively by the District Engineer and the Regional
Administrator to satisfy their mutual concerns about compliance of the
material with the criteria.
General compatibility with the disposal site
35. Once the preceding criteria have been satisfied, the general
compatibility of the dredged material with the proposed disposal site
must be evaluated under Sections 227.9 and 227.10 of the Register. Both
sections are rather subjective criteria, and no specific evaluative
procedures exist for determining compliance with either section. It
should be noted, however, that the available evidence indicates that the
amounts of dredged material usually ocean dumped at one time and place
generally would not create long-term damage caused simply by the volume
of material dumped. Notice from Figure 1 that dredged material excluded
from technical evaluation under paragraph 227.13(b) must meet the
17
-------
requirements of Sections 227.9 and 227.10.
Evaluation of Subpart B results
36. At this point the evaluations required under Subpart B of the
Register will have been conducted. Under Section 227.3, if a dredged
material fails to meet the Subpart B criteria, the permit may be denied.
Need for Ocean Dumping (Subpart C)
37. No material may be ocean dumped unless there is a demonstrated
need to do so under Subpart C. This subpart is in effect an evaluation
of alternative disposal sites in terms of potential environmental
impacts, irreversible commitment of resources, and costs. No disposal
alternative is initially considered more desirable than any other and
the evaluation is to be made on a case-by-case basis. That is, confined
or upland disposal cannot be considered environmentally preferable to
aquatic disposal unless consideration of the potential environmental
impacts (including groundwater contamination, leachate and runoff impacts,
and permanent alteration of the site) show it to be so. Similarly,
ocean disposal should not automatically be considered the most desirable
alternative. As pointed out in Section 227.14, specific quantitative
criteria for evaluating the need for ocean dumping cannot be given, and
this evaluation remains essentially a subjective one.
Impacts on Esthetics, Recreation, and Economics (Subpart D)
38. Before a permit may be granted, the probable impacts on
esthetics, recreational, and economic values must be evaluated, as
described in Subpart D. Although this, too, is a nonquantitative evalu-
ation, consideration of information from the Subpart B technical assess-
ments is required in paragraph 227.18. Despite the qualitative nature
of the required assessment, paragraph 227.19 requires that the results
be expressed, insofar as possible, in quantitative terms.
Impacts on Other Uses of the Ocean (Subpart E)
39. Subpart E is related to the above requirements, but it re-
quires evaluation of specific actual or potential uses of the disposal
site, including but not limited to those listed in paragraph 227.21.
These again are criteria for which specific quantitative tests of
18
-------
compliance cannot be given. However, much information developed in the
Subpart B technical evaluations will be directly relevant to the assess-
ment of potential impacts on living resources and their utilization.
Site Management Considerations
40. The evaluation of the proposed disposal site in relation to
requirements for effective site management must also be considered,
according to paragraph 227.13(a). This is covered in Part 228 of the
Register, of which only paragraph 228.4(e) and Sections 228.9 and 228.12
apply to dredged material. Specific implementation procedures for these
requirements cannot be offered at present, and appropriate techniques to
satisfy the criteria will have to be worked out cooperatively by the
Regional Administrator and the District Engineer on a case-by-case basis.
Decision on the Permit Application
41. It is possible that in some cases adequate information upon
which to base a sound environmental evaluation of a permit under the
criteria will not be supplied. In such cases paragraph 225.2(b) allows
additional information to be requested and the application to be re-
evaluated .
42. Only when dredged material can comply with all the applica-
ble requirements of Parts 227 and 228, as discussed earlier, may a
permit be granted for ocean dumping under paragraph 227.2. The permit
must be denied in all other cases. If the permit is denied but the
dredging is essential and no feasible alternatives are available, a
waiver of the criteria may be sought under Sections 225.3 and 225.4.
19
-------
Appendix A: Reorganization of Section 227.13 from
"Ocean Dumping—Final Revisions of Regulations and Criteria"*
to Incorporate Cross- References
This appendix is appropriate for use by all regulatory elements concerned
with the ocean disposal of dredged material. It is a compilation and
reorganization of those sections of Part 227 of the 11 January 1977 Federal
Register that concern technical evaluation of dredged material proposed for
ocean disposal. There have been no alterations of content, but the sections
have simply been rearranged to incorporate cross-referenced items. This
appendix concerns technical evaluation only and must be used in conjunction
with the other pertinent sections of the Federal Register.
* Environmental Protection Agency, "Ocean Dumping—Final Revisions of Regulations and
Criteria," Federal Register. Part VI, Vol. 42, No. 7, Tuesday, 11 January 1977.
-------
§ 227.1 Applicability.
(a) Section 102 of the Act requires that criteria for the issuance of ocean
disposal permits be promulgated after consideration of the environmental effect
of the proposed dumping operation, the need for ocean dumping, alternatives
to ocean dumping, and the effect of the proposed action on esthetic,
recreational and economic values, and on other uses of the ocean. This Part 227
and Part 228 of this Subchapter H together constitute the criteria established
pursuant to Section 102 of the Act. The decision of the Administrator,
Regional Administrator or the District Engineer, as the case may be, to issue
or deny a permit and to impose specific conditions on any permit issued will
be based on an evaluation of the permit application pursuant to the criteria
set forth in this Part 227 and upon the requirements for disposal site
management pursuant to the criteria set forth in Part 228 of this Subchapter H.
(b) With respect to the criteria to be used in evaluating disposal of dredged
materials, this Section 227.1 and Subparts C, D, E, and G apply in their
entirety. To determine whether the proposed dumping of dredged material
complies with Subpart B, only Sections 227.4, 227.5, 227.6, 227.9, 227.10,
and 227.13 apply. An applicant for a permit to dump dredged material must
comply with all of Subparts C, D, E, G, and applicable sections of B, to be
deemed to have met the EPA criteria for dredged material dumping promulgated
pursuant to Section 102(a) of the Act. If, in any case, the Chief of Engineers
finds that, in the disposition of dredged material, there is no economically
feasible method or site available other than a dumping site, the utilization of
which would result in noncompliance with the criteria established pursuant to
Subpart B relating to the effects of dumping or with the restrictions established
pursuant to Section 102(c) of the Act relating to critical areas, he shall so
certify and request that the Secretary of the Army seek a waiver from the
Administrator pursuant to Part 225.
(c) The Criteria of this Part 227 are established pursuant to Section 102
of the Act and apply to the evaluation of proposed dumping of materials under
Title I of the Act. The Criteria of this Part 227 deal with the evaluation of
proposed dumping of materials on a case-by-case basis from information
supplied by the applicant or otherwise available to EPA or the Corps of
Engineers concerning the characteristics of the waste and other considerations
relating to the proposed dumping.
(d) After consideration of the provisions of Sections 227.28 and 227.29,
no permit will be issued when the dumping would result in a violation of
applicable water quality standards.
§ 227.13 Dredged materials.
(a) Dredged materials are bottom sediments or materials that have been
dredged or excavated from the navigable waters of the United States, and their
disposal into ocean waters is regulated by the U. S. Army Corps of Engineers
using the criteria of applicable sections of Parts 227 and 228. Dredged material
consists primarily of natural sediments or materials which may be contaminated
A1
-------
by municipal or industrial wastes or by runoff from terrestrial sources such
as agricultural lands.
(b) Dredged material which meets the criteria set forth in the following
paragraphs (1), (2), or (3) is environmentally acceptable for ocean dumping
without further testing under this section:
(1) Dredged material is composed predominantly of sand, gravel,
rock, or any other naturally occurring bottom material with particle sizes larger
than silt, and the material is found in areas of high current or wave energy
such as streams with large bed loads or coastal areas with shifting bars and
channels; or
(2) Dredged material is for beach nourishment or restoration and
is composed predominantly of sand, gravel, or shell with particle sizes
compatible with material on the receiving beaches; or
(3) When:
(i) The material proposed for dumping is substantially the same
as the substrate at the proposed disposal site; and
(ii) The site from which the material proposed for dumping
is to be taken is far removed from known existing and historical sources of
pollution so as to provide reasonable assurance that such material has not been
contaminated by such pollution.
(c) When dredged material proposed for ocean dumping does not meet
the criteria of paragraph (b) of this section, further testing of the liquid,
suspended particulate, and solid phases, as defined in Section 227.32, is
required ....
§ 227.32 Liquid, suspended particulate, and solid phases of a material.
(a) For the purposes of these regulations, the liquid phase of a material,
subject to the exclusions of paragraph (b) of this section, is the supernatant
remaining after one hour undisturbed settling, after centrifugation and filtration
through a 0.45 micron filter. The suspended particulate phase is the supernatant
as obtained above prior to centrifugation and filtration. The solid phase includes
all material settling to the bottom in one hour. Settling shall be conducted
according to procedures approved by EPA.
(b) For dredged material, other material containing large proportions of
insoluble matter, materials which may interact with ocean water to form
insoluble matter or new toxic compounds, or materials which may release toxic
compounds upon deposition, the Administrator, Regional Administrator, or the
District Engineer, as the case may be, may require that the separation of liquid,
suspended particulate, and solid phases of the material be performed upon a
mixture of the waste with ocean water rather than on the material itself. In
such cases the following procedures shall be used:
A2
-------
(1) For dredged material, the liquid phase is considered to be the
centrifuged and 0.45 micron filtered supernatant remaining after one hour
undisturbed settling of the mixture resulting from a vigorous 30-minute agitation
of one part bottom sediment from the dredging site with four parts water
(vol/vol) collected from the dredging site or from the disposal site, as
appropriate for the type of dredging operation. The suspended particulate phase
is the supernatant as obtained above prior to centrifugation and filtration. The
solid phase is considered to be all material settling to the bottom within one
hour. Settling shall be conducted by procedures approved by EPA and the
Corps of Engineers.
(2) For other materials, the proportion of ocean water used shall
be the minimum amount necessary to produce the anticipated effect (e.g.,
complete neutralization of an acid or alkaline waste) based on guidance provided
by EPA on particular cases, or in accordance with approved EPA procedures.
For such material the liquid phase is the filtered and centrifuged supernatant
resulting from the mixture after 30 minutes of vigorous shaking followed by
undisturbed settling for one hour. The suspended particulate phase is the
supernatant as obtained above prior to centrifugation and filtration. The solid
phase is the insoluble material settling to the bottom in that period.
§ 227.13(c) Continued
. . .Based on the results of such testing, dredged material can be considered
to be environmentally acceptable for ocean dumping only under the following
conditions:
(1) The material is in compliance with the requirements of
Section 227.6; and. . .
§ 227.6 Constituents prohibited as other than trace contaminants.
(a) Subject to the exclusion of paragraphs (f), (g), and (h) of this section,
the ocean dumping, or transportation for dumping, of materials containing the
following constituents as other than trace contaminants will not be approved
on other than an emergency basis:
(1) Organohalogen compounds;
(2) Mercury and mercury compounds;
(3) Cadmium and cadmium compounds;
(4) Oil of any kind or in any form, including but not limited to
petroleum, oil sludge, oil refuse, crude oil, fuel oil, heavy diesel oil, lubricating
oils, hydraulic fluids, and any mixtures containing these, transported for the
purpose of dumping insofar as these are not regulated under the FWPCA;
(5) Known carcinogens, mutagens, or teratogens or materials
suspected to be carcinogens, mutagens, or teratogens by responsible scientific
opinion.
A3
-------
(b) These constituents will be considered to be present as trace
contaminants only when they are present in materials otherwise acceptable for
ocean dumping in such forms and amounts in liquid, suspended particulate,
and solid phases that the dumping of the materials will not cause significant
undesirable effects, including the possibility of danger associated with their
bioaccumulation in marine organisms.
(c) The potential for significant undesirable effects due to the presence
of these constituents shall be determined by application of results of bioassays
on liquid, suspended particulate, and solid phases of wastes according to
procedures acceptable to EPA, and for dredged material, acceptable to EPA
and the Corps of Engineers. Material shall be deemed environmentally acceptable
for ocean dumping only when the following conditions are met:
(1) The liquid phase does not contain any of these constituents in
concentrations which will exceed applicable marine water quality criteria after
allowance for initial mixing; provided that mercury concentrations in the
disposal site, after allowing for initial mixing, may exceed the average normal
ambient concentrations of mercury in ocean waters at or near the dumping
sites which would be present in the absence of dumping, by not more than
50 percent; and
(2) Bioassay results on the suspended particulate phase of the waste
do not indicate occurrance of significant mortality or significant adverse
sublethal effects including bioaccumulation due to the dumping of wastes
containing the constituents listed in paragraph (a) of this section. These
bioassays shall be conducted with appropriate sensitive marine organisms as
defined in Section 227.27(c) using procedures for suspended particulate phase
bioassays approved by EPA, or, for dredged material, approved by EPA and
the Corps of Engineers. Procedures approved for bioassays under this section
will require exposure of organisms for a sufficient period of time and under
appropriate conditions to provide reasonable assurance, based on consideration
of the statistical significance of effects at the 95 percent confidence level, that,
when the materials are dumped, no significant undesirable effects will occur
due either to chronic toxicity or to bioaccumulation of the constituents listed
in paragraph (a) of this section; and
(3) Bioassay results on the solid phase of the wastes do not indicate
occurrence of significant mortality or significant adverse sublethal effects due
to the dumping of wastes containing the constituents listed in paragraph (a)
of this section. These bioassays shall be conducted with sensitive benthic
organisms using benthic bioassay procedures approved by EPA, or, for dredged
material, approved by EPA and the Corps of Engineers. Procedures approved
for bioassays under this section will require exposure of organisms for a
sufficient period of time to provide reasonable assurance, based on
considerations of statistical significance of effects at the 95 percent confidence
level, that, when the materials are dumped, no significant undesirable effects
will occur due either to chronic toxicity or to bioaccumulation of the
constituents listed in paragraph (a) of this section; and
(4) For persistent organohalogens not included in the applicable
A4
-------
marine water quality criteria, bioassay results on the liquid phase of the waste
show that such compounds are not present in concentrations large enough to
cause significant undesirable effects due either to chronic toxicity or to
bioaccumulation in marine organisms after allowance for initial mixing.
(d) When the Administrator, Regional Administrator or District Engineer,
as the case may be, has reasonable cause to believe that a material proposed
for ocean dumping contains compounds identified as carcinogens, mutagens,
or teratogens for which criteria have not been included in the applicable marine
water quality criteria, he may require special studies to be done prior to issuance
of a permit to determine the impact of disposal on human health and/or marine
ecosystems. Such studies must provide information comparable to that required
under paragraph (c) (3) of this section.
(e) The criteria stated in paragraphs (c) (2) and (3) of this section will
become mandatory as soon as announcement of the availability of acceptable
procedures is made in the FEDERAL REGISTER. At that time the interim
criteria contained in this Section 227.6(e) shall no longer be applicable.
NOTE: The remainder of this paragraph has been deleted since the interim
criteria are superseded by the Implementation Manual.
(f) The prohibitions and limitations of this section do not apply to the
constituents identified in paragraph (a) of this section when the applicant can
demonstrate that such constituents are (1) present in the material only as
chemical compounds or forms (e.g., inert insoluble solid materials) non-toxic
to marine life and non-bioaccumulative in the marine environment upon disposal
and thereafter, or (2) present in the material only as chemical compounds or
forms which, at the time of dumping and thereafter, will be rapidly rendered
non-toxic to marine life and non-bioaccumulative in the marine environment
by chemical or biological degradation in the sea; provided they will not make
edible marine organisms unpalatable; or will not endanger human health or
that of domestic animals, fish, shellfish, or wildlife.
(g) The prohibitions and limitations of this section do not apply to the
constituents identified in paragraph (a) of this section for the granting of
research permits if the substances are rapidly rendered harmless by physical,
chemical or biological processes in the sea; provided they will not make edible
marine organisms unpalatable and will not endanger human health or that of
domestic animals.
§ 227.13(c) Continued
. . .(2)(i) All major constituents of the liquid phase are in compliance
with the applicable marine water quality criteria after allowance for initial
mixing; or. . .
§ 227.31 Applicable marine water quality criteria.
Applicable marine water quality criteria means the criteria given for marine
AS
-------
waters in the EPA publication "Quality Criteria for Water" as published in
1976 and amended by subsequent supplements or additions.
§ 227.29 Initial mixing.
(a) Initial mixing is defined to be that dispersion or diffusion of liquid,
suspended particulate, and solid phases of a waste which occurs within four
hours after dumping. The limiting permissible concentration shall not be
exceeded beyond the boundaries of the disposal site during initial mixing, and
shall not be exceeded at any point in the marine environment after initial
mixing. The maximum concentration of the liquid, suspended particulate, and
solid phases of a dumped material after initial mixing shall be estimated by
one of these methods, in order of preference:
(1) When field data on the proposed dumping are adequate to predict
initial dispersion and diffusion of the waste, these shall be used, if necessary,
in conjunction with an appropriate mathematical model acceptable to EPA or
the District Engineer, as appropriate.
(2) When field data on the dispersion and diffusion of a waste of
characteristics similar to that proposed for discharge are available, these shall
be used in conjunction with an appropriate mathematical model acceptable to
EPA or the District Engineer, as appropriate.
(3) When no field data are available, theoretical oceanic turbulent
diffusion relationships may be applied to known characteristics of the waste
and the disposal site.
(b) When no other means of estimation are feasible,
(1) The liquid and suspended particulate phases of the dumped waste
may be assumed to be evenly distributed after four hours over a column of
water bounded on the surface by the release zone and extending to the ocean
floor, thermocline, or halocline if one exists, or to a depth of 20 meters,
whichever is shallower, and
(2) The solid phase of a dumped waste may be assumed to settle
rapidly to the ocean bottom and to be distributed evenly over the ocean bottom
in an area equal to that of the release zone as defined in Section 227.28. . . .
§ 227.28 Release zone.
The release zone is the area swept out by the locus of points constantly
100 meters from the perimeter of the conveyance engaged in dumping activities,
beginning at the first moment in which dumping is scheduled to occur and
ending at the last moment in which dumping is scheduled to occur. No release
zone shall exceed the total surface area of the dumpsite.
§ 227.29 Continued
. . .(c) When there is reasonable scientific evidence to demonstrate that other
A6
-------
methods of estimating a reasonable allowance for initial mixing are appropriate
for a specific material, such methods may be used with the concurrence of
EPA after appropriate scientific review.
§ 227.13(c) Continued
. . .(ii) When the liquid phase contains major constituents not
included in the applicable marine water criteria, or there is reason to suspect
synergistic effects of certain contaminants, bioassays on the liquid phase of
the dredged material show that it can be discharged so as not to exceed the
limiting permissible concentration as defined in paragraph (a) of
Section 227.27; and. . .
§ 227.27 Limiting permissible concentration (LPC).
(a) The limiting permissible concentration of the liquid phase of a
material is:
(1) That concentration of a constituent which, after allowance for
initial mixing as provided in Section 227.29, does not exceed applicable marine
water quality criteria; or, when there are no applicable marine water quality
criteria,
(2) That concentration of waste or dredged material in the receiving
water which, after allowance for initial mixing, as specified in Section 227.29,
will not exceed a toxicity threshold defined as 0.01 of a concentration shown
to be acutely toxic to appropriate sensitive marine organisms in a bioassay
carried out in accordance with approved EPA procedures.
(3) When there is reasonable scientific evidence on a specific waste
material to justify the use of an application factor other than 0.01 as specified
in paragraph (aX2) of this section, such alternative application factor shall be
used in calculating the LPC.
(b) The limiting permissible concentration of the suspended particulate
and solid phases of a material means that concentration which will not cause
unreasonable acute or chronic toxicity or other sublethal adverse effects based
on bioassay results using appropriate sensitive marine organisms in the case
of the suspended particulate phase, or appropriate sensitive benthic marine
organisms in the case of the solid phase; or which will not cause accumulation
of toxic materials in the human food chain. These bioassays are to be conducted
in accordance with procedures approved by EPA, or, in the case of dredged
material, approved by EPA and the Corps of EngineersJ
An implementation manual is being developed jointly by EPA and the Corps of
Engineers, and announcement of the availability of the manual will be published in
the FEDERAL REGISTER. Until this manual is available, interim guidance on the
appropriate procedures can be obtained from the Marine Protection Branch, WH-548,
Environmental Protection Agency, 4C1 M Street SW, Washington, DC 20460, or the
Corps of Engineers, as the case may be.
A7
-------
(c) "Appropriate sensitive marine organisms" means at least one species
each representative of phytoplankton or zooplankton, crustacean or mollusk,
and fish species chosen from among the most sensitive species documented
in the scientific literature or accepted by EPA as being reliable test organisms
to determine the anticipated impact of the wastes on the ecosystem at the
disposal site. Bioassays, except on phytoplankton or zooplankton, shall be run
for a minimum of 96 hours under temperature, salinity, and dissolved oxygen
conditions representing the extremes of environmental stress at the disposal
site. Bioassays on phytoplankton or zooplankton may be run for shorter periods
of time as appropriate for the organisms tested at the discretion of EPA, or
EPA and the Corps of Engineers, as the case may be.
(d) "Appropriate sensitive benthic marine organisms" means at least one
species each representing filter-feeding, deposit-feeding, and burrowing species
chosen from among the most sensitive species accepted by EPA as being reliable
test organisms to determine the anticipated impact on the site; provided,
however, that until sufficient species are adequately tested and documented,
interim guidance on appropriate organisms available for use will be provided
by the Administrator, Regional Administrator, or the District Engineer, as the
case may be.
§ Section 227.13(c) Concluded
. . .(3) Bioassays on the suspended particulate and solid phases show
that it can be discharged so as not to exceed the limiting permissible
concentration as defined in paragraph (b) of Section 227.27.
(d) For the purposes of paragraph (c)(2), m^jor constituents to be
analyzed in the liquid phase are those deemed critical by the District Engineer,
after evaluating and considering any comments received from the Regional
Administrator, and considering known sources of discharges in the area.
A8
-------
APPENDIX B: DREDGED MATERIAL SAMPLE COLLECTION AND PREPARATION
Introduction
1. The collection and preparation of disposal site water and
dredged material samples for testing is one of the more important
phases of evaluating the impact of dredged material discharge upon
the aquatic environment. Samples that are improperly collected, pre-
served, or prepared will totally invalidate any testing conducted and
will lead to erroneous conclusions regarding the potential impact of
the proposed discharge. Meticulous attention must therefore be given
to all phases of water and sediment sampling, storage, and preparation.
The procedures described herein specify the apparatus and procedures to
use for sampling water and dredged material and preparing the water and
dredged material for chemical analyses and bioassay procedures.
Sample Collection and Preservation
2. Collection and preservation of dredged material and water
samples are discussed in this section. The procedures are designed to
minimize sample contamination and alteration of the physical or chemi-
cal properties of the samples due to freezing, air oxidation, or drying.
Number of samples
3. The number of sediment and water samples to be taken from the
dredging or excavation site for processing must be carefully considered
because of the extremely heterogeneous nature of samples of this type.
The largest source of variation between dredged material samples taken
at a dredging site has been shown to be the vertical and horizontal
distribution of the samples. With this in mind, sediment should be
collected from a minimum of three sampling stations within the dredging
area. The sampling stations should be located throughout the area to
be dredged and should be selected to characterize obviously contaminated
as well as noncontaminated areas. The amount of dredged material or
water collected should be limited to the amount that can be used within
2 weeks after sampling.
B1
-------
Apparatus
4. The following items are required for water and dredged materi-
al sampling and storage.
a.. Noncontaminating sediment grab or core sampler (Smith-
Mclntyre or Van Veen grab, K. B. corer, etc.).
b_. Noncontaminating water sampler (Van Dorn water sampler,
etc.).
Acid-rinsed linear polyethylene bottles for water
samples to be analyzed for metals and nutrients.
(i. Solvent-rinsed glass bottles with Teflon-lined screw-
type lids for water samples to be analyzed for pesticide materials.
e^ Plastic jars or bags for collection of dredged material
samples.
f_. Ice chests for preservation and shipping of dredged
material and water samples.
Water sampling
5. Collection of water samples should be made with appropriate
noncontaminating water-sampling devices. Special care must be taken
to avoid the introduction of contaminants from the sampling devices and
containers. To avoid trace metal contamination, sampling devices should
be constructed of plastic materials. Frior to use, the sampling devices
and containers should be thoroughly cleaned with a detergent solution,
rinsed with tap water, soaked in 10-percent hydrochloric acid (HC1) for
4 hr, and then thoroughly rinsed with metal-free water. Water samples
taken for trace organic analyses should be taken with glass or stainless
steel devices. If plastic devices must be used, they must be cleaned,
aged, and characterized as to the material that may leach from them
into the samples. The sampling devices should be thoroughly cleaned,
following the procedures outlined in the "Manual of Analytical Methods
for the Analysis of Pesticide Residues in Human and Environmental
2
Samples," and then rinsed just before using with the same solvent to
be used in the analysis, most probably hexane.
6. A representative disposal site water sample is obtained by
B2
-------
collecting 1/3 of the sample volume directly below the water surface,
1/3 from mid-depth in the water column, and 1/3 from approximately 1 m
above the sediment surface. The portion of the samples to be used for
pesticide material analyses must be stored in glass or aluminum con-
tainers .
7. The samples should be stored immediately at 2 to 4°C, never
frozen. The storage period should be as short as possible to minimize
changes in the characteristics of the water. It is recommended that
samples be processed within two weeks of collection.
Sediment sampling
8. Sediment samples should be taken with a corer or a grab
sampler in a manner designed to ensure that their characteristics are
representative of the proposed dredging site. Sampling stations should
include known or suspected areas of high contamination as well as more
representative areas. The larger the proposed dredging site, the more
samples will be required for adequate coverage and characterization.
The samples should be placed in airtight linear polyethylene containers.
If organic materials are of primary concern, airtight glass storage con-
tainers should be used. Care should be taken to ensure that the con-
tainers are completely filled by the samples and that air bubbles are
not trapped in the containers. The samples should be stored immediately
at 2 to 4°C. The samples must never be frozen or dried. The storage
period should be as short as possible to minimize changes in the charac-
teristics of the dredged material. It is recommended that the samples
be processed within two weeks of collection.
Liquid Phase Sample Preparation
9. Water and liquid phase samples should be prepared for bio-
assays and/or chemical analysis as soon as possible after collection.
The liquid phase may be used for either chemical analysis as given in
Appendix C or for bioassay testing as detailed in Appendices D and E.
The volume of solution needed for chemical analyses will vary depending
upon the number and type of analyses to be conducted (Appendix C).
Appendices D and E should be consulted to determine the volumes
B3
-------
required for bioassays.
Apparatus
10. The following items are required.
a_. Laboratory shaker capable of shaking 2-1 flasks at
approximately 100 excursion per min. Box-type or
wrist-action shakers are acceptable.
Id. Several l-£ (or larger) graduated cylinders.
c^. Large (15 cm) powder funnels.
d_. Several 2-£ large-mouth graduated Erlenmeyer flasks.
e_. Vacuum or pressure filtration equipment, including vacuum
pump or compressed air source and an appropriate filter
holder capable of accomodating 47-, 105-, or 155-mm-diam
filters.
f_. Presoaked filters with a 0.45-y pore-size diameter.
£. Centrifuge capable of handling six 1.0- or 0.5-£ centri-
fuge bottles and operating at 3000 to 5000 rpm.
h. Plastic sample bottles, 500-ml capacity for storage of
water and liquid phase samples for metal and nutrient
analyses.
i^. Wide-mouth, 1-gal capacity glass jars with Teflon-lined
screw-type lids should be used for sample containers when
samples are to be analyzed for pesticide materials. (It
may be necessary to purchase jars and Teflon sheets
separately; in which case, the Teflon lid liners may be
prepared by the laboratory personnel.)
11. Prior to use, all glassware, filtration equipment, and filters
should be thoroughly cleaned. Wash all glassware with detergent, rinse
five times with tap water, place in a clean 10-percent (or stronger) HC1
acid bath for a minimum of 4 hr, rinse five times with tap water, and
then rinse five times with distilled or deionized water. Soak filters
for a minimum of 2 hr in a 5-M HC1 bath and then rinse 10 times with
distilled water. It is also a good practice to discard the first 50 ml
of water or liquid phase filtered. Wash all glassware to be used in
preparation and analysis of pesticide residues using the eight-step pro-
cedure given in the "Manual of Analytical Methods for the Analysis of
2
Pesticide Residues in Human and Environmental Samples." Flush the
glassware just before using with the same solvent to be used in the
B4
-------
pesticide analyses.
Sample preparation
12. In order to properly prepare liquid phase samples for chemical
analyses or for use in bioassays, the stepwise procedure given below
must be followed.
13. Step 1. Subsample approximately 1 SL of sediment from the well-
mixed original sample. Mix the sediment and unfiltered disposal site
water in a volumetric sediment-to-water ratio of 1:4 at room temperature
(22 + 2°C) . This is best done by the method of volumetric displacement.
One hundred ml of unfiltered disposal site water is placed into a gradu-
ated Erlenmeyer flask. The sediment subsample is then carefully added
via a powder funnel to obtain a total volume of 300 ml. (A 200-ml
volume of sediment will now be in the flask.) The flask is then filled
to the 1000-ml mark with unfiltered disposal site water, which produces
a slurry with a final ratio of one volume sediment to four volumes water.
If the volume of liquid phase required for bioassay or analysis exceeds
700 to 800 ml, the initial volumes should be proportionately increased
(e.g., mix 400 ml of sediment and 1600 ml of disposal site water).
Alternately, several l-£ dredged material/disposal site water slurries
may be prepared as outlined above and the filtrates combined to provide
sufficient liquid phase.
14. Step 2.
a. Cap the flask tightly with a noncontaminating stopper and
shake vigorously on an automatic shaker at about 100 oscillations per
min for 30 min. A polyfilm-covered rubber stopper is acceptable for
minimum contamination.
Jb. During the mixing step given in paragraph 14a, the oxygen
demand of the dredged material may cause the dissolved oxygen concen-
tration in the flask to be reduced to zero. This change can alter the
release of chemical contaminants from dredged material to the disposal
3
site water and reduce the reproducibility of the liquid phase tests.
If it is known that anoxic conditions (zero dissolved oxygen) will not
occur at the disposal site or if reproducibility of liquid phase analyses
B5
-------
is a potential problem, the mixing may be accomplished by using a com-
pressed air mixing procedure instead of the mechanical mixing described
in paragraph 14a. To accomplish air mixing, after the preparation of
the sediment-water slurry, an air-diffuser tube is inserted almost to
the bottom of the flask. Compressed air should be passed through a
deionized water trap and then through the diffuser tube and the slurry.
The flow rate should be adjusted to agitate the mixture vigorously for
30 min. In addition, the flasks should be stirred manually at 10-min
intervals to ensure complete mixing.
• Step 3. After shaking or mixing with air, allow the sus-
pension to settle for 1 hr.
16. Step 4.
a_. If analysis of pesticide or polychlorinated biphenyl (PCB)
materials is desired, carefully decant an appropriate portion of the
supernatant after the settling period. Samples to be analyzed for pesti-
cide or PCB materials must be free of particulates but should not be
filtered, due to the tendency for these materials to adsorb on the
filter. However, particulate matter can be removed before analysis by
high-speed centrifugation at 10,000 times gravity using Teflon, glass,
4
or aluminum centrifuge tubes.
b. If analyses for nonpesticide or non-PCB materials are
desired or if liquid phase bioassays are to be conducted, at the end
of the settling period, carefully decant the supernatant into appro-
priate centrifuge bottles and then centrifuge. The time and rpm's
during centrifugation should be selected to reduce the suspended solids
concentration substantially and therefore shorten the final filtration
process. After centrifugation, vacuum or pressure filter approximately
50 ml of sample through a 0.45-y filter and discard the filtrate.
Filter the remainder of the sample to give a clear final solution.
17. Step 5.
a. Samples to be analyzed for pesticide or PCB materials
should immediately undergo solvent extraction, as described in the
analytical references given in paragraph 3 of Appendix C. The
B6
-------
extract may then be held in clean uncontamlnating containers for periods
up to three or four weeks at -15 to -20°C before the analyses are per-
formed .
b_. Samples for metals analysis should be preserved
immediately after filtration by lowering the pH to < 2 with 3 to 5 ml
of concentrated nitric acid per litre.^ High purity acid, either
purchased commercially or prepared by a subboiling unit, must be used.
c_. Nutrient analyses should be conducted immediately.
Acidification with to pH < 2 and storage at 4°C may allow the
sample to be held for a maximum of 24 hr for ammonia nitrogen, Kjeldahl
nitrogen, and nitrate nitrogen analyses.^ Storage at 4°C will allow
holding of samples to be analyzed for dissolved orthophosphate and total
dissolved phosphorus for up to 24 hr."* Subsamples to be analyzed for
cyanide should be preserved with 2 ml of 10 N sodium hydroxide per litre
of sample (pH > 12)."*
d_. Liquid phase samples to be used in bioassays must not be
preserved or stored. Bioassays should begin as soon as the liquid
phase is prepared.
Disposal Site Water Sample Preparation
18. Disposal site water samples are prepared by following the
filtration and preservation steps discussed in paragraphs 11, 16, and
17.
Suspended Particulate Phase Sample Preparation
19. The suspended particulate phase, which is used exclusively for
bioassays, is prepared in a manner very similar to that for the liquid
phase. The steps given in paragraphs 11, 13, 14, and 15 are followed
exactly. The suspended particulate phase is the liquid and that materi-
al remaining in suspension after the 1-hr settling period (in other words,
the suspended particulate phase is an unfiltered liquid phase). With
some very fine-grained sediments, it may be necessary to centrifuge the
supernatant after the 1-hr settling period. This centrifugation, if
used at all, should be only enough to make the test organisms visible
during the bioassay. The suspended particulate phase bioassay should
begin as soon as the suspended particulate phase is prepared.
B7
-------
Solid Phase Sample Preparation
20. The solid phase of dredged material is used exclusively in
bioassays or bioaccumulation studies as discussed in Appendices F and
G. The solid phase is defined for bioassessment purposes as sediments
of in situ density collected within the dredging site. These sediment
samples should be collected and stored as described in paragraph 8.
The solid phase for use in bioassays should be prepared immediately
prior to beginning the bioassays. Indeed, the solid phase preparation
is an integral part of the bioassay procedure and is described in
detail in Appendix F, "Guidance for Performing Solid Phase Bioassays."
REFERENCES
1. Brannon, J. M., et al., "Selective Analytical Partioning of
Sediments to Evaluate Potential Mobility of Chemical Constituents
During Dredging and Disposal Operations," Technical Report No.
D-76-7, December 1976, Environmental Effects Laboratory, U. S. Army
Engineer Waterways Experiment Station, CE, Vicksburg, Mississippi.
2. U. S. Environmental Protection Agency, "Manual of Analytical
Methods for the Analysis of Pesticide Residues in Human and Environ-
mental Samples," 1974, Environmental Toxicology Division, Research
Triangle Park, North Carolina.
3. Lee, G. F., et al., "Research Study for the Development of Dredged
Material Disposal Criteria," Contract Report No. D-75-4, November
1975, prepared by the Institute for Environmental Sciences,
University of Texas at Dallas, under contract to the U. S. Army
Engineer Waterways Experiment Station, CE, Vicksburg, Mississippi.
4. Fulk, R., et al., "Laboratory Study of the Release of Pesticide and
PCB Materials to the Water Column During Dredging and Disposal
Operations," Contract Report No. D-75-6, December 1975, prepared
by Envirex, Inc., Milwaukee, Wisconsin, under contract to the U. S.
Army Engineer Waterways Experiment Station, CE, Vicksburg,
Mississippi.
5. U. S. Environmental Protection Agency, "Methods for Chemical
Analysis of Water and Wastes," EPA-625-/6-74-003, EPA National
Environmental Research Center, Analytical Quality Control Labora-
tory, Cincinnati, Ohio. (Available from the Office of Technology
Transfer, Washington, D. C., 20460).
B8
-------
APPENDIX C: LIQUID PHASE CHEMICAL ANALYSES
1. Presented herein are procedural references for chemical
analyses of disposal site water and the liquid phase of dredged
material. Samples must be collected, preserved, and prepared according
to directions in Appendix B. Trace metal analyses in the liquid phase
of dredged material from saline waters are both difficult and compli-
cated because of the high salt content. Special analytical methods
such as solvent extraction prior to metal analyses are often required.
Also, because of the comparatively low background concentrations of
some constituents in samples of this type, the number of replicate
analyses of composite liquid phase or disposal site water samples must
be carefully considered.
Apparatus
2. The specific equipment necessary for liquid phase chemical
analyses will vary depending on the chemical constituent(s) to be
analyzed. Referenced procedure manuals should be consulted to deter-
mine specific needs, sample size, proper cleanup procedures for glass-
ware and other apparatus, and possible interferences in the
i ^ 1. 2, 3
analysis.
Procedures
3. The liquid phase may be treated as a water sample and analyzed
as described in the referenced methods. Standard procedures for anal-
ysis for specific constituents other than pesticides and polychlorinated
biphenyl (PCB) materials are given in Table CI and analytical procedures
for pesticides and PCB materials are given in Table C2.
4. Prepare and analyze the liquid phase in triplicate and report
the average concentration of the three replicates as the concentration
of the contaminant of concern in the liquid phase. Report all concen-
trations in milligrams or micrograms per litre.
Interpretation of Results
5. Paragraph 227.29(a)(1) of the Register defines the limiting per-
missible concentration (LPC) of the liquid phase as that concentration
CI
-------
Table CI
Procedural References for Liquid Phase Analytical Methods for
Specific Constituents Other than Pesticides and PCBs
Other
Parameter
Reference 1
Reference 2
Reference 3
References
Inorganic
Constituents
Cyanide (total)
p. 40
Method 413
Method D2036
p. 361
p. 503
Flouride (total)
p. 59
Method 414A and C
Method D1179
_
p. 389
p. 310
Metals (dissolved)
p. 82(4.1.3)
-
-
_
Antimony
p. 94
-
-
-
Arsenic
p. 95
-
-
-
Beryllium
p. 99
-
-
-
Cadmium
p. 101
p. 151
p. 351
_
Chromium
p. 105
p. 151
p. 351
-
Cobalt
p. 107
p. 151
p. 351
-
Copper
p. 108
-
p. 351
-
Iron
p. 110
p. 151
p. 351
-
Lead
p. 112
p. 151
p. 351
Manganese
p. 116
p. 151
p. 351
-
Mercury
p. 118, 138
-
p. 344
.
Nickel
p. 141
-
p. 351
-
Selenium
p. 145
-
-
-
Vanadium
p. 153
-
-
_
Zinc
p. 155
p. 151
p. 351
-
Nitrogen
Ammonia
p. 159
-
-
.
Nitrate-Nitrite
p. 201
Method 419C
-
_
p. 423
Total Kjeldahl
p. 175
Method 421
-
_
p. 437
Phosphorus
Method 6515
Total
p. 249
Method 425C
-
III p. 474
p. 387
Ortho
p. 249
Method 425F
Method D515
_
p. 481
p. 389
Organic Constituents (except chlorinated hydrocarbons)
Amines
Benzidine
Methyl mercury
Oil and grease
Petroleum hydrocarbons
Phenol
Phenols (specific)
p. 229
Step 7.
p. 226
Step 6.
p. 241
Method 510
p. 574
Method D1783
p. 542
Method D2580
p. 548
Step 13.1
Ref. 4
Ref. 5
Step 8.2
Ref. 6
C2
-------
Table C2
Procedural References for Liquid Phase Analytical Methods for
Pesticides and PCB Materials
Parameter
PCBs
Pesticides:
N-Aryl carbamates
barb an
chloropropham
diuron
linuron
monuron
O-Aryl carbamates
baygon
carbaryl (sevin)
metacil
mesural
zectran
Organochlorine
aldrin
DDT
dieldrin
endrin
toxaphene
Organophosphorus
malathion
methyl parathion
parathion
guthion
demeton
diazinon
disyston
Phenoxy acids
2,UD
silvex
2,U,5-T
Triazines
altrazine
propazine
Reference 7
Section 10, A
Section 10, A
Section 10, A
Other References
Reference 8
Reference 9-, Step 9.3
Reference 10, Step 9.3
Reference 11, Step 9.3
Reference 12 , Step 9.3
Reference 13
Reference lU , Step 9»3
C3
-------
ac which none of the constituents of concern will exceed their re-
spective water-quality criteria after allowance for initial mixing.
Whether the LPC would be exceeded can be determined by comparing the
volume of the initial mixing zone to the volume of water required to
dilute the liquid phase sufficiently to meet the water-quality criteria
for the constituent of concern. The appropriate calculations are
illustrated in paragraphs 15 through 20 of Appendix H, "Estimation
of Initial Mixing." The LPC would be exceeded only if the required
dilution volume exceeds the volume of the initial mixing zone.
REFERENCES
1. U. S. Environmental Protection Agency, "Methods for Chemical
Analysis of Water and Wastes," EPA-625/6-74-003, July 1974,
National Environmental Research Center, Analytical Quality
Control Laboratory, Cincinnati, Ohio. (Available from the
Office of Technology Transfer, Washington, D. C. 20460.
2. American Public Health Association, Standard Methods for the
Examination of Water and Wastewater, 14th Ed, American Water
Works Association, Water Pollution Control Federation, APHA,
Washington, D. C., 1975.
3. American Society for Testing and Materials, 1974 Annual Book of ASTM
Standards, Part 31, Water, Philadelphia, Pennsylvania.
4. Anonymous, "Direct Aqueous Injection for Quantification,"
Analytical Chemistry, Vol. 46, 1974, p. 977. (Column packing
is available through Supelao, Inc.3 Bellefonte, PA. Request
Bulletin 738-Carbopack.)
5. U. S. Environmental Protection Agency, "Method for Benzidine
and Its Salts in Wastewater," 1973, Environmental Monitoring
and Support Laboratory, Cincinnati, Ohio.
6. U. S. Environmental Protection Agency, "Methyl Mercury in Sediment,"
1973, Environmental Monitoring and Support Laboratory, Cincinnati,
Ohio.
7. U. S. Environmental Protection Agency, "Analysis of Pesticide
Residues in Human and Environmental Samples," 1974, Environ-
mental Toxicology Division, Research Triangle Park, North
Carolina.
C4
-------
8. U. S. Environmental Protection Agency, "Method for Polchlorinated
Biphenyls (PCB's) in Industrial Effluents," 1973, Environmental
Monitoring and Support Laboratory, Cincinnati, Ohio.
9. U. S. Environmental Protection Agency, "Method for N-Aryl Carbamate
and Urea Pesticides in Industrial Effluents," 1973, Environmental
Monitoring and Support Laboratory, Cincinnati, Ohio.
10. U. S. Environmental Protection Agency, "Method for O-Aryl Carbamate
Pesticides in Industrial Effluents," 1973, Environmental Monitoring
and Support Laboratory, Cincinnati, Ohio.
11. U. S. Environmental Protection Agency, "Method for Organochlorine
Pesticides in Industrial Effluents," 1973, Environmental Monitoring
and Support Laboratory, Cincinnati, Ohio.
12. U. S. Environmental Protection Agency, "Method for Organo-
phosphorus Pesticides in Industrial Effluents," 1973, Environmental
Monitoring and Support Laboratory, Cincinnati, Ohio.
13. U. S. Environmental Protection Agency, "Method for Chlorinated
Phenoxy Acid Herbicides in Industrial Effluents," 1973, Environ-
mental Monitoring and Support Laboratory, Cincinnati, Ohio.
14. U. S. Environmental Protection Agency, "Method for Triazine
Pesticides in Industrial Effluents," 1973, Environmental Monitoring
and Support Laboratory, Cincinnati, Ohio.
C5
-------
APPENDIX D: GUIDANCE FOR PERFORMING LIQUID PHASE AND
SUSPENDED PARTICULATE PHASE ANIMAL BIOASSAYS
Introduction
1. The described bioassay of appropriate sensitive marine organ-
isms can be used as an aid in evaluating the importance of dissolved
chemical constituents released from the sediment during disposal oper-
ations. This procedure can also be used to evaluate the effect of
suspended particulate matter that is present in the water column for
certain periods of time during disposal of dredged material. A series
of experimental treatments and controls are established using the
liquid phase or suspended particulate phase of the dredged material and
disposal site water. The test organisms are added to the test chambers
and incubated under standard conditions for a prescribed period of time.
The surviving organisms are examined at appropriate intervals to de-
termine if the test material is producing an effect. Phytoplankton
bioassays require a somewhat different approach and are described in
Appendix E, "Guidance for Performing Liquid Phase and Suspended Particu-
late Phase Phytoplankton Bioassays."
Apparatus
2. The following items are required for each separate test series,
which consists of one set of control and test aquaria with three repli-
cates of each. Appropriate additional items will be needed for each
additional test series.
a_. Twelve crystallizing dishes (100 mm x 50 mm) to be used
as test containers for zooplankton and larvae.
Jk Covers for the crystallizing dishes. Sheets of window
glass or clear plastic are suitable,
jc. Twelve 10-gallon (37.8-£) all-glass aquaria, 26 cm wide,
51 cm long, and 31 cm deep, to be used as test containers
for crustaceans, molluscs, and fish.
d_. Transfer pipettes with a 0.2- to 0.3-nm bore size and
D1
-------
rubber bulbs; transfer pipettes with 7- to 9-mm bore size.
Fine-mesh dip nets.
f_. Facility for maintaining constant temperature and ap-
propriate photoperiod in the test containers. Any
incubator that allows control of the temperature within
+ 1°C and provides acceptable lighting will suffice.
Cool-white flourescent lighting located above the test
units at a distance of approximately 0.5 to 1 m should
be used.
A light box with illumination from below for ease in
counting zooplankton and larvae.
Species Selection
3. Liquid phase and suspended particulate phase bioassays must
utilize appropriate sensitive marine organisms as described in para-
graph 227.27(c) of the Register (see Appendix A). The sensitivity of
all bioassays is dependent primarily on the selection of appropriate
species.
4. If at all possible the test species should be collected from
a reference area near the disposal site and similar to it in water
quality and substrate sedimentology , but with no recent history of dis-
posal. They should be the same species or be closely related to those
species that naturally dominate biological assemblages in the vicinity
of the disposal site in the season of the proposed operation. Experi-
ence has shown that with reasonable care it is possible to collect test
organisms from wild populations and maintain them under controlled
conditions with low mortality. However, a preliminary study of the
ability of field-collected test organisms to acclimate to laboratory
conditions is highly desirable.
5. If it is not practical to use the dominant species collected
from near the disposal site, test species may be selected from Table D1
if they are chosen so that, insofar as possible, they are related phylo-
genetically and/or by ecological requirements to the dominant appropri-
ate sensitive marine organisms expected in the area of the disposal
D2
-------
site at the time of the proposed operation. Commercially important
organisms from the vicinity of the disposal area may also be included
if desired. In Table Dl, species are not listed in order of preference,
except that the fish of group I are the most desirable for bioassay pur-
poses, and those in group III are considered generally less likely to be
sensitive indicators of potential effects.
6. All liquid phase and suspended particulate phase bioassays
should include a species of mysid shrimp of the genus Mysidopsis or
Neomysis. This will provide an internal standard in all bioassays and
form a basis for quality assurance in the regulatory program.
7. It is recommended that juvenile forms, particularly of fish,
be utilized because of their generally greater sensitivity than adults.
The wet weight of individual test specimens should not be greater than
3 g. Molluscs generally are relatively resistant to many toxicants, and
therefore are often undesirable for bioassays, but they are very useful
for bioaccumulation studies. If used in bioassays, they should be less
than 2 cm long. To avoid predation, it probably will be necessary to
conduct the bioassay with potential predator and prey species isolated
from each other. For example, fish and zooplankton or larvae must be
separated to avoid predation. The identity of all test species must be
verified by experienced taxonomists. If the bioassay animals are also
to be used in estimating bioaccumulation potential, species selection
should consider the factors discussed in paragraphs 5, 6, and 7 of
Appendix G, "Guidance on Assessing Bioaccumulation Potential."
8. Whatever the source of the animals, collection and handling
should be as gentle as possible. Transportation to the laboratory
should be in well-aerated water from the animal collection site in which
the animals are held at the temperature and salinity from which they
were obtained. Animals should be held in the laboratory no more than
two weeks before bioassays are begun. During this period they must be
gradually acclimated to the salinity and temperature at which the bio-
assay will be conducted. Methods for collecting, handling, acclimating,
and sizing bioassay organisms given in "Bioassay Procedures for the
D3
-------
Table D1
Recommpnded Appropriate Sensitive Marine Organisms for Use in
Liquid Phase and Suspended Particulate Phase Bioassays*
Zooplankton
Copepods, Aaartia sp.
Larvae of reconmended
crustacean or mollusc
species
Crustacean or Mollusc
Mysid shrimp, Mysidopsis sp.**
Neomyaie sp**
Grass shrimp, Palaemonetes sp.
Palaemon sp.
Commercial shrimp, Penaeus sp.
Sand shrimp, Cvangon sp.
Oceanic shrimp, Pandalus sp.
American lobster, Homarus amerioanus
Blue crab, Callineotes sapidus
Cancer crab, Cancer sp.
Amphipods, AmpeHsca sp.
Paracphoxus sp.
Cumacean, Diaaty lap-is sp.
Macoma clam, Macoma sp.
Nucula clam, Nueula sp.
Yoldia clam, Yoldia sp.
Surf clam, Spisula. eolidlssima
Hard clam (quahog), Mereenaria sp.
Ocean quahog, Arotioa islandiaa
Scallop, Argopectin sp.
Aequipeatin sp.
Gemma clam, Germa gemma
Edible mussel, Mytilns edul-ie
Fish
Group I
Silversides, Menidia sp.
Pinfish, Lagodon rhombiodes
Spot, Leiostomus xanthurus
Shiner perch, Cymatogaster aggregata
Group II
English sole, Parophrys vetulus
Flounder, Platichthys sp.
Paraliehthya sp.
Limanda sp.
Group III
Sheepshead minnow, Cyprinodon
variegatus
Mummichog, Fundutus heteroalitus
Killifish, Fundutus sp.
* Lists are not in order of preference or desirability except for the groups of fish.
** All liquid phase and suspended particulate phase bioassays should include one of these species.
-------
Ocean Disposal Permit Program" and "Standard Methods for the Examina-
2
tion of Water and Wastewater" should be followed in all matters for
which no guidance is given here.
Sample Collection and Preservation
9. Sediment and water samples are collected and stored and the
liquid phase and suspended particulate phase are prepared as described
in Appendix B, "Dredged Material Sample Collection and Preparation."
Water collected from the disposal site should be used if at all possi-
ble. Otherwise uncontaminated seawater or an artificial sea salts
mixture (such as that given on page 32 of Reference 1) of the proper
salinity may be used.
Experimental Conditions
10. Liquid and suspended particulate phase bioassays should be
conducted at a salinity near that expected at the disposal site at the
time of the proposed operation. Experimental temperature should be held
stable within + 1°C of a temperature approximating that expected at the
disposal site in the season of the proposed operation. Recommended
experimental temperatures are given on a seasonal basis for various zoo-
geographic areas in the following tabulation.
Test
Temperature, C
Zoogeographic
Areas
Summer Winter
CE Division
EPA Region
20 5
New England
I
North Atlantic
II*
III
25 12
South Atlantic
IV
Lower Mississippi Valley
VI
Southwestern
10 10
North Pacific
X
South Pacific
IX**
25 25
Pacific Ocean
IX**
*Puerto Rico and Virgin Islands are in EPA Region II,
but should use temperatures recommended for Region IV.
**Mainland portions of Region IX should use South Pacific
Division temperatures; Pacific island portions of Region
IX should use Pacific Ocean Division temperatures.
D5
-------
11. Dissolved oxygen should not be allowed to fall below A ppm,
unless there is reason to believe that depression to lower levels
would occur for a substantial period of time in the field during the
proposed disposal operation or if lower levels occur naturally at the
2
site. Light intensity should be approximately 1200 yw/cm using cool-
white flourescent bulbs with a 14-hr light and 10-hr dark cycle. At
the beginning and end of the bioassay, the temperature, salinity, dis-
solved oxygen, and pH in the test containers should be measured and
reported.
Experimental Procedure
12. Glassware must be extremely clean. Wash all glassware with
detergent, rinse five times with tap water, place in a clean 10-percent
HC1 acid bath for a minimum of 4 hr, rinse five times with tap water,
and then rinse five times with distilled water.
13. Establish treatment levels using disposal site water and
liquid phase or suspended particulate phase of the material, prepared
as described in Appendix B. A minimum of three replicates of each
experimental and control condition must be used. More replicates should
be used whenever possible, as this will increase the sensitivity and
reliability of the test. The final liquid volume in each dish is 200
ml and in each aquarium is 30 1.
13. The following concentrations of the test material are recom-
mended as a minimum, with more being desirable whenever possible.
Percent Percent
Liquid Phase Disposal Site Water
10 90
50 50
100 0
Percent Suspended Percent
Particulate Phase Disposal Site Water
10 90
50 50
100 0
The following controls should be used for each phase:
D6
-------
£i. 100-percent fresh culture water of the type in
which the animals have been held prior to testing.
b^. If the bioassays are conducted with disposal site
water,it is advisable to establish an additional
control of 100-percent disposal site water.
14. Ten organisms are exposed in each test dish or aquarium.
Individual organisms must be randomly assigned to treatments. Make
every attempt to collect animals of approximately equal size. Use a
pipette to transfer zooplankton and larvae from the laboratory culture
vessel to the test containers. Care must be taken during the transfer
process to ensure that air is not trapped on the zooplankton and
larvae. Place the pipette under the surface of the liquid in the test
container and gently release the liquid and animal into the test
solution. Juvenile and adult crustaceans, molluscs, and fish are
transferred to the test containers in fine-mesh nets. Submerge the
net in the test container and gently evert it, releasing the animals.
During this process, transfer the minimum amount of culture medium
possible with each animal and use a different pipette or net for each
concentration of test solution. The utmost care should be taken when
transferring any of the animals from holding facilities to the exposure
containers to avoid damaging the organisms. Discard any animals that
are dropped or physically abused during the transfer. Never touch
animals by hand. References 1 and 2 provide detailed instructions on
handling and transfer procedures.
15. Cover the dishes and incubate the test containers in an
appropriate test chamber. Positioning of the test containers holding
various concentrations of test solution must be randomized. The test
medium is not replaced during the 96-hr experimental period. No aera-
tion is supplied (unless indicated by the considerations in paragraph
11), and the test medium is not stirred. Therefore, some sedimentation
will take place during suspended particulate bioassays, and at the end
of the test only very fine particles will remain in suspension.
16. Observations should be made at 0, 4, 8, 24, 48, 72, and 96 hr.
Animals are counted visually at each observation time with the aid of a
D7
-------
light box or dissecting microscope. Take care to minimize stresses on
the animals during counting. Counting should be done quickly and the
animals immediately returned to the test containers. Death is the end
point, so the number of living organisms is recorded. Death is deter-
mined by lack of movement after a gentle swirl of the dish or gentle
touching of a sensitive part with a probe. All crustaceans, both larval
and adult, molt at regular intervals, shedding a complete exoskeleton.
Care should be taken not to count an exoskeleton as a dead animal. Dead
animals may decompose or be eaten between observations. Therefore,
always count living, not dead, animals. Remove dead organisms and molted
exoskeletons at each observation with a pipette or forceps. Care must
be taken not to disturb living organisms and to minimize the amount of
liquid withdrawn.
Data Analysis and Interpretation
17. The criteria require that liquid and suspended particulate
phase bioassay results be interpreted in view of the mixing and dilution
expected at the disposal site. According to Section 227.13 of the
Register, dredged material can be considered environmentally acceptable
for ocean disposal only if bioassay results and initial mixing estimates
indicate that the limiting permissible concentration (LPC) will not be
exceeded (Section 227.27). Therefore, bioassay results cannot be
interpreted until initial mixing calculations are performed, as de-
scribed in Appendix H, "Estimation of Initial Mixing." Procedures in
this section apply to both liquid phase and suspended particulate phase
bioassays of all appropriate sensitive marine organisms.
Data presentation
18. Complete survival data in all test containers at each obser-
vation time should be presented as shown in Table D2. The species must
be identified by scientific name. If greater than 10-percent mortality
occurs in the controls, all data must be discarded and the experiment
repeated. Control mortalities of 20-percent may be acceptable in zoo-
plankton and larval bioassays. Unacceptably high control mortality
indicates the presence of important stresses on the organisms other
D8
-------
Table D2
Hypothetical Results of a Bioassay of the Liquid Phase
or Suspended Particulate Phase of a Dredged Material*
Time of Observation - Number of Survivors
Exposure Condition
Replicate
0 hr
4 hr
8 hr
24 hr
48 hr
72 hr
96 hr
Culture water control
1
10
10
10
10
10
10
10
2
10
10
10
8
8
8
8
3
10
30
10
30
10
30
10
28
10
28
10
28
10
28
100-percent test medium
1
10
10
10
8
7
5
4
2
10
10
9
7
6
4
2
3
10
30
10
30
10
29
8
23
6
19
4
13
3
9
50-percent test medium
1
10
10
10
9
9
7
6
2
10
10
10
10
9
8
6
3
10
30
olo
—l|fO
10
30
10
29
9
27
7
22
6
18
10-percent test medium
1
10
10
10
10
9
9
9
2
10
10
10
9
9
9
9
3
10
30
10
30
olo
10
29
10
28
10
28
10
28
* Appropriate sensitive marine organisms must be used, and table must include the scientific
name of species tested.
-------
than the material being tested, such as injury or disease, stressful
physical or chemical conditions in the test containers, or improper
handling, acclimation, or feeding. If less than 10-percent (or 20-
percent) mortality occurs in the controls, the data may be evaluated.
Statistical analysis
19. To assess the possibility of unacceptable adverse impacts in
the water column, it is necessary to statistically compare the 96-hr
survival in the appropriate control to survival in the 100-percent test
medium and then to determine the LPG. If mortalities are similar in
both controls, the culture water control is appropriate for comparison
to the 100-percent test medium. Higher mortality in the disposal site
water control indicates a potentially adverse effect of the water at
the disposal site. In this case the disposal site control is the appro-
priate one for comparison with the 100-percent test medium in order to
estimate what, if any, additional effect might be caused by the proposed
disposal.
20. It is possible that the liquid and suspended particulate phases
of some dredged materials will cause no mortality, and total survival in
the test medium may be equal to or higher than survival In the controls.
If so, visual inspection of the data is adequate and no statistical
analyses are needed. Such cases have been documented and in no way
reflect on the quality of the bioassay, simply indicating an absence of
lethal effects of the dredged material. The LPC cannot be precisely
specified in such cases because the acutely toxic concentration cannot
be determined. However, since the acutely toxic concentration is
known to be at least 100-percent test medium concentration, the LPC
could be exceeded only if the calculations from the appropriate part of
Appendix H predicted dilution by a factor of 0.01 or less during initial
mixing.
21. If survival in the appropriate control is higher than that in
the 100-percent test medium after 96 hr, these sets of data must be
compared statistically by use of the t-test as illustrated below using
the data from Table D2. Before calculation of t, it is necessary to
D10
-------
determine whether the variances of the two sets of data are homogeneous.
This is determined by Cochran's test for the homogeniety of variances,
in which C is calculated as the ratio of the largest variance to the
sum of all the variances.
s2
C = -SyS. = . 0.5708 (Dl)
where
ES
2
S = larger variance of either the control data or the
ni£ix
100-percent test medium data, calculated as in the
example of paragraph 24
2
ES = sum of both variances
22. This C-value is evaluated by comparing it to the tabulated
C-value given in the table that is Enclosure 1 to this appendix. In
the table, k is the number of treatment variances summed in the de-
nominator (2 in this case), and v is one less than the number of
observations contributing to each variance (3 - 1 = 2 in this case).
Therefore, the tabulated value of C in this example is 0.9750.
23. If the calculated C-value is smaller than the tabulated C-
value, as it is here, the calculated value is not significant at the 95-
percent confidence level, and the variences may be considered homogene-
ous. If the calculated C-value is larger than the tabulated C-value,
the variances are not homogeneous. In such cases, all data should be
transformed in order to achieve homogeniety of variances. Such trans-
formations are performed on each datum by obtaining either the natural
logarithm of (X + 1) or the arcsin V"x, where X is the datum. In
order to use the arcsin Vx transformation, the data must be in the
form of a percent expressed as a decimal fraction (i.e., 0.80 survival,
not 80-percent survival). Recalculate the C-value using data transformed
by either of these methods. If variances are now found to be homogeneous,
use the transformed data in all t-calculations. If variances are still
nonhomogeneous, t is calculated by using the original data, with a
different evaluation given in paragraph 26.
Dll
-------
24. The t-value for the 96-hr control and 100-percent test medium
data in Table D2 is calculated as follows:
Number of Survivors
Replicate
1
2
3
sum of data = EX
- EX
mean X =
n
— 2
sum of squares SS = E(X-X)
c2 ss
variance S = —r-
n-1
'Xc * X100>
Control
10
8
"JO
= 28
9.33
2.67
1.33
9.33 - 3.00I
100% test medium
4
2
_3
9
3.00
2.00
1.00
6.33
¦
2 2
Sc + S100
>F
33 + 1.00
V0.7767
7.18 (D2)
n
where:
X -
1 c
1001
Sc and S100
= absolute value of mean of control minus mean
of the 100-percent test medium data
= variances for control and 100-percent test
medium data, respectively
n = number of data points in each set
25. This t-value is evaluated by comparing it to the tabulated
t-value given in Reference 3 at the 0.05-probability level with the
appropriate degrees of freedom (df); in this case, 2(n-l) * 4. It is
important that the tabulated t q,. value be obtained from a table labeled
"one-tailed t values" or "t values, sign considered," or some similar
designation. Alternatively, the appropriate t-value may be obtained
from a standard table at the 0.1-probability level. In this example,
the appropriate t value is:
t.05(4) - 2,13
Since the calculated t-value is larger than the tabulated t-value,
the difference between the control survival and the 100-percent test
medium survival is statistically significant at the 95-percent confi-
dence level.
D12
-------
26. If homogeneous variances could not have been obtained by trans-
formation of the data, the calculations given above could have been
performed on the original data. However, the tabulated t-value would
have to have been obtained for (n-1) = 2 df, instead of 2(n-l) = 4 df,
thereby raising the tabulated t-value and making a difference less
likely to be detected.
27. If no statistical difference at the 95-percent confidence
level had been shown between survival in the control and test medium,
the situation with regard to the LPC would be identical to that described
in paragraph 20. If no difference at the 95-percent confidence level
is shown between survival in the control and test medium, no effect of
the liquid or suspended particulate phase could be predicted for the
disposal site, even if no dilution occurred for 96 hr. This obviously
will not actually occur at any aquatic disposal site. Thus, when no
differences are detected between control and test survival after 96 hr,
the analysis may be considered complete at this point with no indication
of potential impact.
28. However, some dredged material may produce data such as the
example from Table D2, which showed a statistically significant reduc-
tion in survival after 96-hr exposure to 100-percent test medium. In
such cases it is necessary to compare bioassay results to the mixing
and dilution expected at the disposal site in order to determine whether
the LPC would be exceeded. Only then can a prediction be made of the
likelihood of adverse effects in the water column if the disposal occurs.
Limiting permissible concentration
29. The likelihood of adverse effects is evaluated by first con-
structing a time-concentration mortality curve from the bioassay data,
which can be compared graphically to the time-concentration relation-
ship for dilution of the material as calculated in Appendix H, "Esti-
mation of Initial Mixing." A time-concentration mortality curve is
constructed from the bioassay data by calculating the LC50 (lethal con-
centration to 50 percent of the sample) for each observation time. This
is possible only when 50-percent or greater mortality actually occurs in
D13
-------
the highest concentration of test medium. Thus for the data in Table
D2, an LC50 can be calculated for the 72- and 96-hr observations, but
not for earlier observations. Calculation of LC50 values can be per-
2
formed by a variety of methods; where verified computer programs are
4
not available, the method of Litchfield and Wilcoxon is recommended.
A sample calculation using the data from Table D2 is given in the
following paragraphs.
30. Mortality of at least 50 percent was first observed after
72 hr. The 72-hr LC50 is calculated by arranging the 72-hr experi-
mental data from Table D2 as shown in the first three columns of the
following tabulation.
Percent Dead
Percent
Dose
Dead/
Tested
Observed
Expected
Observed minus
Expected
Concentration
to X
10
2/30
6.7
2.0
4.7
0.110
50
8/30
26.7
30.0
3.3
0.005
100
17/30
56,7
57.0
0.3
0.000
0.115
x 10
2
Overall contribution to X " 1.15
31. Special so-called "probability paper" is then used to plot
observed percent dead on the probability axis against concentration of
test medium, as with the closed circles and solid line in Figure Dl.
A line appearing to fit the data is then drawn through the plotted
points. Column 4 of the preceding tabulation is the percent dead
"predicted" at the test concentrations by the line just drawn. Column
5 is the absolute value of the difference between columns 3 and 4.
2 2
Column 6, the contribution to Chi ( X )» is obtained from Nomograph 1
4
in the paper by Litchfield and Wilcoxon. The individual contributions
2
to X are summed and multiplied by 10 to obtain the overall contribu-
2
tion to x • The "goodness of fit" of the line drawn above to the data
2
is then tested by comparing the calculated overall contribution to X
D14
-------
PERCENT DEAD
Figure Dl. Graphical presentation of 72-hr and 96-hr mortality data from Table D3
(NOTE: Probability paper must be used.)
-------
to the tabulated value at the 95-percent confidence level with n - 2 df.
In this case, 1.15 is less than the tabulated value of 3.84 (df = n - 2
4
=3-2=1) in Litchfield and Wilcoxon's Table 2. Therefore the line
is considered to fit the data adequately. If the calculated value had
exceeded the tabulated value, the line would not be considered an
acceptable representation of the data, and another line would have to be
tried until an acceptable fit was obtained.
32. Once a satisfactory line is obtained, the LC16, LC50, and LC84
values (lethal concentration to the stated percent of the sample) for
this observation time are read from the graph. In this case:
LC16 = 30-percent test medium
LC50 = 84-percent test medium
LC84 = 123-percent test medium
33. The slope S of the line is then calculated as:
LC84 LC50 123 84
s , LC50 ^ LCI6 . _84_30 . 2 u (M)
34. It is then necessary to determine "VN' where N' is the total
number of test animals represented by the observed data points falling
between 16 and 84 percent expected effects. In this case, N' « 60,
VIP" = V60 = 7.75
35. The next step is calculation of FLC50, the factor by which
the LC50 is manipulated to obtain the 95-percent confidence limits
about the LC50. 9 11
2.77
FLC50 - (S) (2.13)7*75 = (2.13)*36 = 1.31 (D4)
where 2.77 is constant. The exponential calculation can be solved from
4
Litchfield and Wllcoxon's Nomograph 2. The upper confidence limit (UCL)
and lower confidence limit (LCL) about the LC50 at the 95-percent confi-
dence. level are then determined as follows:
UCL - (LC50) x (FLC50) - (84%) * (1.31) - 110% (D5)
LCL = (LC50) t (FLC50) - (84%) * (1.31) - 64% (D6)
36. According to these calculations, the estimate of the concen-
tration of test material required to kill 50 percent of the test
organisms after 72-hr exposure is 84-percent test medium. That is, the
D16
-------
calculated 72-hr LC50 equals 84 percent, and we can say with 95 percent
confidence that the true 72-hr LC50 lies between 64- and 110-percent test
medium.
37. The same process is used to calculate the 96-hr LC50 and its
confidence limits, as illustrated by the open circles and broken line
in Figure Dl and shown in Table D3. According to these calculations,
Table D3
Calculation of 96-hr LC50 for Data from Table D2
Percent Dead/ Percent Dead Observed minus Contribution
Dose Tested Observed Expected Expected to
10
2/30
6.7 4.0 2.7
0.019
50
12/30
40.0 42.0 2.0
0.002
100
21/30
70.0 69.0 1.0
0.001
0.022
x 10
2
Overall contribution to X
= 0.22
df » n-2 * 3-2 ¦ 1, tabulated X = 3.84
LC16 ¦ 22% test medium
LC50 - 60% test medium
LC84 = 117% test medium
LC84 , LC50 117 . 60
„ LC50 LC16 60 " 22
Slope S = ^ - - 2—
= 2.34
N' - 60, VF - V?0 - 7.75
2.77
FLC50 - (S) N' - (2.34)7*75 -
(2.34)*36 - 1.35
UCL
- (LC50) x (FCL50) - (60%) x
(1.35) - 81%
LCL
- (LC50) * (FLC50) - (60%) *
(1.35) - 44%
the 96-hr LC50 is 60-percent test medium* and we can say with 95 percent
confidence that the true 96-hr LC50 ilea between 44- and 81-percent test
medium.
D17
-------
38. The LC50 estimates and their 95-percent confidence limits for
each observation are then plotted against time, as in Figure D2. This
illustrates the relationship of concentration and exposure time causing
50-percent mortality in the bioassay and is an estimate of conditions
required to produce a similar effect in the field. To determine whether
the LPC might be exceeded in the field, this time-concentration
mortality curve is graphically compared to the expected dilution
curve from Appendix H (Figure D2). The best available mixing esti-
mate, as described in Section 227.29 of the Register and discussed
in Appendix H, should be used to derive the time-concentration
relationship for dilution to be compared to the time-concentration
mortality curve. The initial mixing example used here was taken from
Appendix H, paragraphs 24 through 28. It assumes complete lack of
knowledge concerning mixing at the disposal site and utilizes a
hypothetical disposal operation and the arbitrary mixing calculation
of paragraph 227.29(b)(1) of the Register.
39. Paragraphs 227.29(a) and 227.27(a) of the Register state
that a concentration of 0.01 (or other factor) of the toxic concen-
tration of the liquid phase shall never be exceeded beyond the
boundaries of the disposal site and may be exceeded within the dis-
posal site only during the 4 hr following dumping. The suspended
particulate phase is treated similarly, except that the application
factor is not included and instead the Register specifies that
"unreasonable effects" are forbidden. To help ensure that such effects
do not occur and for the sake of uniformity of interpretation, it is
recommended that the application factor of 0.01 of the toxic concen-
tration (or other factor as specified in paragraph 227.27(a)(3)) be
applied to suspended particulate as well as liquid phase bioassay
interpretations.
40. In Figure D2, illustrating the data from Table D2, both the
4-hr and long-term requirements of the LPC are met. After 4 hr, the
toxic concentration cannot be precisely specified but is greater than
100 percent of the original suspended particulate phase concentration,
D18
-------
Figure D2. Comparison of time-concentration mortality curve for
data from Table D3 with estimated dilution curve
D19
-------
and during initial mixing the predicted dilution is by a factor of more
than 1600, to 0.06 percent of the original suspended particulate phase
concentration. Since the dilution curve and mortality curve continue
to diverge, the LPC requirement that a concentration of 0.01 of the
toxic concentration shall not be exceeded is met both at the end of and
beyond the 4-hr initial mixing period. Therefore, the bioassay would
be considered to have given no indication that the material might pro-
duce any environmentally unacceptable impacts in the water column.
41. Figure D3 is a hypothetical case illustrating a situation
where the LPC would not be met. It should be emphasized that a situ-
ation as severe as this, both in terms of high mortality and low
dilution, has never been documented for either the liquid or suspended
particulate phase of dredged material. This hypothetical situation is
purely for illustrative purposes. In Figure D3, the LPC is exceeded
after the 4-hr initial mixing period and at 8 and 24 hr because the
concentration predicted by the dilution curve is greater than 0.01 of
the lower 95-percent confidence limit about the time-concentration
mortality curve. At 48 hr the LPC is satisfied, since the predicted
concentration is less than 0.01 of the lower 95-percent confidence
limit about the toxic concentration. However, at 72 hr, the LPC is
again exceeded. Both the 4-hr and long-term considerations of the LPC
must be met to satisfy the criteria. Therefore, this hypothetical
situation does not meet the LPC, and if a bioassay gave similar results,
it would be considered to have shown the material to have a real
potential for causing environmentally unacceptable impacts in the water
column.
42. Procedures for using the bioassay animals to estimate the
potential for bioaccumulation of contaminants from the liquid or sus-
pended particulate phases of dredged material are discussed in
Appendix G, "Guidance for Assessing Bioaccumulation Potential."
D20
-------
Figure D3. Comparison of hypothetical time-concentration mortality
curve with hypothetical dilution- curve
D21
-------
REFERENCES
1. U. S. Environmental Protection Agency, "Bioassay Procedures for the
Ocean Disposal Permit Program," EPA-600/9-76-010, 1976, EPA
Environmental Research Laboratory, Office of Research and Develop-
ment, Gulf Breeze, Florida.
2. American Public Health Association, Standard Methods for the
Examination of Water and Wastewater. 14th Ed. 1975.
Water Works Association, Water Pollution Control Federation
Washington, D. C.
3. Rohlf, F. J., and Sokal, R. R.", Statistical Tables. 1969,
W. H. Freeman Company, San Francisco, California.
4. Litchfield, J. T.,Jr., and Wilcoxon, F., "A Simplified Method of
Evaluating Dose-Effect Experiments," Journal of Pharwwcoloqy anrt
Experimental Therapeutics, Vol. 96, 1949, pp 99-117.
D22
-------
Enclosure 1
Critical Values for Cochran's Test*
Values given are for the statistic (largest s'J/CZsi1), where each of the k values of s1 has v degrees of freedom.
percentile 95
1
2
3
4
5
6
7
8
9
10
16
36
144
00
2
0.9985
0.9750
0.9392
0.9057
0.8772
0.8534
0.8332
0.8159
0.8010
0.7880
0.7341
0.6602
0.5813
0.5000
s
0.9669
0.8709
0.7977
0.7457
0.7071
0.6771
0.6530
0.6333
0.6167
0.6025
0.5466
0.4748
0.4031
0.3333
4
0.9065
0.7679
0.6841
0.6287
0.5895
0.5598
0.5365
0.5175
0.5017
0.4884
0.4366
0.3720
0.3093
0.2500
5
0.8412
0.6838
0.5981
0.5441
0.5065
0.4783
0.4564
0.4387
0.4241
0.4118
0.3645
0.3066
0.2513
0.2000
e
0.7808
0.6161
0.5321
0.4803
0.4447
0.4184
0.3980
0.3817
0.3682
0.3568
0.3135
0.2612
0.2119
0.1667
1
0.7271
0.5612
0.4800
0.4307
0.3974
0.3726
0.3535
0.3384
0.3259
0.3154
0.2756
0.2278
0.1833
0.1429
8
0.6798
0.5157
0.4377
0.3910
0.3595
0.3362
0.3185
0.3043
0.2926
0.2829
0.2462
0.2022
0.1616
0.1250
9
0.6385
0.4775
0.4027
0.3581
0.3286
0.3067
0.2901
0.2768
0.2659
0.2568
0.2226
0.1820
0.1446
0.1111
10
0.6020
0.4450
0.3733
0.3311
0.3029
0.2823
0.2666
0.2541
0.2439
0.2353
0.2032
0.1655
0.1308
0.1000
12
0.5410
0.3924
0.3264
0.2880
0.2624
0.2439
0.2299
0.2187
0.2098
0.2020
0.1737
0.1403
0.1100
0.0833
15
0.4709
0.3346
0.2758
0.2419
0.2195
0.2034
0.1911
0.1815
0.1736
0.1671
0.1429
0.1144
0.0889
0.0667
20
0.3894
0.2705
0.2205
0.1921
0.1735
0.1602
0.1501
0.1422
0.1357
0.1303
0.1108
0.0879
0.0675
0.0500
24
0.3434
0.2354
0.1907
0.1656
0.1493
0.1374
0.1286
0.1216
0.1160
0.1113
0.0942
0.0743
0.0567
0.0417
30
0.2929
0.1980
0.1593
0.1377
0.1237
0.1137
0.1061
0.1002
0.0958
0.0921
0.0771
0.0604
0.0457
0.0333
40
0.2370
0.1576
0.1259
0.1082
0.0968
0.0887
0.0827
0.0780
0.0745
0.0713
0.0595
0.0462
0.0347
0.0250
60
0.1737
0.1131
0.0895
0.0765
0.0682
0.0623
0.0583
0.0552
0.0520
0.0497
0.0411
0.0316
0.0234
0.0167
120
0.0998
0.0632
0.0495
0.0419
.0.0371
0.0337
0.0312
0.0292
0.0279
0.0266
0.0218
0.0165
0.0120
0.0088
to
0
0
0
0
0
0
0
0
0
0
0
0
0
0
* By permission from C. Eisenhart, M. W. Hastay, W. A. Wallis, Techniques of Statistical
Analysis3 chap. 15. McGraw-Hill Book Company, New York, 1947.
-------
APPENDIX E: GUIDANCE FOR PERFORMING LIQUID PHASE AND SUSPENDED
PARTICULATE PHASE PHYTOPLANKTON BIOASSAYS
Introduction
1. Paragraph 227.27(c) of the Register (see Appendix A) includes
"phytoplankton or zooplankton" as one of the three groups of appropriate
sensitive marine organisms to be used in bioassays. Phytoplankton bio-
assays can give information on the potential availability of stimulatory
or inhibitory materials associated with the sediment proposed for
dredging. However, because of the extremely dynamic and variable nature
of normal phytoplankton assemblages and because of the rapid mixing and
dilution that takes place in the water column, it is widely felt that
effects on phytoplankton are generally of minimal environmental concern
at ocean sites for dredged material disposal. In addition, phytoplankton
bioassays using the suspended particulate phase are extremely difficult
to conduct and interpret because of interferences and predation on the
test species by indigenous protozoans in the dredged material being
tested. The presence of suspended particulates significantly interferes
with the determination of response in many cases, leading to a recom-
mendation against attempts to use suspended particulate phase phyto-
plankton bioassaVs. For these reasons, unless there is particular con-
cern about effects on phytoplankton by the disposal operation in ques-
tion, it is recommended that zooplankton, rather than phytoplankton,
bioassays be employed to fulfill this requirement of the criteria. This
approach would generally provide the most useful Information on potential
effects of the disposal being evaluated.
2. If the special circumstances of the case warrant a phyto-
plankton bioassay, it is conducted by establishing a series of treat-
ments and controls using the liquid phase and filtered disposal site
water. The experimental units are then inoculated with test organisms
and held under a specified set of test conditions while a sampxing
program is conducted to determine response.
El
-------
Apparatus
3. The following items are required:
a. Thirty 500-ml Erlenmeyer flasks made of Pyrex or Kimex
glass.
b. Plastic "beakers or stainless steel caps to cover the 500-ml
Erlenmeyer flasks.
c. Facility for growing algae at constant temperature, illumi-
nation, and shaking rate. Any incubator that allows temperature^control
within +2°C, light intensity of approximately 1100 to 1500 yw/cm using
cool-white flourescent bulbs, and a shaking rate of 110 rpm will suffice.
d. Equipment required for evaluation of response. Require-
ments will depend on whether cell counts, CU uptake, productivity, or
chlorophyll values are the responses to be measured.
Species Selection
4. Phytoplankton should be collected from the disposal site and
isolated into axenic cultures for use in the bioassays when this is
permitted by practical considerations and the expertise of the experi-
menter. Otherwise, the species listed in the following tabulation are
recommended and may be purchased for laboratory culture as indicated.
Methods for collecting and culturing algae are given in "Standard
Methods for the Examination of Water and Wastewater," "Bioassay Pro-
cedures for the Ocean Disposal Permit Program," and "Marine Algal
Assay Procedure: Bottle Test."
Species Source
m i on USEPA Environmental Research Center
Skeletoned sp. Cotvallls, Oregon 37330
or
USEPA Environmental Research Center
Narragansett, Rhode Island 02874
Chloroooeeum CMilford "C") 819 Department of Botany
lolotella sp. 1269 Culture Collection of Algae
Porphyridiim sp. 637 Indiana University
* " Bloomington, Indiana 41701
^ x USEPA Environmental Research Center
Cyolotella sp. 1269 narragansett, Rhode Island 02874
E2
-------
Sample Collection and Preservation
5. Sediment and water samples are collected and stored and the
liquid phase (or suspended particulate phase) is prepared as described
in Appendix B, "Dredged Material Sample Collection and Preparation."
Experimental Conditions (Liquid or
Suspended Particulare Phase)
6. Procedures for the algal assay for marine disposal sites are
similar to those described in Reference 3. This reference gives details
of the procedure and rationale and must be used in conjunction with the
guidance provided here.
3
7. Grow stock algal cultures in synthetic nutrient medium. Start
new cultures each week by transferring 0.5 ml of a one-week-old culture
to 100 ml of fresh medium using aseptic technique. Grow stock cultures
at approximately 18-20°C under continuous cool-white flourescent light-
2
ing at an intensity of approximately 1500 yw/cm and shake continuously
at 110 rpm. If shaking tables are not available, swirl all flasks at
regular intervals at least twice daily. Acclimation of the stock algal
cultures should begin at least two weeks prior to the actual test.
Salinity of the test water should be approximately that expected at the
disposal site. If test species are not maintained at the proper salini-
ty, they should be transferred to medium of appropriate salinity follow-
ing procedures for adjusting salinity given in Reference 3. The concen-
tration of nutrients in the growth medium should be reduced to 20 per-
cent of the stock growth medium during the acclimation period. The
algae should also be acclimated to the temperature given in paragraph 10
of Appendix D. The rate of temperature change should not exceed 2°C
every 24 hr. Photoperiod should be 14 hr dark and 10 hr light during
the acclimation period.
8. Use 500-ml Erlenmeyer flasks covered with beakers or stainless
steel caps for culture vessels. Wash all glassware with nonphosphate
detergent, rinse with tap water, place in a clean 10-percent HC1 acid
bath for a minimum of 4 hr, and then rinse five times with distilled
water.
E3
-------
Experimental Procedure
9. Establish treatment levels using the liquid or suspended
particulate phase, disposal site water, and an inoculum of the test
organism to produce a total liquid volume of 100 ml in 500-ml Erlenmeyer
flasks when cell counts are the parameter of interest. A greater
volume will be required for some of the techniques required for measur-
14 12^
ing other responses, such as C uptake or chlorophyll. ' ' Establish
at least three replicates of each of the following treatment levels and
controls:
Percent Percent
Liquid Phase Disposal Site Water
100 0
50 50
10 90
Controls: 100-percent disposal site water
100-percent synthetic algal growth medium
10. Inhibition of growth could be the result of lack of required
nutrients or the availability of toxicants. As an aid in determining if
toxic chemicals are available to the phytoplankton from the liquid or
suspended particulate phase being tested, nutrient additions are useful.
Adjust the concentration of a stock solution of growth medium such that
when 1 ml is added to the following flasks, the final concentration of
the nutrients in each flask is equivalent to 10 percent of the stock
growth medium. The following treatments should receive nutrient addi-
tions :
Percent Percent
Liquid Phase Disposal Site Water
100 0
50 50
10 90
Control: 100-percent disposal site water
Also, establish a set of control flasks that contain only 10 percent
of the nutrients of the stock growth medium.
11. Prepare the inoculum by centrifuging and washing stock
E4
-------
culture cells with sterile artificial seawater of appropriate salinity
without nutrients. Adjust the inoculum cell concentration by dilution
with sterile seawater; then pipette the inoculum into the test water to
give a starting concentration in the test waters of 1000 cells per ml.
12. Distribute the flasks randomly in incubation chambers. Set
the temperature at the level given in paragraph 10 of Appendix D (+2°C),
2
lighting intensity at approximately 1100 to 1500 uw/cm using cool-
white flourescent bulbs on a 14 hr dark and 10 hr light photoperiod,
and the shaking rate at 110 rpm throughout the assays. Test salinity
should be approximately that expected at the disposal site. It is
important that all test containers are exposed to the same conditions.
Continue the assays until the maximum cell number occurs in each treat-
ment level. This does not necessarily occur on the same day for each
treatment. Cell numbers can be used to determine cell volume as de-
scribed in Reference 3 and are a suitable method for reporting results
and comparing effects among treatment levels.
13. Determine the effect of the test solution on the algae by
comparing the response in the controls to that in the flasks containing
the test solution. This may be done by comparing cell counts, cell
14
volume, C uptake, ATP levels, or chlorophyll values. Procedures for
these methods for measuring algal response may be found in References 1,
2, and 3. Whatever method of measurement is chosen, observations
should be made at 24-hr intervals.
14. The differences measured (e.g., cell counts) can be compared
at different times during the bioassay depending upon the type of
information needed. The maximum standing crop can be compared when
controls and treatments have reached the maximum biomass. This param-
eter is helpful in predicting total effects in situations where there
is concern that frequent use of the disposal site may affect water
quality for extended periods of time. Shorter term effects can be
3
compared by calculating the maximum specific growth rate between the
controls and experimental treatments. Additional information about
potential short-term effects can be gained by comparing the measured
ES
-------
parameter at daily intervals during the bioassay. If there is a lag in
the initiation of growth, comparing daily measurements will show this
and could indicate short-term problems. For example, it is conceivable
that the control and experimental cultures will all reach the same
maximum biomass, but their rate of attaining that biomass may vary be-
cause of differences in the onset of rapid growth or different mav^.Tn
specific growth rates.
Data Analysis and Interpretation
15. Phytoplankton bioassay data are analyzed in the manner de-
scribed for animal bioassays in the "Data Analysis and Interpretation"
section of Appendix D. The single difference is that in phytoplankton
bioassays, both increased and decreased responses in the test medium
relative to the controls (stimulation and toxicity) are potentially of
interest. Therefore, the calculated t-value is compared to the tabulated
value at the 0.05-probability level from a "two-tailed" or "sign-ignored"
table, rather than a "one-tailed" or "sign-considered" table.
16. The interpretation of phytoplankton bioassay results must
consider mixing and dilution, and it must be determined whether the
limiting permissible concentration (LPC) would be violated if the pro-
posed disposal occurred. This is also done by the method presented in
the "Data Analysis and Interpretation" section of Appendix D. In the
case of phytoplankton, it is the effective concentration to 50 percent
of the sample (EC50), rather than the lethal concentration (LC50), that
is calculated. The method of calculation is exactly the same and only
the notation is changed.
E6
-------
REFERENCES
1. American Public Health Association, Standard Methods for the
Examination of Water and Wastewater, 14th ed., 1975, American
Water Works Association, Water Pollution Control Federation,
Washington, D. C.
2. U. S. Environmental Protection Agency, "Bioassay Procedures
for the Ocean Disposal Permit Program," EPA-600/9-76-010, 1976,
EPA Environmental Research Laboratory, Office of Research
and Development, Gulf Breeze, Florida.
3. U. S. Environmental Protection Agency, "Marine Algal Assay
Procedure: Bottle Test," 1974, EPA National Environmental
Research Center, Corvallis, Oregon.
E7
-------
APPENDIX F: GUIDANCE FOR PERFORMING SOLID PHASE BIOASSAYS
Introduction
1. This bioassay of appropriate sensitive benthic marine organ-
isms can aid in assessing the potential environmental impact of the
solid phase of dredged material proposed for ocean disposal and acts as
an indirect indicator of chemical toxicity of the sediment. It provides
exposure conditions approximating those that would be experienced by
animals living near the boundaries of the disposal site. Several benthic
species are allowed to establish themselves in an appropriate reference
sediment and are then covered with a layer of the dredged material being
evaluated. Survival in the dredged material relative to that in the
reference sediment control is used as the primary blotic response
criterion.
2. The objective is to determine the potential impact of the
solid phase on benthos at and beyond the disposal site boundaries. The
concept of a disposal site Implies that conditions within that site may
be adverse, but that conditions beyond its boundaries cannot be.
Therefore, this bioassay does not duplicate the depths of sediment
deposition that may cover animals directly under the disposal vessel,
but rather it approximates the conditions found within or at the dis-
posal site boundaries. The bioassay is designed to determine whether
a biological effect is likely, but the bioassay cannot be used to deter-
mine the cause of the observed effects. Indeed, if an adverse effect
may occur outside the disposal site, it matters little from a regulatory
viewpoint whether that effect is due to the physical presence of the
sediment or is due to some chemical constituent(s) associated'with the
sediment carried beyond the site. Therefore, it is important to realize
that this benthic bioassay measures the total impact of the dredged
material. That Impact may be due to an unrecognized pollutant or to the
synergistic effects of many pollutants, none of which may have an ex-
ceptionally elevated concentration. At the present technical state-of-
Fl
-------
the-art, it is not possible to determine by any known chemical analysis
which pollutant(s) may be the causative agent(s).
Aquarium System
3. The exact dimensions of the test aquaria are not critical,
2
but their bottom area should not be less than 1000 cm nor their volume
less than 20 I. Standard 10-gal (37.8-A) all-glass aquaria 26 cm wide,
51 cm long, and 31 cm deep are satisfactory. Five aquaria will be
needed for the controls and for each dredged material sampling site
tested.
4. Filtered seawater of approximately the same temperature,
salinity, and dissolved oxygen as the water near the bottom at the dis-
posal site should flow into each aquarium at a rate that will replace
the aquarium volume at least once every 4 hr. The flow should be di-
rected to achieve good mixing without disturbing a layer of sediment on
the aquarium bottom. Water leaves the aquarium through a perforated
standpipe covered with a 0.5-ram nylon screen. If a continuous-flow
seawater system is not available, animals can be tested in static water
aquaria provided that 75 percent of the seawater volume is replaced 1
and 48 hr after the test is begun and at 48-hr intervals thereafter.
The frequency of changing shound be increased if the control animals
appear stressed.
Pr>i 1 fr.tion of Sediments and Test Organisms
5. Collect sediments, water, and test species from the field with
an appropriate benthic sampler such as the Smith-Mclntyre or Van Veen
grab. Sediment should be placed in clean nonmetallic containers and
maintained at 2 to 4°C from the time of collection until the bioassay
begins. Sediment samples must never be frozen or dried. Detailed
guidance for collection of sediment and water samples is given in
Appendix B. The bioassay must be initiated within two weeks after the
sediment and faunal collections. Field-caught test species and the
reference sediment must be obtained from an uncontaminated area in the
vicinity of the disposal site. This reference sediment must have sedi-
mentological characteristics similar to the disposal site and should
F2
-------
be an approximation of the sediment that would be found at the disposal
site if no disposal had ever taken place there. In the likely event
that sediment conditions vary substantially within the proposed dredging
site, sediment samples from more than one location must be tested. Thus,
the bioassay will include at least two and probably more treatments,
i.e., the reference substrate control and sediments from one or more
locations within the dredging site. Five replicate aquaria are es-
tablished for each treatment, including the controls.
6. The quantity of sediment needed for the bioassay is dependent
on the size of the test aquaria, as discussed in paragraph 3. A 30-mm
layer of reference sediment is placed on the bottom of all replicates
of all treatments, including the controls. A 15-mm layer of the dredged
material in question is placed on top of the 30-mm reference sediment
layer in all test replicates, but not the controls. An additional 15-mm
layer of the reference sediment is placed on the controls. Sediments
for a single treatment should be mixed to ensure homogeneity, and aliquots
taken for the bioassay aquaria. If standard 10-gal aquaria are used, a
minimimum of 4.5 I of reference sediment and 2.5 I of dredged material
should be collected for each aquarium.
Species Selection
7. Solid phase bioassays must be conducted with appropriate
sensitive benthic marine organisms. Paragraph 227.27(d) of the Register
(see Appendix A) defines this to mean at least three species, consisting
of one filter-feeding, one deposit-feeding, and one burrowing species.
These are broad overlapping general categories and it is recommended
that the species be selected to include a crustacean, an infaunal bivalve,
and an infaunal polychaete. Infaunal amphipods seem to be among the most
sensitive crustaceans and, for this reason, are among the preferred
organisms for solid phase bioassays. All solid phase bioassays should
include a species of mysid shrimp of the genus Mysidopsie or Neomyeie.
This will provide an internal standard in all bioassays and form a basis
for quality assurance in the regulatory program.
8. The sensitivity of this and all bioassays is dependant
F3
-------
primarily on the selection of appropriate species. If at all possible,
the test species should be collected from the area in which the reference
substrate is collected. They should be the same species or closely
related to those species that naturally dominate benthic assemblages in
the vicinity of the disposal site in the season of the proposed operation.
Experience has shown that with reasonable care, it is possible to collect
test organisms from wild populations and maintain them under control
conditions with low mortality. However, a preliminary study of the
ability of field-collected test organisms to acclimate to laboratory
conditions is highly desirable.
9. If it is not practical to use the dominant species collected
from near the disposal site, test species may be selected from Table F1
if they are chosen so that insofar as possible they are related phylo-
genetically and/or by ecological requirements to the dominant appro-
priate sensitive benthic marine organisms expected in the area of the
disposal site at the time of the proposed operation. Species are not
listed in Table F1 in any order of preference or desirability. Com-
mercially important organisms from the vicinity of the disposal site
may also be included if desired. The considerations of paragraph 7
must be kept in mind when selecting solid phase bioassay species from
any source.
10. This solid phase bioassay uses five species, each represented
by 20 individuals, in each replicate aquarium. It is recommended that
juvenile forms, particularly of molluscs and large crustaceans, be
utilized because of their generally greater sensitivity than adults.
The wet weight of individual test specimens should not be greater than
3 g. Molluscs are often most useful in bioaccumulation studies but
should be less than 2 cm long if used in bioassays. To avoid predation,
it probably will be necessary to conduct the bioassay with potential
predator and prey species isolated from each other. The identity of all
test species should be verified by experienced taxonomists. If the bio-
assay animals are also to be used in estimating bioaccumulation potential,
species selection should consider the factors discussed in paragraphs
F4
-------
Table F1
Recommended Appropriate Sensitive Benthic Marine Organisms for Use In Solid Phase Bioassays*
Crustacean
Infaunal Bivalve
Mysid shrimp, Myeidopeis sp.** (D)
Neomysis sp.** (D)
Infaunal amphipods,
Ampelieaa sp. (F, D)
Faraphoxus sp. (F, D, B)
Grass shrimp, Palaemonetea sp. (D)
Palaemon sp. (D)
Commercial shriwp, Panaens sp. (D)
Sand shrimp, Crangcm sp. (D)
Oceanic shriivp, Bccndahis sp. (D)
Blue crab, Callineetea 8apidua (D)
Cancer crab, Caneer sp. (D)
Cumacean, Dicuttylopie sp. (F, D, B)
Diaatylia sp. (F, D, B)
Lampvope sp. (F, D, B)
Macoma clam, Manama sp. (F, D)
Nucula clam, Nuaula sp. (F, D)
Surf clam, SpieuXa solidieeirrta (F)
Hard clam (quahog), Meraenaria sp. (F)
Ocean quahog, Arctica islandica (F)
Gemma clam, Gerrma genrna (F)
Littleneck clam, Ppototheaa staminea (F)
Cockle, Clinoaardium rcuttali (F)
Infaunal Polychaete
Neanthes sp. (D, B)
Nereis sp. (B)
Nepthys sp. (B)
Glyaera sp. (B, D)
Urechis sp. (B, F)
Magetcma sp. (B, D)
Ouenia sp. (B, D)
Diopatra sp. (B, D)
Gtyoinde sp. (B)
Mote: Parenthetical notations as follows: F - filter-feeding species
D - deposit-feeding species
B - burrowing species
*Lists are not in order of preference or desirability
** A11 solid phase bioassays should include one of these species
-------
5, 6, and 7 of Appendix G, "Guidance for Assessing Bioaccumulation
Potential."
11. Test organisms should be collected from the region of the
disposal site or cultured in the laboratory. If the organisms
are collected from the field with the reference substrate, grab samples
for faunal collections should be gently sieved through a 1.0-mm screen,
and the animals placed in buckets containing a 2- to 3-cm layer of sedi-
ment and several litres of seawater. Whatever the source of the animals
collection and handling should be as rapid and gentle as possible.
12. Transportation to the laboratory should be in well-aerated
water from the collection site in which the animals are held at the
temperature and salinity from which they were obtained. Benthic animals
should be held in the laboratory in aquaria in which approximately 30 ma
of reference sediment has been placed. This sediment should contain no
other animals and should be from an uncontaminated source similar to
the disposal site in sedimentological characteristics. Animals should
be held in the laboratory for no more than two weeks before bioassays
are begun. During this period they must be gradually acclimated, if
necessary, to the salinity and temperature at which the bioassay will
be conducted.
13. Methods for collecting, handling, acclimating, and sizing bio-
assay organisms given in "Bioassay Procedures for the Ocean Disposal
Permit Program,"1 and "Standard Methods for the Examination of Water
2
and Wastewater" should be followed in all matters for which no guidance
is given here.
Experimental Conditions
14. Solid phase bioassays should be conducted at a salinity approxi-
mating that expected at the disposal site in the season of the proposed
operation. Water collected from the disposal site should be used if at
all possible. Otherwise uncontaminated seawater, or an artificial sea
salts mixture such as that given on page 32 of Reference 1, of the
proper salinity may be used. Experimental temperature should be held
stable within + 1°C of a temperature approximating that expected at the
F6
-------
disposal site in the season of the proposed operation. Recommended
experimental temperatures are given in the following tabulation on a
seasonal basis for various zoogeographic areas.
Test
Temperature
, °c
Zoogeographic Areas
Summer Winter
CE Division EPA
Region
20
5
New England
North Atlantic
I
xx*
III
25
12
South Atlantic
Lower Mississippi Valley
Southwestern
IV
VI
10
10
North Pacific
South Pacific
X
IX**
25
25
Pacific Ocean
IX**
*Puerto Rico and Virgin Islands are in EPA Region II,
but should use temperatures recommended for Region IV.
**Mainland portions of Region IX should use South Pacific
Division temperatures; Pacific island portions of Region
IX should use Pacific Ocean Division temperatures.
Bioassay Procedure
15. The reference substrate, and perhaps the dredged material being
tested, may initially contain live organisms of the same species to be
used in the bioassay. These must be removed by wet sieving the sediment
through a 1.0-mm screen using the smallest amount of seawater possible.
The water and sediment must all be retained in a settling container.
Place the material retained on the screen in a sorting tray, remove the
animals, and return the remainder to the settling container. Allow the
sediment to settle for 6 hr, decant the seawater without disturbing the
sediment surface, and then mix the sediment to ensure homogeneity. The
animal-free dredged material is then returned to its storage containers
and held under the conditions specified in paragraph 8 of Appendix B
for approximately 48 hr until needed. The animal-free reference sedi-
ment is used at once as described in the following paragraph. It is
recognized that the screening and sedimentation procedure described in
F7
-------
this paragraph may result in some alteration of the biological
availability of any contaminants present. Although the degree of
alteration is unknown, its influence on test results is felt to be
minimal. This is the least disruptive method by which the necessary
task of removing indigenous animals from the sediments can be ac-
complished .
16. Partially fill each aquarium with seawater and then add enough
reference sediment to produce an even 30-mm layer on the bottom. After
1 hr turn on the seawater and allow it to run for 2 hr before any
animals are added. In a static water system, the seawater is replaced
after the 30-mm layer of reference sediment has settled for 1 hr, being
careful not to resuspend the deposited material.
17. While the reference sediment is settling in the test aquaria,
sediments in the animal-holding tanks can be gently siphoned and sieved
through a 1.0-mm screen to recapture the test organisms. The utmost
care should be taken when handling any of the animals to avoid damaging
the organisms. Discard any animals that are dropped or physically
abused during the transfer. References 1 and 2 provide instructions on
handling and transfer procedures. Specimens of each of the five species
are randomly divided among finger bowls equal in number to all aquaria
in the bioassay. Each bowl will contain 20 individuals of each species
After the water over the reference sediment has been cleared as describe
ed in paragraph 16, the test organisms are released from the bowls to
the aquaria and allowed to acclimate for 48 hr. In a static system,
75 percent of the water in the aquaria may be replaced 24 hr after the
animals are introduced.
18. During the acclimation period, dead specimens can be removed
from the test aquaria and replaced with healthy individuals. It is
difficult to determine the exact mortality of infaunal species without
disturbing the sediment layer. However, if apparent mortalities exceed
10 percent of the seeded specimens of any species, this test must be
discontinued and a new one begun. Species selection, collection, and
holding techniques must then be reexamined in an effort to reduce
F8
-------
pretesting mortality in the new test. The bioassay procedure assumes all
the original animals are alive when the dredged material is introduced,
and any undiscovered dead animals in the reference sediment will there-
fore give a false impression of the effects of the dredged material.
19. After the 48-hr acclimation period, the animals should be
established in the reference sediment. The dredged material is divided
into aliquots sufficient to produce a 15-mm layer on top of the 30-mm
reference sediment layer in the test aquaria. An additional 15-mm layer
of reference sediment is placed on the controls. The temperature of the
sediment aliquots must be approximately that of the seawater in the
aquaria. Turn the water off and remove a seawater volume slightly greater
than the dredged material volume to be introduced. Treatments must be
randomly assigned to the aquaria. Each bioassay will consist of five
aliquots of control sediment and five aliquots from each sampling site
within the proposed dredging area. The 15-mm layer is deposited by
evenly distributing the sediment aliquot over the water surface. Many
sediments can be poured onto the surface if they are mixed with a small
volume of seawater. Some crustaceans, such as mysid shrimp, will not
survive the physical disruption of the sediment addition and must be
placed in the aquaria immediately after the test sediment addition.
After allowing 1 hr for settling, the seawater is turned on again. In
a static water system, 75 percent of Che seawater is replaced 1 and 48
hr after the 15-mm sediment aliquot is added and at 48-hr intervals
thereafter.
20. The bioassay continues for 10 days, during which dally records
should be kept of obvious mortalities, formation of tubes or burrows,
and unusual behavior patterns* Daily levels of salinity, temperature,
and dissolved oxygen coritent of aquaria water should be reported.
Gentle aeration or increased flow rate should be used to keep dissolved
oxygen concentration above 4 ppm unless there are reliable data to
indicate that lower dissolved oxygen levels would occur for a sub-
stantial period of timein the field during the proposed disposal opera-
tion or that lower levels occur naturally at the site.
-------
21. After 10 days, turn off the flow of water and siphon the sedi-
ments through a 0.5-mm screen. Mix the material retained on the screen
with some seawater and search it thoroughly for animals. Consider
animals alive if they show any response to gentle probing of a sensitive
part. Sublethal effects such as partial paralysis should also be re-
corded. For many benthic species, an appropriate sublethal response
criterion is the inability to burrow in sediments or to excavate burrows.
Specimens not recovered must be considered dead. All crustaceans molt
at regular intervals, shedding a complete exoskeleton. Care should be
taken not to count an exoskeleton as a dead animal. Dead animals may
decompose or be eaten between observations. Therefore, always count
living, not dead, animals. A sample of recovered specimens not needed
for further analysis should be preserved in formalin if needed for
verification of species identification. If animals from the bioassay
are to be used in estimating bioaccumulation potential, the survivors
should be gently and rapidly counted and treated as discussed in
Appendix G, beginning with paragraph 12.
Analysis and Interpretation of Results
22. According to Section 227.13 of the Register, dredged material
can be considered environmentally acceptable for ocean disposal only if
bioassay and mixing results indicate that the limiting permissible con-
centration (LPC) will not be exceeded (Section 227.27). The primary
objective of the bioassay is to determine if there is a statistically
significant decrease in mean survival of all species in the dredged
material treatment(s) relative to the control.
23. It is important to realize that a statistically significant
effect in a bioassay does not necessarily imply that an ecologically
Important Impact would occur in the field. This must be kept in mind
when interpreting results, particularly in cases where a difference of
small magnitude between survival in the control and test sediments is
shown to be statistically significant. At present there is no quanti-
tative method for estimating the magnitude of such a difference that
might reliably be assumed to predict the occurence of adverse impact on
F10
-------
animals in the field. However, there is a general feeling among many
scientists that differences between control and treatment survival of
10 percent are necessary in most cases before predictions of
probable impact can be made. Of course, regardless of the magnitude
of the difference between mean survival levels, if the means are not
shown to be statistically different, they must be regarded as equal.
24. The statistical example given later analyzes total mortality
of all species. The sensitivity of this procedure may be increased, if
desired, by blocking the data on species using the method given in
3
Table 11.7 on page 327 of Sokal and Rohlf. Survival of individual
species can be analyzed by the same statistical tests as the combined
survival of all five test species. The relative sensitivity of the
different species could reflect phylogenetic susceptibility to certain
toxicants. If differences in mean survival are not significant, analysis
of sublethal responses such as paralysis, inability to burrow, or bio-
accumulation may indicate potentially unacceptable impact. Such responses
may also be analyzed by the statistical method presented below.
Data presentation
25. Present data in a table giving the scientific name of the test
species, the number of animals seeded, and the percent of animals re-
covered alive from each aquarium. If greater than an average of 10-
percent mortality occurs in the controls, all data must be discarded
and the experiment repeated. The 10-day test period represents a major
portion of the life span of some species such as mysid shrimp, and unless
the test is begun with juveniles, mortality greater than 10-percent may
be expected from natural causes. Unacceptably high control mortality
indicates the presence of important stresses on the organisms other than
the material being tested, such as injury or disease, stressful physical
or chemical conditions in the test containers, improper handling or
acclimation, or perhaps an adverse impact from an unsuitable or contami-
nated reference sediment.
26. If less than 10-percent mortality occurs in the controls, the
data may be evaluated. It is possible that the solid phase of some
Fll
-------
dredged materials will produce no mortality, and total survival in the
dredged material may be equal to or higher than survival in the reference
substrate controls. If so, visual inspection of the data is adequate,
and no statistical analyses are needed. Such cases have been docu-
mented and in no way reflect on the quality of the bioassay, simply
indicating an absence of lethal effects of the dredged material.
Statistical analysis
27. If survival in the reference substrate control is higher than
that in the dredged material, the data must be compared statistically.
The following example is a hypothetical case in which dredged material
from three sampling stations in the dredging site were analyzed, giving
a total of four treatments including the reference substrate. The
hypothetical data are shown in Table F2.
Table F2
Hypothetical Results of a Solid Phase Dredge Material
Bioassay Using Three Samples of Dredged Material
Total Surviving Animals,
Replicate
Dredged
Material
V
Sample
(n =
5)
Control
1
2
3
1
85
71
94
79
2
90
67
88
72
3
92
69
94
83
4
96
61
90
67
5
92
84
93
77
sum of data * EX
-
455
352
459
378
- EX
mean, X - —
-
91.0
70.4
91.8
75.6
2
sum of squared data - EX
- 41
,469
25,068
42,165
28,732
corrected sum of squares,
CSS - EX2 -
n
-
64.0
287.2
28 .8
155.2
_2 CSS
variance, S *
Wt
16.0
71.8
7.2
38.8
F12
-------
28. An analysis of variance (ANOVA) is used to compare the mean
survival in the reference substrate control to the mean survival in the
dredged material samples. In cases where it is felt that adequate in-
formation may be obtained from one sampling station in the dredging site,
the data may be analyzed by a t-test analogous to that discussed in
paragraphs 19 through 26 of Appendix D.
29. Before an ANOVA can be performed, it is necessary to determine
whether the variances of the data sets are homogeneous. This is deter-
mined by Cochran's test for the homogeniety of variances. The C-value
is calculated' as the ratio of the largest variance to the sum of all
variances. 2
" T&t *
where
2
= largest variance among the data sets
max
ES^ « sum of all the variances
30. This C-value is evaluated by comparing it to the tabulated
C-value given in the table that is Enclosure 1 to Appendix D. In the
table, k is the number of treatment variances summed in the denomina-
tor (4 in this case), and v is one less than the number of observa-
tions contributing to each variance (5 - 1 ¦ 4 in this case). There-
fore, the tabulated C-value in this example is0.6287.
31. Since the calculated C-value is smaller than the tabulated
C-value, the calculated value Is not significant at the 95-percent
confidence level, and the variances may be considered homogeneous. If
the calculated C-value is larger than the tabulated C-v*iue# the
variances are not homogeneous. In this case before any ANOVA calcula-
tions are performed, a transformation shouxd be performed on all data
in order to achieve homogeniety of variances. This may oe done oy
obtaining either the natural logarithm of ( X + 1 ), or tfie arcaln
VT, where X is the datum. If t&e arcfin Y1T transformation
is to be used, the data must first be converted to percents anfl .ex-
pressed as decimal fractions (i.e., 0.92 survival, not 92 percent
-------
survival). Recalculate the C-value using data transformed by either of
these methods. If variances are now found to be homogeneous, use the
transformed data in all ANOVA calculations. If variances are still non-
homogeneous, an approximate test of the equality of means given by Sokal
3
and Rohlf in their Box 13.2 should be used.
32. ANOVA equations and calculations for the data of Table F2 are
given in Table F3. The values on the third line of the table (Total)
should be the same whether they are calculated by the equation or ob-
tained by summing the corresponding treatment and error values, thus
providing an easy means of checking the accuracy of the calculations.
The calculated F-value is evaluated by comparison with the tabulated
F-value from Reference 4 at the 0.05-probability level with the appro-
priate df. The df's are those given for the treatments and error,
respectively, in Table F3. The tabulated F-value with 3 and 16 df is
shown at the bottom of Table F3. Since the calculated F-value exceeds
the tabulated value, there is a statistical difference between mean
survival among the four sets of data. If the calculated F-value had
been equal to or less than the tabulated value, there would be no
statistical differences between survival in the reference substrate
controls and any of the dredged material samples. In that case, the
analysis would be complete at this point with no indication of potential
adverse impact of the solid phase.
33. When the calculated F-value exceeds the tabulated value, it
is then necessary to determine which dredged material means differ
significantly from the reference substrate control mean. This may be
done by the Student-Newman-Keuls multiple-range test given by Sokal and
3
Rohlf in their Box 9.9. Least significant ranges (LSR) used in this
process are the product of the pooled standard error of the group mean
-------
Table F3
ANOVA Equations and Calculations for the Data from Table F2
Degree of
Source of Freedom Sum of Squares (SS) Mean Square (MS)
Variation Equation Value Equation Value Equation Value Equation Value
Treatmentst (a-1) 3 I (£X)2 - 1762.0 SS 587.33 MS„ . . 17.56*
n En treatment treatment
a-1 MS
error
Error a(n-l) 16 ICSS 535.2 SS 33.45
error
a(n-l)
Total (an)-l 19 E(EX2) --^f^- 2297.2
2.n
snts a * 4
24
-------
the number of means (K) = 2, 3, and 4 are:
K
QCRohlf and Sokal, Table U ) = 2.998 3.649 4.046
S_ = 2.59 2.59 2.59
X
LSR = QS_ = 7.76 9.45 10.48
X
35. The multiple-range test is completed by arranging the four
treatment means in increasing order and then comparing the difference
between means with the LSR for the number of means (K) in the range
separating the two being compared. That is, for two adjacent means
K = 2; for comparing means separated by one mean, there is the
intervening mean and the two being compared, so that K = 3. It is
necessary to compare each dredged material mean to the control mean,
but not to compare treatment means among themselves. Such comparisons
between treatment means are not necessary for permitting decisions, but
could provide useful managerial information by distinguishing sediments
causing a great impact from those causing a smaller but still statisti-
cally significant impact. The comparison of treatment means to the
control for the above example is given below.
Treatment Means Computed from Table F2
X, X, X ^ _ X
1 3 control 2
70.4 75.6 91.0 91.8
Mean Comparison
K
LSR
Difference Between
Means
2
7.76
X - X, - 91.0 - 70,4 -
C 1
20.6*
2
7.76
X_- X - 91.8 - 91.0 -
2 c
0.8 n.s.
3
9.45
X - X - 91.0 - 75.6 -
C J
15.4*
Note: Entry of * indicates differenc between
means is significant at the 0.05-probability
level; n.s. Indicates difference is cot
significant
wm
-------
36. When the difference between two means is greater than the LSR,
the difference between those means is statistically significant at the
0.05-probability level. Therefore, the multiple-range test has shown
that the mean survival in dredged material samples 1 and 3 is statisti-
cally lower than survival in the reference substrate control, while the
survival in dredged material sample 2 is not statistically different
from that in the control. The difference between survival in the con-
trol and samples 1 and 3 is greater than the minimum difference gener-
ally considered to indicate the probability of biologically important
effects, as discussed in paragraph 23. Had the treatment and control
means shown a statistically significant difference of only a few percent,
the prediction of important effects would be much more tenuous.
Limiting permissible concentration
37. The LFC of the solid phase of dredged material is defined in
paragraph 227.27(b) of the Register as that concentration of solids that
will not cause "unreasonable effects" beyond the disposal site boundary.
Paragraphs 227.29(a) and (b)(2) clearly imply that the initial disper-
sion of the solid phase that occurs within 4 hr after disposal is to be
considered in determining whether the LFC would be exceeded. At pre-
sent there are no objective methods for considering initial
dispersion in the interpretation of solid phase bioassay data. There-
fore, this guidance takes the environmentally protective approach that
the LFC of the solid phase is operationally determined by the results
of the solid phase bioassays. If the difference in mean survival
between animals in the control and test sediments is statistically signi-
ficant and greater than 10 percent, as in this example, the LFC would
be exceeded, and the bioassay would have shown the naterial to have a
real potential for causing environmentally unacceptable impacts on
benthic organisms. This method of interpretation based on statistically
significant mean differences of at least 10 percent should be used only
with the solid phase bioassay technique. The level of 10 percent Is
subject to revision if warranted by further studies and experience, but
is the level presently considered most realistic for environmental
nr
-------
protection purposes by those most familiar with solid phase dredged
material bioassays.
REFERENCES
1. U. S. Environmental Protection Agency, "Bioassay Procedures for the
Ocean Disposal Permit Program," EPA-600/9-76-010, 1976, EPA
Environmental Research Laboratory, Office of Research and Develop-
ment, Gulf Breeze, Florida.
2. American Public Health Association, Standard Methods for the
Examination of Water and Wastewater, 14th ed., 1975. American
Water Works Association, Water Pollution Control Federation,
Washington, D. C.
3. Sokal, R. R., and Rohlf, F. J., Biometry, 1969, W. H. Freeman
Company, San Francisco, California,
4. Rohlf, F. J-, and Sokal, R. R., Statistical Tables. 1969.
W. H. Freeman Company, San Francisco, California.
f 18
-------
APPENDIX G: GUIDANCE FOR ASSESSING BIOACCUMULATION POTENTIAL
Introduction
1. The ocean disposal criteria require that the potential for
bioaccumulation of contaminants from dredged material be evaluated in
the technical assessment of permit applications. This requires pre-
dicting whether there is a cause-and-effect relationship between an
animal's presence in the area influenced by the dredged material and a
significant elevation of its tissue content or body burden of contami-
nants above that in similar animals not influenced by the disposal of
dredged material. That is, it must be predicted whether an animal's
exposure to the influence of the dredged material is likely to cause
a meaningful elevation of contaminants in its body.
2. A variety of laboratory research methods for measuring bio-
accumulation are presently undergoing modification and evaluation as
regulatory tools. All such methods require one or two months or more
for completion and provide no quantitative method for considering field
conditions such as mixing in the interpretation of the results, as re-
quired by the Register. Field sampling programs overcome the latter
difficulty since the animals are exposed to the conditions of mixing
and sediment transport actually occurring at the disposal site in
question. The former difficulty is also overcome if organisms already
living at the disposal site are utilized in the bioaccumulation studies.
The use of this approach for predictive purposes is technically valid
only where there exists a true historical precedent for the proposed
operation being evaluated. That is, it can be used only in the case of
maintenance dredging where the quality of the sediment to be dredged is
considered not to have deteriorated or become more contaminated since
the last dredging and disposal operation. In addition, the disposal
must be proposed for the site at which the dredged material in question
has been previously disposed or for a site of similar sediment type
supporting a similar biological community.
G1
-------
3. Considering these limiting conditions and following the pro-
cedure given below, it is possible to assess bioaccumulation by animals
that have spent major portions of their life in or on a sediment very
similar to the sediment in question under the physical and chemical con-
ditions actually occurring at the disposal site. Caged animals of
suitable species may also be placed at appropriate stations in and around
the disposal site, but this will require a substantial exposure time
before analysis. If the conditions discussed above cannot be met in the
field, a general approximation of bioaccumulation potential may be ob-
tained as described later in this appendix from animals used in the
suspended particulate or solid phase bioassays.
Field Assessment of Bioaccumulation Potential
Apparatus
4. The following is a general description of the major items
required. Additional miscellaneous equipment will have to be furnished.
a. A vessel capable of operating at the disposal site and
equipped to handle benthic sampling devices. Navigation
equipment must be sufficient to allow precise positioning.
b. Sampling devices such as a Smith-Maclntyre or other benthic
grab. Corers are less satisfactory since they sample a
smaller surface area and have a greater penetration than
is needed.
£. Stainless steel screens of 1-mm mesh to remove animals
from the sediment.
d. Tanks sufficient for transporting the animals to the
laboratory in collection site water.
-------
species must be collected at all stations since comparisons of bio-
accumulation cannot be made across species lines.
6. For each species at each station, a minimum of several grams
of tissue, as indicated in the references given in paragraph 20, must
be collected to provide sufficient sample to allow measurement of
chemical concentrations. In samples that do not contain sufficient
tissue, it will be impossible to quantify the amount of contaminant
present. Since data in the form of "concentration below detection
limits" is not quantitative, it is vital that sufficient tissue to
allow definitive measurement of concentration be collected for each
species at each station. It is also important that exactly equal masses
of tissue be analyzed for each station. If possible, several samples
of sufficient size for analysis should be collected at each sampling
station in order to provide a statistical estimate of variability in
tissue content of the contaminants of concern. The collection of more
than one sample per station, however, may prove impossible in practice
if small organisms must be used or if suitable organisms are not
abundant at the disposal site. In such cases the use of caged animals,
as discussed in paragraph 10, may be advisable.
7. It is desirable to select the largest appropriate species so as
to minimize the numbers and collection effort required. However, highly
mobile epifauna (such as crabs, lobsters, shrimp, and fish) should not
be used, since their location when collected cannot be related to their
body burden at the time of collection in any potential cause-and-effeet
manner. Therefore, relatively immobile species that are fairly large,
such as bivalves, some gastropods, large polychaetes, etc., ere the
most desirable organisms. Any relatively immobile species collectable
in sufficient numbers at all stations may be used, but tne required
collection effort increases sharply as organism size decreases.
Sampling design and conduct
8. Sufficient tissue to obtain definitive body burden values nut
be collected from each of at least three stations vicaia urn aispoaax
site boundaries and from each of at least tlx stations outside the
G3
-------
disposal site. The stations outside the site must be located in areas
with a substrate sedimentologically similar to that within the disposal
site. These stations outside the disposal site will serve two purposes.
If the direction of net bottom transport at the site is known, at least
three stations should be located in a substrate similar to that within
the site and in the path of transport away from the site. The data
from these stations will provide an indication of uptake of any con-
taminants transported out of the disposal site. At least three stations
must also be located in an uncontaminated sediment sedimentologically
similar to that within the site, but in a direction opposite that of the
net bottom transport. Data from these sites will provide a reference
level of contaminants in tissues to which those levels found in and down-
stream from the disposal site may be compared. If the direction of net
bottom transport is not known, at least six stations surrounding the
disposal site should be established in sediments sedimentologically
similar to those within the disposal site.
9. In all cases it is mandatory that several stations be sampled,
rather than collecting all of the animals at one station. This will
provide a measure of the variability that exists in tissue concentra-
tions in the animals in the area. Samples from all stations should be
collected the same day if possible and in any case within four days.
10. If caged animals placed around the disposal site are utilized
instead of free animals living there naturally, all the considerations
of paragraphs 8 and 9 must be evaluated in selecting the samping
stations, including the sedimentological similarity of the substrate at
all stations. The cages must be designed and positioned such that the
animals are able to burrow or establish their natural relationship to
the sediment in order to truly evaluate the influence of the dredged
material on bloaccumulatlon potential. Cages should not be constructed
of metal or coated with material that may leach the contaminants of
concern. They must be anchored and marked on the surface so that they
can be reliably located and recovered.
11. When the collection vessel has been positioned, repeated
G4
-------
collections are made at the same spot until an adequate sample is
obtained. The sediment obtained by the sampler is hosed through l-mm
stainless steel screens, and the retained individuals of the desired
species are placed in holding tanks. In all cases no animal with any
indication of injury should be retained.
12. Return the animals to the laboratory, being careful to label
the samples clearly and keep them separated and to maintain nonstressful
temperature and dissolved oxygen levels. In the laboratory, maintain
the samples in clean water in separate containers. No sediment is placed
in the containers and the animals are not fed. Any organisms that die
must be immediately discarded. Fecal material is siphoned from the
aquaria twice daily until little more is produced, indicating that all
material has been voided from the digestive tracts. This probably will
be completed within 2 to 3 days after collection, and sooner with small
animals. A more desirable procedure, if animals are large enought to
make it practical, is to excise the digestive tracts soon after collec-
tion rather than allowing the animals to excrete their contents. It is
necessary to empty or remove the digestive tracts since material there-
in may well contain inert constituents and the contaminants of concern
in forms that do not become biologically available during passage through
the digestive tract. Such material would also probably be unavailable
while passing through the digestive tract of any predator that might
have ingested the animals being analysed* Therefore, since the diges-
tive tract content has not been incorporated into the tissue, it would
give an- artifically high indication of bioaccuaulation if it were in-
cluded in the analysis.
13. The shells or exoskeletons of molluscs or crustaceans ere
removed and not included in the analysis. Thaaa
contain low levels of contaminants and would contribute weight but
little contaminants if they were included in the analysis. This would
give an artifically low indication of bioaccuaulation.
Analysis and interpretation
14. Preparation and analysis of tissues are by the procedures
65
-------
given in the "Chemical Analysis" section of this appendix. The section
on "Data Analysis and Interpretation" gives guidance on these matters.
Laboratory Assessment of Bioaccumulation Potential
Sampling design and conduct
15. This approach should be taken only in those cases where a true
historical precedent for the proposed operation does not exist (as
discussed in paragraph 2). The considerations of paragraphs 5, 6, and
7 should be kept in mind when selecting bioassay species to be used for
laboratory assessments of bioaccumulation potential.
16. Animals from solid or suspended particulate phase bioassays
may be used, but it is considered unlikely that important bioaccumula-
tion would occur at the disposal site from the latter phase, since
animals would be exposed to it for such short periods due to dilution.
At the end of the bioassay, surviving animals from the replicate con-
trols are treated in a manner corresponding to the separate reference
samples in the field assessment outlined earlier. Survivors from the
replicate sediment-exposure aquaria correspond to the samples from the
disposal site. In the case of suspended particulate bioassays,
survivors from the first replicate of all test medium concentrations
are pooled to make one sample corresponding to a disposal site sample;
survivors from the second replicate of all test medium concentrations
are pooled to make the second disposal site sample, etc.
17. At the end of the bioassay, each sample is placed in separate
aquaria in clean, sediment-free water to void the digestive tracts, as
discussed in paragraph 12. Each replicate from the bioassay is treated
as if it was a sample from the field assessment discussed earlier. If
very snail animals are to be analyzed, more than the minimum number
specified for the bioassay may have to be used, or more replicate aquaria
may be established in the bioassay. The considerations of paragraph 13
also apply to bioassay organisms used in assessing bioaccumulation
potential.
66
-------
Analysis and interpretation
18. Preparation and analysis of tissues are by the procedures
given in the "Chemical Analysis" section of this appendix. The section
on "Data Analysis and Interpretation" gives guidance on these matters.
Chemical Analysis
Constituents to be assessed
19. The chemical constituents to be assessed for bioaccumulation
are those constituents deemed critical by the District Engineer and
Regional Administrator after considering known inputs to the sediment
to be dredged. The following constituents, discussed in Section 227.6
of the Register, are of particular concern and should be assessed for
bioaccumulation whenever the District Engineer and Regional Adminis-
trator have any reason to believe they may be of concern in the sediment
in question.
_a. Organohalogen compounds (PCB's, DDT, etc.)
_b. Mercury and its compounds
c_. Cadmium and its compounds
Petroleum hydrocarbons
e. Known or suspected carcinogens, mutagens, or teratogens, (this
is a very poorly defined group of materials for which specific
analytical procedures are not generally available*)
Procedures
20. Referenced standard procedures for specific constituents are
given in Table Gl. These references should be consulted for detailed
guidance on amount of tissue required for analysis of each constituent
of concern, methods of sample preparation and analysis, and data pre-
sentation.
Data Analysis and Interpretation
21. Complete tissue concentration data for all samples should be
presented as in Table G2. A separate analysis Mist be conducted for
each chemical constituent and each animal species. This example
utilizes laboratory bioaccumulation data from analysing the survivors
of the hypothetical solid phase bioassay presented in Appendix V, The
-------
Table Gl
Procedural References for Analytical Methods for
Tissue Analyses of Organic Materials
Other
Material Reference 1 Reference 2 References
BHC Section 211, 212 Section 5A
Heptachlor " " " 3, 4
DDD, DDE, DDT " " " *" 5, 6, 7
Chlordane " " " " 8
Dieldrin " 7
Endrin " " " " 9
Toxaphene " " " " 4
PCB Sections 211, 212, 251 " 10
Mirex Section 211,212 " 11
, - If II II fl
Methoxychlor
Mercury and
its compounds 12
Cadmium and
its compounds 12
Petroleum
hydrocarbons:
Aliphatic 13
Aromatic 13
G8
-------
control and the dredged material samples from three sites were each
replicated five times, corresponding to the five replicates used here.
Small organisms were used; in one case, tissue concentration of the
constituent of concern was below detection limits. Such data are non-
quantitative and cannot be used in statistical analyses. However, the
arbitrary but environmentally protective assumption made in such cases
is that the actual concentration in the sample was only slightly less
than the detection limit, and the detection limit is used as if it was
the datum.
Table G2
Hypothetical Results* of a Laboratory Assessment
of Bioaccumulation Potential
Tissue
Concentration,
ppm (wet wt)
Replicate
(n = 5)
Control
Dredged Material Sample
12 3
1
0.15
0.27
0.25
0.15
2
0.08
0.42
0.38
0.12
3
0.38
0.24
0.52
0.24
4
<0.05
0.37
0.47
0.14
5
0.23
0.49
0.61
0.30
sum of data, IX
s*
0.89
1.79
2.23
0.95
- IX
mean, X * —
' n
=
0.18
0.36
0.45
0.19
2
sum of squared data ¦ IX
=
0.2287
0.6839
1.0703
0.2041
corrected sum of squares.
CSS - EX2 -
n
»
0.0703
0.0431
0.0757
0.0236
e2 CSS
variance, S = —r
' n-1
m
0.0176
0.0108 0.0189
0,0059
* The constituent measured and the animal species used in the assess-*
ment must be identified.
22. To determine whether there is an indication of bloaccuSKtlatlon
potential, it is necessary to make statistical comparisons of the tissue
concentrations in the controls to those in animals exposed to tha
69
-------
dredged materials. It is possible that in some cases the mean tissue
concentration in one or more of the dredged material samples may be less
than or equal to that in the controls. Such cases have been documented
and in no way reflect adversely on the quality of the evaluation, but
simply give no indication of bioaccumulation potential for the constit-
uent, species, and sediment sample in question.
23. If tissue concentration in any of the dredged material
samples is higher than that in the controls, the data must be compared
statistically. An analysis of variance (ANOVA) is used to compare the
mean tissue concentration in animals from the reference substrate con-
trol to the mean tissue concentration in animals exposed to each dredged
material sample. Before an ANOVA can be performed, it is necessary to
use Cochran's test to determine whether the variances of the data sets
are homogeneous. This is determined by calculating the C-value, defined
as the ratio of the largest variance to the sum of all the variances. In
this case: 2
Smax _ 0.0189 _ n _r
C ZT 0532 " °"3553 <«>
i.i b
where
2
S = largest variance among the data sets
max
2
2S = sum of all the variances
The calculated C-value is evaluated by comparing it to the C-value
given in the table in Enclosure 1 to Appendix D. In the table, k is
the number of treatment means summed in the denominator (4 in this case)
and v is one less than the number of observations contributing to each
variance (5 - 1 ¦ 4 in this case). Therefore, the tabulated value for
C in this example is 0.6287.
24. Since the calculated C-value is smaller than the tabulated
C-value, the calculated value is not significant at the 95-percent
confidence level, and the variances may be considered homogeneous. If
the calculated C-value is larger than the tabulated C-value, the
variances are not homogeneous. In such cases, before any ANOVA calcu-
t
lations are performed, a transformation should be performed on all data
G10
-------
in order to achieve homogeniety of variances. The transformation is
performed on each datum by obtaining the natural logarithm of ( X + 1 ),
where X is the datum. Recalculate the C-value using the transformed
data. If variances are now found to be homogeneous, use the transformed
data in all ANOVA calculations. If the variances are still non-
homogeneous, an approximate test of the equality of means given by
14
Sokal and Rohlf in their Box 13.2 should be used.
25. ANOVA equations and calculations for the data of Table G2 are
given in Table G3. The values on the third line of the table (Total)
should be the same whether they are calculated by the equation or
obtained by summing the corresponding treatment and error values, thus
providing an easy means of checking the accuracy of the calculations.
The calculated F-value is evaluated by comparison with the tabulated
F-value*^ at the 0.05-probability level with the appropriate degrees of
freedom (df). The df's are those given for the treatments and error,
respectively, in Table G3. The tabulated F-value with 3 and 16 df's
is shown at the bottom of Table G3. Since the calculated F-value ex-
ceeds the tabulated value, there is a statistical difference between
mean tissue concentrations among the four sets of data. If the calcu-
lated F-value had been equal to or less than the tabulated value, there
would be no statistical differences between tissue concentration in the
reference substrate controls and any of the dredged material samples.
In that case, the analysis would be complete at this point with no
indication of potential bioaccumulation from the dredged material in
question.
26. When the calculated F-value exceeds the tabulated value, it
is then necessary to determine which dredged material means differ
significantly from the reference substrate control mean. This may be
done by the Student-Newman-Keuls multiple-range test given by Sokal and
Rohlf in their Box 9.9.^ Least significant ranges (LSR) used in this
process are the product of the pooled standard error of the group mean
15
S_ and the studentized ranges Q given in Rohlf and Sokal'a Table U.
X
Gil
-------
Table G3
ANOVA Equation and Calculations for the Data of Table G2
Source of df Sum of Squares SS Mean Square MS F
Variation Equation Value Equation Value Equation Value Equation Value
Treatmentst (a-1) 3 I
(EX)2 (EIX)2 0.2573 SStreatment 0.0858 MStreatment 6.45*
n In a-1 MS
error
Error a(n-l) 16 ECSS 0.2127 SS _ nioo
error 0.0133
a(n-l)
2
Total (an)-l 19 I(£X2) - 0.4700
hn
t Number of treatments a = 4
* F.05(3,16) = 3,24
-------
S_ , JMS err°r . JOMM . 0_0516 (G2)
x
where the terms are taken from Table G3.
27. At the 0.05 level of significance, the Q and LSR values for
K = 2, 3, and 4 items are:
K
2 3 4
Q(Rohlf and Sokal, Table U15) = 2.998 3.649 4.046
S_ (equation G2) = 0.0516 0.0516 0.0516
X LSR = QS_ = 0.1547 0.1883 0.2088
X
28. The multiple-range test is completed by arranging the four
treatment means in increasing order and then comparing the difference
between means with the LSR for the number of means K in the range
separating the two being compared. That is, for two adjacent means
K = 2 and for comparing means separated by one mean, there is the
intervening mean and the two being compared, so that K = 3. It is
necessary to compare each dredged material mean to the control mean
but not to compare treatment means among themselves. Such comparisons
between treatment means are not necessary for permitting decisions, but
could provide useful managerial information by distinguishing sediments
with high bioaccumulation potential from those with a lesser but still
statistically significant bioaccumulation potential. The comparison of
treatment means to the control for the above example is given in the
following tabulation.
Treatment Means from Table G2
X „ , X- X. x9
control 3 1 Z
0.18 0.19 0.36 0.45
G13
-------
K
LSR
Difference Between Means
2
0.1547
X3 ~ = 0.19 — 0.18 = 0.01 n.s.
3
0.1883
X - X = 0.36 - 0.18 ® 0.18 n.s.
1 c
X - X « 0.45 - 0.18 = 0.27*
2 c
4
0.2088
Note; Entry of n.s. indicates difference is not signif-
icant at the 0.05-probability level; * indicates
difference is significant
29. When the difference between two means is greater than the LSR,
the difference between those means is statistically significant at the
0.05-probability level. Therefore, the multiple-range test has shown
that the mean tissue concentration of the constituent of concern in ani-
mals exposed to dredged material sample 2 is statistically higher than
the corresponding concentration in animals exposed to the control sedi-
ment. Tissue concentrations of this constituent in animals exposed to
dredged material samples 1 and 3 were not statistically higher than in
the control animals.
30. The ANOVA calculations and mean comparison given above may be
used for data analysis in all cases involving two or more treatments,
provided that the same number of samples occurs in each treatment. The
ANOVA calculations for studies in which the same number of samples does
not occur in each treatment are given by Sokal and Rohlf in their Box
9.1.1^ Unequal numbers of replicate samples may occur in field evalua-
tions where the direction of net bottom transport is not known and
samples outside the disposal site are located in all directions from the
site. In such cases, those stations outside the site having the highest
tissue concentrations cannot arbitrarily be assumed to lie in the
direction of net bottom transport, unless this is also indicated by
independent evidence. Otherwise the only analysis possible is to com-
pare the mean tissue concentration at the stations within the disposal
site to the pooled mean tissue concentration at all stations outside
the site. That is, two samples containing different numbers of
G14
-------
observations will be compared. When the direction of net bottom trans-
port is known, three mean tissue concentrations will be compared. These
will be from samples within the site, samples outside the site in the
direction of net bottom transport, and samples outside the site and not
influenced by net transport from the site.
31. In the example given in paragraphs 21 through 29, by compari-
son to the control animals, animals in one of the dredged material
samples had elevated tissue concentrations of the constituent of con-
cern, and those in the other two samples did not. Therefore, there is
a potential for bioaccumulation of this chemical by this species of
animal from sediments at one site in the dredging area.
32. At present there are very little data for marine species upon
which to base an evaluation of the meaning of a specific concentration
of a particular contaminant in the species in question. The only such
levels that are fixed from a regulatory viewpoint are those levels set
by the Food and Drug Administration for fish and shellfish for human
consumption. Therefore, this guidance recommends the environmentally
protective approach of assuming that any statistically significant
differences in tissue concentrations between control and exposed
organisms are a potential cause for concern. It should be kept in mind,
however, that at present tissue concentration of most constituents in
most species cannot be quantitatively related to biological effects.
Therefore, in making the final assessment of bioaccumulation, the
District Engineer and the Regional Administrator must objectively con-
sider the magnitude of bioaecuawlatlcn shown, the toxicological signifi-
cance of the material(s) bioaccumulated (I.e., arsenic would be of
greater concern than iron), the proportion of sediment sampling sites
which produced uptake, the number of different constituents bioaccumulated
from the sediment in question, the position is human sad nonhuman food
webs of the species showing uptake, the presence of motile species at
the site that might serve as transportation vectors removing bioaccumu-
lated materials from the disposal area, and other factors relevant to
the particular operation in question.
-------
REFERENCES
1. Food and Drug Administration, Pesticide Analytical Manual*. 1968,
U. S. Dept. of Health, Education and Welfare, Washington, D. C.
(looseleaf).
2. Thompson, J. F. (editor), Manual of Analytical Methods, "Analysis
of Pesticide Residues in Human and Environmental Samples," 1972,
EPA, National Environmental Research Center, Research Triangle
Park, North Carolina.
3. Schimmel, S. C., Patrick, J. M., Jr., Forester, J., "Heptachlor
Toxicity to and Uptake by Several Estuarine Organisms," Journal
of Toxicology and Environmental Health, 1:955-965, 1976.
4. Goodman, L. R., et al., "Effects of Heptachlor and Toxaphene on
Laboratory-Reared Embryos and Fry of the Sheepshead Minnow,"
Archives of Environmental Contamination and Toxicology (in press).
5. Nimmo, D. R., Wilson, A. J., Jr., and Blackman, R. R., "Localiza-
tion of DDT in the Body Organs of Pink and White Shrimp," Bulletin
of Environmental Contamination and Toxicology, 5(4):333-341, 1970.
6. Duke, T. W., and Wilson, A. J., Jr., "Chlorinated Hydrocarbons in
Livers of Fishes from the Northeastern Pacific Ocean," Pesticides
Monitoring Journal, 5(2):228-232, 1971.
7. Butler, Philip A., "Organochlorine Residues in Estuarine Molluscs,
1965-1972." Pesticides Monitoring Journal, 6(4):238-362, 1973.
8. Parrish, P. R., et al., "Chlordane: Effects on Several Estuarine
Organisms," Journal of Toxicology and Environmental Health.
1:485-494, 1976.
9. Schimmel. S. C., et. al., "Endrin: Effects on Several Estuarine
Organisms," Proceedings of the 28th Annual Conference of the
Southeastern Association of Game and Fish Commissioners, pp:187-193,
1974.
* Volume I (2nd Edition, 1968, and subsequent revisions) contains
directions and associated background information for multi-residue
methods used by FDA to analyze food and feed samples collected under
its surveillance programs. Volume XI (1967 and subsequent revisions)
contains methods that can be used to analyze for single compounds.
Each volume is revised continuously to reflect appropriate changes and
additions to the methodology and Information.
G16
-------
10. Duke, T. W., Lowe, J. I., and Wilson, A. J., Jr., "A Poly-
chlorinated Biphenyl (AroclorR 1254) in the Water, Sediment, and
Biota of Escambia Bay, Florida," Bulletin of Environmental
Contamination and Toxicology, 5(2):171-180, 1970.
11. Lowe, J. I., et al., "Effects of Mirex on Selected Estuarine
Organisms," Transactions of the 36th North American Wildlife and
Natural Resources Conference, 171-186, 1971.
12^ Goldberg, E. D. (editor), Strategies for Marine Pollution
Monitoring, 1976, Wiley Interscience.
13. Warner, J. S., "Determination of Aliphatic and Aromatic Hydro-
carbons in Marine Organisms," Analytical Chemistry, 48:578-583,
1976.
14. Sokal, R. R., and Rohlf, F. J., Biometry, 1969, W. H. Freeman
Company, San Francisco, California.
15. Rohlf, F. J., and Sokal, R. R., Statistical Tables, 1969,
W. H. Freeman Company, San Francisco, California.
SELECTED BIBLIOGRAPHY
1. Federal Working Group on Pesticide Management, "Guidelines on
Analytical Methodology for Pesticide Residue Monitoring,"
Washington, D. C.
2. Horwitz, W. (editor), Official Methods of Analysis of the
Association of Official Analytical Chemists**. 12th ed., 1975,
Association of Official Analytical Chemists, Washington, D, C.
3. Reichel, W. L. (compiler), Analytical Manual for Organochlorine
Compounds, U. S. Department of the Interior, Fish and Wildlife
Service, Patuxent Wildlife Research Center, Laurel, Maryland.
(Manual developed for internal use.)
4. Stalling, D. L., "Analyses of Organochlorine Residues in Fish:
Current Research at the Fish-Pesticide Laboratory," Methods in
Residue Analysis, Vol. IV, Proceedings of the 2nd International
IUPAC Congress of Pesticide Chemistry, Tel Aviv, Isreal, 1971,
Gordon and Breach Science Publishers, New York, New York.
** This manual is revised every four years and annual supplements of
changes in the official methods are issued in March of the inter-
vening years.
G17
-------
Tindel, R. C. (compiler), Handbook of Procedures for Pesticide
Residue Analysis, Technical Paper No. 65, 1972, U. S. Department
of the Interior, Fish and Wildlife Service, Bureau of Sport
Fisheries and Wildlife, Washington, D. C.
U. S. Environmental Protection Agency, Laboratory Manual, Report
PML-2, 1975, Pesticides Monitoring Laboratory, Bay St. Louis,
Mississippi. (Draft report marked for internal use only.)
G18
-------
APPENDIX H: ESTIMATION OF INITIAL MIXING
Introduction
1. The Register recognizes the fact that the oceanic environment
is physically dynamic and that materials dumped into it will be dis-
persed, mixed, and diluted to some degree. Therefore, all evaluative
procedures must be interpreted in light of the initial mixing expected
at the disposal site. Initial mixing is defined (paragraph 227.29(a)
of the Register) to be that dispersion or diffusion of liquid, sus-
pended particulate, and solid phases of a waste that occurs within 4 hr
after disposal. The limiting permissible concentration (Section 227.27)
shall not be exceeded beyond the boundaries of the disposal site during
the 4-hr initial mixing period and shall not be exceeded at any point
in the marine environment after initial mixing. A series of methods,
discussed in the order of preference shown in Section 227.29 of the
Register, may be used to estimate the maximum concentration of the liquid
and suspended particulate phases found at the disposal site after initial
mixing. Since no objective method has been devised for incorporating
initial mixing of the solid phase into the interpretation of bioassay
results, no calculations are presented here for estimation of the
initial mixing of the solid phase.
Initial Mixing Calculations
Mathematical models using specific
field data (227.29(a)(1))
2. The first and most preferred method requires the use of
comprehensive field data relevant to the proposed disposal operation
in conjunction with an appropriate mathematical model for adequate
prediction of initial mixing and dispersion. Bowever, the amount of
field data necessary for adequate prediction ofdispersion and dif-
fusion is substantial, and such predictions require a detailed under-
standing of tides, currents, waves, water column stratification, and
climatic conditions at the disposal site.
HI
-------
3. Description of the WES mathematical models. The Dredged
Material Research Program (DMRP) of the U. S. Army Engineer Waterways
Experiment Station (WES) has modified two numerical models, now being
field verified, to predict the short-term fate of dredged material dis-
charged in the marine environment. One model simulates an instantaneous
discharge from a rapidly emptying barge or hopper. The other simulates
a continuous fixed or moving jet discharge from a slowly emptying vessel.
The models can be applied to disposal sites in enclosed bodies of water,
sites with depth variations, sites whose flow regimes vary in three
dimensions and in time, and those disposal sites where ambient density
varies in time.
4. It should be noted that neither adequate calibration nor
verification of the models has been completed at this time. However,
from limited demonstrations and calibrations, the models have been
shown conceptually to be capable of predicting the dynamic physical
processess associated with various dredged material disposal operations
in the marine environment. The models will not be generally available
until the calibrations and verifications necessary to ensure the accu-
racy of model predictions have been concluded. This is expected to be
completed by early 1978. The availability of the models for general
use will be announced. At that time the WES models will become the
preferred means of estimating initial mixing for most disposal opera-
tions whose size or potential impact warrant this level of sophistica-
tion. The release zone method described in paragraphs 10 through 28 may
still be acceptable for small projects of little anticipated impact.
5. In consideration of those who are planning a field data-
collection program in anticipation of use of the WES mathematical models
or who may think adequate data are available for input to the models
discussed in paragraph 3, a brief description of the two models is
given with specific requirements of the necessary input data for
optimal model utilization. Both models characterize the behavior of
released dredged material with a convective descent phase in which the
cloud has a high density relative to the disposal site water and is
H2
-------
dominated by gravitational forces; a dynamic collapse phase in which
horizontal spreading dominates, usually initiated when the descending
cloud impacts the bottom; and a long-term dispersion phase, which is
dominated by the ambient currents and turbulent diffusion at the disposal
site, rather than the forces of the disposal operation.
6. WES model input requirements. Ocean disposal of dredged
material is usually made by barges or scows and hopper dredges. When
a barge releases its material in a stationary mode (i.e., not underway),
the disposal can be assumed to be an instantaneous dump for modeling
purposes and the instantaneous model can be used to simulate this opera-
tion with the proper input parameters. Moving barges having to open
several doors to release all the material would be best described by
the moving jet model. The moving jet model also best describes most
hopper dredge disposal operations in which one or two doors are usually
opened at a time and up to thirty minutes may be required to remove all
the material. The characteristics of each specific disposal operation
will determine the model appropriate to its simulation and thus will
determine the model input data required. Adequate input data can be
obtained only with a comprehensive understanding of the factors affect-
ing the dredging and disposal operations and by a thorough field sampling
program based on this understanding.
7. Input data required to run the models can be categorized as (a)
data describing the actual disposal operation, (b) characterization of
the dredged material, (c) a description of the ambient environment, and
(d) model coefficients.
a. Disposal data. For the instantaneous or stationary dis-
charge model, the grid position of the barge on the horizontal grid,
the radius of the initial cloud, the depth below the surface where the
material is released, and the initial velocity of the cloud are re-
quired. Generally the radius of the initial cloud will be determined
by the total volume of the barge. The fixed or moving jet discharge
model requires the initial position of the discharge, the vessel course
and speed, the orientation and depth of the discharge point in the
H3
-------
water column, the radius of the initial jet, the flow rate, and the
total time required for complete discharge.
Characterization of dredged material. The models will
accept up to twelve solid fractions (grain sizes), a fluid component,
and a conservative chemical constituent (e.g., ammonia) if desired.
The concentration, density, fall velocity, void ratio, and an indicator
of cohesion must be input for each solid fraction. If a conservative
chemical constituent is to be used, its initial concentration in the
liquid phase and a background concentration in the disposal site water
must be given. In addition, the bulk density and aggregate void ratio
of the dredged material must be determined. It is not necessary to in-
put all twelve solid fractions to adequately simulate the dispersion
processes during disposal, especially when there is a cohesive fraction
included. An important but difficult value to obtain prior to dredging
is the bulk density of the total volume of dredged material in the barge.
If large volumes of water are dredged with the actual material, the bulk
density values will be substantially reduced.
c. Description of ambient environment. An ambient density
profile must be supplied and, at each horizontal grid point, water depth
and a current velocity profile are required. The level of sophistication
here is optional, as there are three different fcfrms of velocity input,
the depths may be constant or variable in space, and the density pro-
file may vary with time or remain constant.
d. Model coefficients. The models contain recommended
average values for fourteen coefficients, which the user should change
only if justified by case-specific data.
Similar field data and
modeling (227.29(a)(2))
8. The second method of initial mixing estimation permitted by the
Register allows field data determined for a material of similar charac-
teristics to be used in conjunction with an appropriate model. There
may be certain similarities between dredged material disposal operations
in different regions of the country that may allow the use of similar
H4
-------
Input data to simulate a proposed disposal operation; however, the
similarities have not yet been documented that could justify this
method for prediction of dredged material dispersion. Certainly, there
is no justification for using input data developed for other waste
material in attempts to predict dredged material dispersion.
Theoretical relationships (227.29(a)(3))
9. When no field data are available, the Register permits con-
sideration of theoretical oceanic turbulent diffusion relationships in
order to estimate initial mixing. The state-of-the-art of dredged
material dispersion theory does not presently allow the use of this
method for adequate prediction of initial mixing processes.
Release zone method (227.29(b))
10. Since none of the preceding three methods are feasible until
the models are verified (at which time the models will become the
generally preferred method), the release zone method of estimating
initial mixing must be used in the interim. The liquid and suspended
particulate phases of the dredged material may be assumed to be evenly
distributed at the end of the 4-hr initial mixing period over a column
of water bounded on the surface by the locus of points constantly 100 m
from the perimeter of the conveyance engaged in dumping activities,
beginning at the first moment in which dumping commences and ending at
the last moment (the release zone) and extending to the ocean floor,
thermocline, or halocline if one exists, or to a depth of 20 m, which-
ever is shallower.
11. In order to calculate the initial mixing zone vising the re-
lease zone method, a few preliminary determinations have to be made.
First, one must determine the appropriate depth value: is the thermo-
cline or halocline, the ocean bottom, or 20 m the shallower value? For
the following example calculation, it was assumed that the depth of the
bottom was 30 m and that there was no density stratification, so 20 m
is the appropriate depth value.
12. Next, one must determine the mode of disposal; is the disposal
vessel moving or stationary? This example assumes that a disposal vessel
H5
-------
60 m long and 18 m wide was moving at a speed of one m/sec and took 100
sec to release all its volume of dredged material. With these data an
initial mixing zone volume may be calculated using the following
equation:
V = 7r(100)2d + 200 w d + (200 + w)(u t + fc) d (HI)
m
where
tt = 3.1416
d = appropriate depth value (here 20 m)
w = width of the disposal vessel
Z = length of the disposal vessel
u = speed of the disposal vessel in metres per second
t = time in seconds required to empty disposal vessel
during discharge
13. By equation HI, the volume of the example initial mixing zone
would be:
V = (3.1416) (100m)2(20 m) + 200 m (18 m) (20 m)
m
+ (200 m + 18 m) [(1 m/sec)(100 sec) + 60 m] 20 m
V = 1,397,920 m3
m '
14. If the discharge is instantaneous or from a stationary vessel,
equation HI reduces to:
V = fr(100)2d + 200 w d -I- (200 + w) 1 d (H2)
m
where the terms are defined as for equation HI.
Application to Limiting Permissible Concentration (LPC)
Liquid phase - water-quality
criteria (227.27(a)(1))
15. The LPC of the liquid phase for constituents for which appli-
cable water-quality criteria have been established is that concentration
at which none of the constituents of concern will exceed the criteria
after allowance for initial mixing. It is possible to predict whether
the LPC will be exceeded by the method given in the following example.
16. In this example, the liquid phase was assumed to have a
measured concentration of ammonia (the constituent of concern) of 30
H6
-------
mg/Z and the disposal site water to have a measured concentration of
0.1 mg/%. The water-quality criterion for the constituent of concern
(ammonia) must be determined from the most recent edition of the EPA
publication "Quality Criteria for Water." If the water temperature were
assumed to be 15°C and the pH of the water to be 8.0, then the water-
quality criterion for total ammonia would be found to be 0.75 mg/Jl.
17. The dilution factor D (the amount by which the liquid phase
must be diluted to meet the water-quality criterion) can be determined
from the following equation:
~ _ Ce~ Cs _ 30 - 0.75 _ ., .
C - C " 0.75 - 0.1 ~ 45,0 (H3)
s a
where
Ce = liquid phase concentration of the constituent
of interest (ammonia) = 30 mg/Jl
C = water-quality criterion for the constituent
S of interest = 0.75 mgJI
C = ambient disposal site water concentration of
constituent of interest ¦ 0.1 mg/£
Note that if the liquid phase concentration Cg is less than the water-
quality criterion C , no calculation is necessary since no dilution is
s
required to meet the criterion. If the ambient disposal site water
concentration C is greater than the water-quality criterion C , water
3 S
quality at the disposal site violates the criterion regardless of the
proposed disposal operation, and the criterion cannot be achieved by
dilution.
18. The volume of the liquid phase V can be calculated by equa-
w
tion H4. For purposes of this calculation, the bulk density of the
dredged material may be assumed to be 1.5, the particle density 2.6,
and the density of the liquid phase 1.0. These approximations should
be used unless these parameters have actually been measured for the
dredged material in question.
Vw - pb 1 pd(V ) ¦ <3058 a) - 2102 *3
w d
H7
-------
where
= bulk density (1.5)
P, = particle density (2.6)
a
Pw = density of liquid phase (1.0)
VT = total volume of disposal vessel
(here assumed to be 3058 m^ or 4000 yd3)
19. The volume of disposal site water necessary to dilute the
discharged liquid phase to acceptable levels can be found using the
equation:
Vol - D Vw = 45 (2102 m3) = 94,590 m3 .(H5)
where
Vol = required volume of disposal site water
D = dilution factor = 45.0 (equation H3)
V = the volume of liquid phase in the discharge = 2102 m
W (equation H4)
20. In this example, ammonia would not exceed the LPC, since the
3
volume of the initial mixing zone (1,397,920 m from paragraph 12 and
equation HI) exceeded the volume of disposal site water necessary to
dilute the liquid phase to the water-quality criterion for the constit-
3
uent of interest (94,590 on from paragraph 19 and equation H5). Note
that these calculations must be performed for each constituent of
concern, since the dilution factor D (equation H3) will be site specific
and different for every constituent. The LPC is met only if the appli-
cable water-quality criteria are met by all constituents of concern.
Liquid phase - no water-quality
criteria (227.27(a)(2))
21. If bioassays are conducted with the liquid phase, the above
approach must be modified, since the constituent(s) causing effects in
bioassays cannot be identified, and therefore their concentrations in
the liquid phase or disposal site water cannot be measured. The LPC
applicable to liquid phase bioassay interpretation is the concentration
that, after initial mixing, will not exceed a toxicity threshold of 0.01
of the acutely toxic concentration. The liquid phase bioassay pro-
cedures of Appendices D and E require exposure of organisms to various
R8
-------
dilutions, expressed in percent of original liquid phase concentration.
In order to predict whether the LPC will be exceeded, it is necessary
that the dilution expected at the disposal site after initial mixing
also be expressed in terms of percent of original liquid phase concen-
tration. This may be done by comparing the" dilution calculated by
equation H6 to the bioassay results.
22. The volume of the initial mixing zone is calculated as in the
example above, using equation Hi or H2 as appropriate. In this case it
3
was found to be 1,397,920 m . The volume of the liquid phase contained
in the discharge vessel is then calculated by equation H4; in this
3
example, it was found to be 2102 m . The percent of the original liquid
phase concentration found at the disposal site after initial mixing C
w
may be calculated as:
V 3
C - (100) 2102 n (100) - 0.15% (H6)
M m 1,397,920 w?
where
V ¦ volume of liquid phase released in the discharge
W (equation H4)
=* volume of the initial mixing zone (equation Hi)
23. According to the solution of equation H6, in this example the
original concentration of the liquid phase was diluted by a factor of
667, so that the concentration after initial mixing was only 0.15 per-
cent of the original liquid phase concentration at the instant of re-
lease. In order to predict whether this would exceed the LPC, it is
necessary to determine whether this concentration is higher or lower
than 0.01 (or other factor) of the acutely toxic concentration. This is
done by graphically comparing the dilution curve to the time-concentration
mortality curve as described in paragraphs 38 through 40 of Appendix D.
Suspended particulate phase (227.27(b))
24. Initial mixing of the suspended particulate phase is estimated
in a manner similar to that described in paragraphs 21 through 23 for the
liquid phase without water-quality criteria. First the volume of the
initial mixing zone is calculated, using equation HI or H2 as appropri-
3
ate. In this example the initial mixing zone volume is 1,397,920 m .
H9
-------
25. The volume of suspended particulate phase contained in the
disposal vessel must then be determined. Since it is impractical to
calculate the volume directly, the environmentally protective assump-
tions are made that all silt and clay-sized particles are contained in
the suspended particulate phase and that they would remain in
suspension during the 4-hr initial mixing period. If adequate data are
available for the operation in question to demonstrate that this assump-
tion is invalid, the most accurate estimate of the percent of material
that would remain -in suspension should be incorporated in the calcula-
tions. The volume of suspended particulate phase in the discharge V
can be calculated as:
>
Vsp = (VT " V 100
sp
where
3
VT = total volume of discharge vessel (3058 m )
¦ volume of liquid phase in the discharge
(2102 m^ from equation H4)
w
Pc « percent clay in the dredged sediment
Pg - percent silt in the dredged sediment
26. In this example, assumed to be from harbor maintenance dredg-
ing and to have 50 percent clay and 40 percent silt, the volume of sus-
pended particulate phase in the discharge would be:
Vsp - (3058 m3 - 2102 m3) (40IqQ50^ - (956 m3)(0.90) - 860 m3
27. The percent of the original suspended particulate phase
concentration found at the disposal site after initial mixing C is
sp
calculated from a slight modification of equation H6 as:
V 3
Csp " V52- (100) (100) * °*06*
m 1,397,920 m3
where
V ¦ volume of suspended particulate phase in the
sp discharge (equation H7)
V ¦ volume of the Initial mixing zone (equation Hi)
m
28. According to the solution of equation H8, the original sus-
H10
-------
pended particulate phase concentration was diluted by a factor of 1667,
so that the concentration at the disposal site after initial mixing was
only 0.06 percent of the original suspended particulate phase concen-
tration at the instant of release. In order to predict whether this
would exceed the LPC, one must determine whether C as calculated in
sp
the preceding example is higher or lower than 0.01 (or other factor) of
the acutely toxic concentration. This may be done by graphical compari-
son of the time-concentration mortality curve and the dilution curve,
as discussed in paragraphs 38 through 40 of Appendix D.
Hll
------- |